University of Southern California Dissertations and Theses

Interaction between 11β-hydroxylation and respiration In bovine adrenocortical mitochondria

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Interaction between 11β-hydroxylation and respiration In bovine adrenocortical mitochondria

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Content INTERACTION BETW EEN 11P-HYDROXYLATION
A N D RESPIRATION IN BOVINE
ADRENOCORTICAL MITOCHONDRIA
by
Susan.B anks Oldham
A D is s e r ta tio n P re s e n te d t o th e
FACULTY OF THE GRAD U ATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In P a r t i a l F u lf illm e n t of th e
R equirem ents f o r th e D egree
DOCTOR OF PHILOSOPHY
(B io ch e m istry )
Ja n u a ry 1968
UNIVERSITY OF SOUTHERN CAUFORNIA
THE GRADUATE SCHOOL
UNIVERSITY HARK
LOS ANOELES, CALIFORNIA S0007
This dissertation, written by
__________________
under the direction of h.sz... Dissertation Com
mittee, and approved by all its members, has
been presented to and accepted by the Graduate
School, in partial fulfillment of requirements
for the degree of
D O C TO R OF P H IL O S O P H Y
_________
Dean
Date. Jan u ary. 1968
DISSERTATION COMMITTEE
Ch
ACKNOWLEDGEMENTS
I would lik e to e x p re ss s in c e re g r a titu d e to
D r. Boyd H ard in g , my re s e a rc h a d v is o r, f o r h is guidance
and s u p p o rt. My a p p re c ia tio n i s a ls o extended to
D r. John Mehl and D r. A rnold B ro d ie f o r t h e i r h e lp a s
members o f my D is s e r ta tio n Com m ittee.
I would a ls o lik e to th a n k D r. W illiam L an d g raf,
C h ie f, EPR L a b o ra to rie s , V arian A s s o c ia te s , P a lo A lto ,
f o r h is a s s is ta n c e w ith th e EPR s tu d ie s .
A word o f s p e c ia l th an k s b elo n g s to th e p eople
w ith whom I have worked th e p a s t two y e a rs — t o M iss
J u l i a B e ll, M iss Carmen A le ja n d ro , and M rs. F ran ces
Nakamura, whose w illin g a s s is ta n c e h as been g r e a tly
a p p re c ia te d ; to M rs. M arilyn S c h ie fe lb e in , who has
sp e n t numerous h o u rs draw ing and p h o to g rap h in g f ig u r e s
f o r me in a d d itio n t o b ein g e n d le s s ly h e lp f u l as
s e c r e ta r y t o us a l l ; and to D r. Low ell W ilson, who has
been e v e r ready w ith th e a d v ic e , encouragem ent and
w illin g e a r of a f r ie n d a s w e ll a s a c o o p e ra tiv e
a s s o c i a t e . They and D r. H arding have made my g rad u a te
s tu d ie s a very e n jo y a b le e x p e rie n c e .
G ra te fu l acknowledgement i s made to th e D e p art
ment o f B io ch e m istry , U n iv e rs ity o f Southern C a lif o r n ia ,
f o r su p p o rt from U .S. P u b lic H e alth S e rv ic e T ra in in g
G rant funds th ro u g h o u t my g ra d u a te s tu d ie s .
—S . B. 0 .
ii
DEDICATION
I d e d ic a te t h i s d i s s e r t a t i o n to my husband,
Bob, w ith o u t whose en th u siasm and su p p o rt g rad u a te
stu d y would have been im p o ssib le .
TABLE OF CONTENTS
A CK N O W LED G EM EN TS ............................................................................................
DEDICATION .........................................................................................................
LIST OF TABLES ................................................................................................
LIST OF ILLUSTRATIONS . ..............................................................................
C h ap ter
! I . INTRODUCTION .......................................................................................
| I I . MATERIALS A N D M ETH O D S .................................................................
j P re p a ra tio n of A d re n o c o rtic a l M ito ch o n d ria
In c u b a tio n M edia
I S o u rces of Im p o rta n t R eagents
A b so rp tio n S p e c tra
; K in e tic S tu d ie s
! P o la ro g ra p h ic
S p e c tro p h o to m e tric
F lu o ro m e tric
i
!
H P -H y d ro x y la tin g A c tiv ity
P y rid in e N u c le o tid e s
P re s e n ta tio n o f D ata
D e fin itio n o f M etab o lic S ta te s
Approach t o S tead y S ta te Method o f C a lc u la tin g
R ate C o n stan ts
I I I . PROPERTIES OF THE STEROID llp-HYDROXYLASE SYSTEM . .
I H is to r ic a l Background
A ssessm ent o f M agnitude o f A c tiv ity
C hapter
IV . PROPERTIES OF CY TO C H R O M E P-450 ........................................ .
i
! H i s t o r i c a l Background
S p e c tr a l and EPR E f fe c ts o f S u b s tra te s and
E le c tro n Donors
V. HYDROXYLATION AS A MITOCHONDRIAL FUNCTION ...................
H is to ry o f E n e rg y -lin k e d F u n c tio n s o f
M ito ch o n d ria
R e s p ira to ry C o n tro l
R everse E le c tro n T ra n sp o rt
C alcium A ccum ulation
j P y rid in e N u c le o tid e T ran sh y d ro g en atio n
F u n c tio n s o f A d re n o c o rtic a l M ito ch o n d ria and
I T h e ir E f fe c ts on H y d ro x y latio n
i
| R e s p ira to ry C o n tro l
P y rid in e N u c le o tid e R eduction by S u c c in a te
: P y rid in e N u c le o tid e R eduction by S u lf id e
| p lu s ATP
; C alcium A ccum ulation
A sco rb ate O x id a tio n
j P y rid in e N u c le o tid e T ran sh y d ro g en atio n
I V I. CLASSIC INHIBITORS OF CORTICOSTEROID BIOSYNTHESIS. .
; H i s t o r i c a l Background
j S p e c tr a l and K in e tic S tu d ie s
j
! V II. DISCUSSION ...........................................................................................
y m . SUMMARY................................................................................................
I
Li s t of r e f e r e n c e s ......................................................................................
LIST OF TABLES
T able Page
i
I . S te r o id E f fe c ts on C yanide I n s e n s i t i v e Oxygen
j U t i li z a t io n ............................................................. . . . . . 28
i
j I I . 110-Hydroxy l a t io n S up p o rted by S u lf id e and ATP . . . 85
I
I
j l l l . A sco rb ic A cid -S u p p o rted H y d r o x y l a t i o n .............................. 98
i
j IV . The E f f e c t of U n co u p ler on th e R a te of
| 11 0 -H y d ro x y latio n ...................................................................... 110
i
V. Com parison of R a te s o f P y rid in e N u c le o tid e
R ed u ctio n and C o r t is o l P r o d u c t i o n ................................. 129
vi
26
37
39
42
44
46
48
50
52
67
69
LIST OF ILLUSTRATIONS
Com parison o f Oxygen U tiliz e d by R e s p ira tio n and
lip -H y d ro x y la tio n ....................................................................
The D iffe re n c e Spectrum Produced by 11-Deoxy-
c o r t i s o l in A erobic M ito c h o n d r ia .......................... . .
The D iffe re n c e Spectrum Produced by A ^-Pregnenolone
in A erobic M ito ch o n d ria .......................................................
The D iffe re n c e Spectrum Produced by S e le c tiv e
R ed u ctio n o f th e H ydroxylase System ............................
L iq u id N itro g en T em perature D iffe re n c e Spectrum of
Reduced Minus O xidized H ydroxylase System . . . .
A K in e tic S tudy o f R eduction o f Cytochromes b and
P-450 and th e 1 1 -D e o x y co rtiso l-In d u c e d O xid atio n
o f Cytochrome P-450 ................................................................
C o rre la tio n o f H y d ro x y latio n o f 1 1 -D e o x y co rtiso l
w ith th e O x id atio n S ta te o f Cytochrome P-450 . . .
D em onstration o f Oxygen R equirem ent f o r 11-Deoxy-
c o rtis o l- I n d u c e d D iffe re n c e Spectrum ...........................
The EPR S p e c tra ( F i r s t D e riv a tiv e ) of Bovine
A d re n o c o rtic a l M ito ch o n d ria a t 77° K ...........................
Comparison o f F lu o re sc e n c e Produced by S u c c in a te
and M alate ......................................................................................
Com parison o f F lu o re sc e n c e Produced by M alate and
a -K e to g lu ta ra te p lu s M alonate .........................................
vii
12. The E ffe c t o f ADF on P y rid in e N u c leo tid e
F lu o rescen ce and R e s p ira tio n ............................................. 72
13. E ffe c ts o f Rotenone and Amytal on S u c c in a te
R eduction o f P y rid in e N u c leo tid e .................................... 76
i
I 14. I n i t i a l Time C ourse of S u c c in a te -S u p p o rted
| H y d r o x y l a t i o n .............................................................................. 78
i 15. Dependence o f H ydroxylation on M alate
| C o n cen tratio n .............................................................................. 81
!
16. E ffe c t o f S u lfid e and ATP on F lu o rescen ce and
Oxygen E l e c t r o d e ........................................................................ 82
!
' 17. E ffe c t o f Calcium on S uccin ate-P ro d u ced F lu o re s
cence in th e P resen ce and Absence o f Phosphate . • 87
18. E ffe c t o f Calcium on F lu o rescen ce and
! H y d r o x y l a t i o n .............................................................................. 90
! 19. F lu o rescen ce Produced by Ascorbate-QMPD ....................... 93
20. E ffe c t o f P en tach lo ro p h en o l (PCP) on F lu o rescen ce
and Oxygen U tiliz a tio n in th e P resen ce o f
Ascorbate-TMPD ............................................................... 95
i
I
21. E ffe c t o f 1 1 -D eo x y co rtiso l on F lu o re sc en c e Produced
| by Ascorbate-QMPD ...................................................................... 97
j 2 2 . The E ffe c t o f H ydroxylation on th e O x id atio n S ta te
| of P y rid in e N u c leo tid e s in th e P resen ce of
| S u c c in a te , M alate, and a -K e to g lu ta ra te p lu s
| M a lo n a te ............................................................................................ 106
j 23. R e c ip ro c a l P lo ts o f th e E ffe c t o f M ethopyrapone on
j llp -H y d ro x y la tio n o f 1 1 -D eo x y co rtiso l ........................ 116
J 24. E ffe c ts o f Amphenone on R ates o f R eduction and
O xidation o f Cytochrome P-450 118
viii
F ig u re Page
2 5 . E f f e c t o f o , p ’-DDD on th e R a te s o f R ed u ctio n and
O x id a tio n o f Cytochrom e P-450 120
26# E f f e c t o f o ,p ’-DDD on P y rid in e N u c le o tid e
F lu o re sc e n c e ................................................................................... 122
2 7. Pathw ays o f E le c tro n T ra n s p o rt in A d re n o c o rtic a l
M ito ch o n d ria and R e a c tio n s o f Cytochrome P-450
w ith S t e r o i d ................................................................................... 126
ix
i
I
CHAPTER I
INTRODUCTION
S te r e o s p e c if ic h y d ro x y la tio n s predom inate in th e s e r ie s
o f enzymic re a c tio n s by which horm onally a c tiv e s te r o id s a re
formed from c h o le s te r o l in th e a d re n a l c o rte x . These re a c tio n s
u t i l i z e m o lecu lar oxygen and reduced n ico tin am id e d in u c le o tid e
phosphate (NADPH) in th e fo llo w in g sto ic h io m e try :
H + + A H 2 + N ADPH + 0 2 — * A H O H + NADP+ + H20
Mason has c l a s s i f i e d such re a c tio n s a s mixed fu n c tio n o x id a
tio n s ( 1) . The mechanism of t h i s p ro ce ss i s la r g e ly unknown, and
only re c e n tly have th e enzyme system s in v o lv ed been i d e n t i f i e d .
The f i r s t c lu e as to th e n a tu re o f th e s e mixed fu n c tio n
o x id ases was th e fin d in g by Ryan and E ngel t h a t carbon monoxide
in h ib ite d s te r o id C-21 h y d ro x y latio n by a d re n o c o rtic a l m icro-
somes ( 2 ) . E stabrook and co-w orkers o b tain ed ev id en ce f o r th e
'presence o f a carbon m onoxide-binding pigm ent in a d re n a l m icro-
jsomes ( 3 ) , and u sin g th e photochem ical a c tio n spectrum tec h n iq u e
th e y showed t h a t t h i s pigm ent was th e oxygen a c tiv a tin g enzyme o f
1
!th e C-21 h y d ro x y lase sy stem . A pigm ent w ith s im ila r s p e c t r a l
!p r o p e r tie s when combined w ith CO had been p re v io u s ly o b serv ed in
i
; l i v e r m icrosom es ( 4 , 5 ) . Omura and S a to had c h a r a c te r iz e d th e
l i v e r pigm ent a s a hem oprotein and b ecau se o f th e a b so rp tio n
maximum o f i t s CO-complex a t 450 m|i, named i t P-450 ( 6 , 7 ) . When
th e p h o to ch em ical a c tio n sp ectru m te c h n iq u e was a p p lie d t o
s e v e r a l d ru g m e ta b o liz in g a c t i v i t i e s o f l i v e r m icrosom es, P-450
was a ls o found t o be th e te r m in a l o x id a se o f th e s e m ixed fu n c tio n
o x id a se system s ( 8) .
P r i o r t o th e o b s e rv a tio n o f H ard in g e t a l . t h a t P-450 was
p re s e n t in m ito c h o n d ria o f a d r e n o c o r tic a l t i s s u e ( 9 ) , t h i s ty p e
o f mixed fu n c tio n o x id a se was th o u g h t t o be p u re ly m icrosom al.
In f a c t ,,re tic u lo c h ro m e ,, was su g g e ste d a s an a l t e r n a t i v e name f o r
P -4 5 0 . However, 1 1 0 -h y d ro x y la tio n was re c o g n iz e d t o be c a ta ly z e d
i
jby P-450 when i t was found t o be i n h ib i t e d by carbon monoxide and
I
jthe d e g re e o f i n h ib i t io n was shown t o c o r r e l a t e w ith fo rm atio n o f
th e QO-complex ( 1 0 ) . S in c e th e n th e c o n v ersio n o f c h o le s te r o l t o
p reg n e n o lo n e, w hich in v o lv e s a t l e a s t two h y d ro x y la tio n s a t C-20
iand C-22 (1 1 , 12, 1 3 ), has been found t o be in h ib i t a b l e by carbon
i
monoxide ( 1 4 ) . In a d d itio n C-18 h y d ro x y la tio n has been shown by
jp h o to a c tiv a tio n to in v o lv e P-450 ( 1 5 ) . These a c t i v i t i e s a re a l l
a s s o c ia te d w ith th e m ito c h o n d ria l f r a c t io n (1 6 , 17, 1 8 ). W hether
•the P-450 i s i d e n t i c a l in th e s e v a rio u s system s o r w hether a
|
•s p e c ific P-450 i s a s s o c ia te d w ith each system i s s t i l l n o t c le a r .
i
jThe 110-hydroxylase system was s e le c te d f o r stu d y as i t s a c t i v i t y
|is h ig h e r in bovine a d re n a l tis s u e th an i s th e C-18 h y d ro x y lase,
I
Jand th e low endogenous c o n c e n tra tio n s of th e a p p ro p ria te sub-
• s tr a te s and t h e i r s o l u b i l i t y p r o p e rtie s make i t f a r e a s i e r th a n
jthe c h o le s te r o l system to work w ith .
i S ince carbon monoxide bin d s only to th e reduced form of
i
•the hem oprotein, th e appearance of a b so rp tio n a t 450 m jx can be
used t o determ ine th e r a t e o f P-450 re d u c tio n . Numerous s tu d ie s
u sin g p a r t i a l l y p u r if ie d 110-h y d ro x y lase p re p a ra tio n s had shown
t h a t N A D PH was th e prim ary e le c tro n donor f o r t h i s r e a c tio n ; but
when i n t a c t m ito ch o n d ria were used, th e r a te of re d u c tio n of
P-450 by N A D PH was very much slow er th an by v a rio u s K rebs c y c le
in te rm e d ia te s ( 9 ) . In o rd e r t o stu d y f a c to r s c o n tr o llin g th e
r a te s o f m ito c h o n d ria l h y d ro x y la tio n s in th e i n t a c t p a r t i c l e , th e
i
{pathways by which e le c tro n s a re tra n s p o rte d to P-450 needed t o be
(in v e s tig a te d .
I
! P o s sib le r e la tio n s h ip s between h y d ro x y latio n and r e s p i r a -
I
jtio n in a d re n a l m ito ch o n d ria had been suggested by e a r l i e r
i
{studies o f Brownie and G rant (1 9 ), b u t no f u r th e r e f f o r t had been
I
{made to pursue th e r a th e r in tr ig u in g id e a t h a t th e two o x id ase
activities might be functionally integrated. Preliminary studies
i
in d ic a te d t h a t th e r a t e s o f th e s e two p ro c e s s e s w ere o f com para-
b le m ag n itu d e, and a tte m p ts were made to stu d y th e e f f e c t of
h y d ro x y la tio n upon th e r a t e o f r e s p i r a t i o n . The u se o f s p e c tr o -
p h o to m e tric te c h n iq u e s t o d e te c t s te a d y s t a t e changes in th e
r e s p i r a t o r y com ponents was c o m p lic a te d by anomolous absorbancy
changes w hich were l a t e r shown t o be a s s o c ia te d w ith th e hydrox-
i
y la s e sy stem . D ir e c t flu o ro m e tric m easurem ent o f m ito c h o n d ria l
su sp e n sio n s coupled w ith enzym atic a ssa y o f p y rid in e n u c le o tid e
in m ito c h o n d ria l e x tr a c t s have been more s a t i s f a c t o r y . They i n
d ic a te t h a t th e h y d ro x y la se system i s lin k e d t o th e r e s p ir a to r y
c h ain by a p y rid in e n u c le o tid e tra n s h y d ro g e n a s e , and th ro u g h i t
r e s p i r a t o r y fu n c tio n s e x e r t c o n s id e ra b le in flu e n c e on th e r a t e o f
h y d r o x y la tio n •
D uring th e r a t h e r e x te n s iv e i n v e s tig a tio n o f th e s p e c tr a l
p r o p e r tie s o f th e h y d ro x y lase system in an a tte m p t t o r a t i o n a l i z e
th e absorbancy changes o b serv ed in th e k i n e t i c s t u d i e s , th e redox
p r o p e r tie s o f P-450 were exam ined. C o n clu sio n s rea ch e d from
sp e c tro p h o to m e tric m easurem ents were t e s t e d and su p p o rte d by EPR
i
i
i
m easurem ents. W ith th e in fo rm a tio n o b ta in e d from th e s e s tu d ie s ,
p o s s ib le mechanisms o f a c tio n f o r s e v e r a l c l a s s i c i n h i b i t o r s o f
lip -h y d r o x y la tio n have been e x p lo re d .
I
CHAPTER I I
MATERIALS A N D M ETH O D S
P re p a ra tio n o f A d re n o c o rtic a l M ito ch o n d ria
S te e r a d re n a l g lan d s were o b ta in e d from a l o c a l m eat
p ack in g p la n t w ith in 30 m inutes of th e d e a th o f th e an im al and
p la c e d on i c e . Subsequent o p e ra tio n s were done a t 4 -6 ° C. The
g lan d s were trim m ed o f f a t and c o n n ec tiv e t i s s u e , dem edulated,
d e c a p s u la te d , and m inced. A 10% homogenate o f c o rte x was p re
p ared by hand u sin g a Tenbroek g la s s hom ogenizer. Of s e v e r a l
hom ogenizing m edia t r i e d , i t was found 0 .2 5 M su c ro se c o n ta in in g
US bovine serum album in y ie ld e d m ito ch o n d ria w ith th e g r e a te s t
d e g re e o f r e s p ir a to r y c o n tr o l. The album in was p a r t i c u l a r l y
h e lp f u l in s t a b i l i z i n g th e m ito c h o n d ria a g a in s t p ro g re s s iv e
u n co u p lin g th ro u g h o u t th e d ay . The s u p e rn a ta n t o f an i n i t i a l
1000 g x 10 m inutes c e n tr if u g a tio n was spun a t 8,700 g x 10 min
u te s t o o b ta in th e m ito c h o n d ria l p e l l e t . T h is sedim ent was
iresuspended in ap p ro x im ately o n e -th ird th e i n i t i a l volume o f
i
hom ogenizing medium and resed im en ted a t 19,000 g x 10 m in u te s.
The washed m ito ch o n d ria were suspended in a sm a ll volume o f th e
5
'same medium ( u s u a lly 6 -1 0 mg n itro g e n /m l) f o r su b seq u e n t s t u d i e s . :
; ' i
C are was ta k e n t o e x c lu d e th e c e n t r a l e ry th r o c y te b u tto n from t h e ;
m ito c h o n d ria l se d im e n t. T h is was m a te r ia lly a id e d by u s in g
c l e a r , p o ly c a rb o n a te c e n tr if u g e tu b e s . A ll th e s tu d ie s t o be 1
d is c u s s e d em ployed f r e s h ly p re p a re d m ito c h o n d ria .
i
In c u b a tio n M edia
The medium u t i l i z e d f o r m ost o f th e k i n e t i c s t u d i e s was
!
an i s o t o n ic p h o sp h a te b u f f e r s i m il a r t o t h a t d e s c rib e d by Chance
and W illia m s ( 2 0 ) . I t c o n ta in s 13 m M Na2HP0 4 , 3 iriM KH2PO4 ,
6 m M MgCl2 , and 96 m M KC1 (pH 7 . 2 ) . F o r o b ta in in g d if f e r e n c e
s p e c tr a t h i s medium was f r e q u e n tly d i lu t e d to 50% w ith 0 .2 5 M
su cro se,. The su c ro s e seemed t o red u ce th e s e t t l i n g o f m ito
c h o n d ria in th e c u v e tte s , b u t i t was n o t used in k i n e t i c s t u d ie s
b ecau se i t q u ite s t r i k i n g l y d e c re a se d th e r e s p i r a t o r y r a t e s
;w hile in c r e a s in g th e tim e r e q u ir e d t o o b serv e a re sp o n se t o ADP
or o th e r a g e n ts . I n e x p e rim e n ts r e q u ir in g a p h o s p h a te -fre e
medium, a medium was em ployed w hich c o n ta in e d 0 .2 5 M m a n n ito l,
0 .0 7 M s u c ro s e , and 0 .0 2 M T r i s (2 -am in o -2 -h y d ro x y m eth y 1 -1 ,3 -
p ro p a n e d io l) (pH 7 .4 ) ( 2 1 ) . !
Calbiochem :
Sigma Chem ical C o .:
New E ngland N u clear:
S o u rces of Im p o rta n t R eagents
S te r o id s , NADP(H), g lu c o s e - 6-P dehydro
g e n a se , g lu ta m ic a c id dehydrogenase,
b o v in e serum album in, adrenochrom e
NAD(H), a lc o h o l dehydrogenase, ADP,
ATP, an tim y cin A, ro te n o n e , m alic a c id ,
a - k e t o g lu t a r i c a c id
N u tr itio n a l B io ch em ical: sodium s u c c in a te , a s c o rb ic a c id
4 - 14C la b e lle d s t e r o id s : 11-deoxy-
c o r t i s o l , 11-d e o x y c o rtic o s te ro n e ,
A ^ -a n d ro ste n e -3 ,1 7 -d io n e
M ethopyrapone ^ 2 -m e th y l-l, 2 - b is ( 3-
p y rid y l)-1 -p ro p a n o n e ] , amphenone B
[3 , 3 -b is(p -a m in o p h e n y l)-2-butanone]
o ,p f -DDD [ l- ( o - c h lo r o p h e n y l) -1 -
(p -c h lo ro p h e n y l) - 2 , 2-d ic h lo ro e th a n e ]
KCN, Na2S
TMPD (N,N,N*jN’-te tra m e th y l-p -p h e n y l-
e n e d ia m in e ), p e n ta c h lo ro p h e n o l
C iba:
A ld ric h Chem ical C o .:
M a llin c k ro d t:
Eastman Chem ical C o.:
A b so rp tio n S p e c tra
A bsorption s p e c tr a w ere m easured w ith a Cary model 14
re c o rd in g sp e c tro p h o to m e te r equ ip p ed w ith a s l i d e w ire showing
0 .1 absorbance as a f u l l s c a le d e fle c tio n (1 0 in c h e s) and a !
j ;
;’’s c a tte r e d tra n s m iss io n ” attachm ent w ith a No. 7664 q u a rtz end-
; i
window p h o to m u ltip lie r tu b e . A q u a rtz S y lv an ia ’’Sun Gun”
o p e ra ted a t 90 to 110 v o lts was employed as a l i g h t so u rc e . The
wave le n g th e r r o r o f t h i s in stru m e n t was le s s th a n 0 .8 £ in th e
're g io n o f th e spectrum under stu d y . Low te m p e ra tu re s tu d ie s were
perform ed w ith a L inde CD-189 liq u id n itro g e n Dewar su p p lie d by
th e A pplied P h y sics C o rp o ra tio n , M onrovia, C a lif o r n ia . The c o ld ;
f in g e r was lin e d and covered w ith b la c k p ap er in o rd e r to
m inim ize r e f le c tio n o f s c a tte r e d l i g h t , and th e windows o f th e
c o ld f in g e r was p o s itio n e d in th e sam ple beam o f th e Cary Spec
tro p h o to m e te r. The in stru m e n t was o p e ra te d in th e REF p o s itio n ,
in which a re g u la te d D C v o lta g e i s a p p lie d a c ro s s th e measurement
s lid e w ire in p la c e o f th e re fe re n c e s ig n a l. The re fe re n c e beam
was excluded from th e sample com partm ent. In th e s e s tu d ie s th e
lamp o r dynode v o lta g e o f th e sp ectro p h o to m eter was a d ju s te d to
o b ta in a s l i t w idth o f le s s th a n 0 .1 0 mm. A ll s p e c tr a were
o b tain ed w ith scanning r a te s o f e i t h e r 5 o r 10 ft p e r second ex cep t
;those in th e low tem p eratu re stu d y , which were scanned a t 0 .5 o r
2.5 ft p e r second.
! D iffe re n c e s p e c tra were made acco rd in g t o th e p ro ced u re
of Chance and W illiam s (2 0 ). A ll s p e c tr a l d a ta a re p re s e n te d as
9
i
i
r e p lo ts made by s u b tr a c tin g th e a p p ro p ria te b a se l i n e a t i n t e r -
i
i
v a ls of 2 .5 o r 5 m p, from th e spectrum under c o n s id e ra tio n . S ince
'th e p o in ts used f o r th e r e p lo tte d c u rv e s co rresp o n d ed c lo s e ly t o
th e lin e s shown, th e y were n o t rep ro d u ced in th e f i n a l f ig u r e s in
I
;order t o s im p lify th e d ra w in g s.
K in e tic S tu d ie s
P o la ro a ra p h ic
Oxygen u t i l i z a t i o n was m easured w ith th e v ib r a tin g p l a t i
num e le c tr o d e and a re fe re n c e s i l v e r anode which a re com m ercially
a v a ila b le from American In stru m e n t Company as an attac h m en t f o r
th e Aminco-Chance S p e c tro p h o to m e te r. The e le c tro d e i s p o la riz e d
t
a t - 0 .6 V and v ib r a te s a t 60 cps w ith an am p litu d e w hich can be
i
iv a rie d . I t was c a lib r a te d f o r l i n e a r i t y w ith oxygen te n s io n by
i
m easuring i t s resp o n se when p la c e d in s o lu tio n s e q u ilib r a te d w ith
'gas m ix tu res c o n ta in in g d i f f e r e n t p e rc e n ta g e s o f oxygen and was
found t o be q u ite l i n e a r . The e r r o r r e s u l ti n g from m ixing w ith
th e a ir - s a t u r a t e d i n t e r f a c i a l la y e r has been e s tim a te d by Chance
jand W illiam s (22) t o become s i g n i f i c a n t when r a t e s o f r e s p ir a tio n
i
|f a l l below 0 .1 [iM/sec a t l e s s th a n h a lf atm osphere c o n c e n tra tio n
jof oxygen.
I 1 0
I
| A low r e s is ta n c e sh u n t was in tro d u c e d in to th e c i r c u i t
c o n tr o llin g th e am plitude o f v ib r a tio n . T h is was sw itched i n to
th e c i r c u i t when a d d itio n o f re a g e n ts were made from a sy rin g e
t
i
th ro u g h a diaphragm in th e to p of th e e le c tro d e h o u sin g . The
r e s u ltin g am plitude o f th e e le c tro d e s ig n if ic a n tly reduced th e
s t i r r i n g tim e . F or s tu d ie s in which cyanide was used a G ilso n
Oxygraph which has a K C l-calom el anode was employed as cyanide
in te r a c te d w ith th e s i l v e r anode of th e American In stru m en t e le c
tro d e system .
S pectrophotom et r i c
Measurement of changes in d i f f e r e n t i a l absorbancy between
two w avelengths were made u sin g an Aminco-Chance D ual W avelength
S p ectrophotom eter and reco rd ed on Texas In stru m en t s e r v o / r i t e r ( R)
I I tr ip le - c h a n n e l re c o rd e r which i s a v a ila b le from American
in stru m e n t Company. The s e n s i t i v i t y employed was g e n e ra lly 5 o r
i0% tra n s m iss io n f u l l s c a le (4 .5 in c h e s ) . The v ib r a tin g p latin u m
e le c tro d e was o p e ra tin g sim u lta n eo u sly in th e s e experim ents f o r ,
in a d d itio n t o i t s use f o r s t i r r i n g of re a g e n ts , i t s low am p litu d e
V ib ra tio n h elp ed keep th e m ito ch o n d ria suspended and reduced
a r t i f a c t s a ttr ib u te d to p ro g re s siv e s e t t l i n g d u rin g an e x p erim en t.
1 1 ;
| F lu o ro m e tric
The flu o re s c e n c e produced by p y rid in e n u c le o tid e was
i
; m easured u sin g Am erican In s tru m e n t F lu o re sc e n c e A ttachm ent |
|d e s ig n e d f o r u se w ith th e Aminco-Chance S p e c tro p h o to m e te r. The
m icro p h o to m eter in th e system (c a ta lo g u e number 10-265) c o n ta in s
a b la n k - s u b tr a c t p o te n tio m e te r w hich in c re a s e d th e s e n s i t i v i t y o f
th e in stru m e n t t e n - f o l d . The l i g h t from th e xenon lamp was
f i l t e r e d by a C orning 7-60 f i l t e r w hich peaks a t 360 mp,. The
seco n d ary f i l t e r used was a g e la ti n f i l t e r p ro v id ed w ith th e
a tta c h m e n t w hich c u ts o f f l i g h t l e s s th a n 405 iq i. The c o lle c tio n
o f flu o re s c e n c e by t h i s f i l t e r , w h ile r a t h e r n o n - s e le c tiv e ,
in c re a s e d th e s e n s i t i v i t y ab o u t t h r e e - f o l d o v e r t h a t o b ta in e d
u s in g a W ratten g e la ti n f i l t e r No. 45 w hich peaks a t 460 m|i. The
r e l a t i v e changes in flu o re s c e n c e o f m ito c h o n d ria t r e a t e d w ith
m a la te , 11- d e o x y c o r tis o l, and a n a e ro b io s is were e s s e n t i a l l y th e
same w ith e i t h e r f i l t e r .
llS -H v d ro x v la tin g A c tiv ity
Most h y d ro x y la tio n e x p erim e n ts were c a r r ie d o u t in a
c u v e tte where th e s p e c tro p h o to m e tric o r flu o re s c e n c e p r o p e r tie s
and r e s p i r a t io n o f th e m ito c h o n d ria were sim u lta n e o u sly b e in g
s tu d ie d . 4 -14C -L ab e lle d 1 1 -d e o x y c o rtis o l d is s o lv e d in e th a n o l was
added by s y rin g e in sm a ll volum es (15-30 |j,l) • D uring th e
experim ent a liq u o ts c o n ta in in g a minimum o r 8 me e ach were j
! j
! removed by a sy rin g e and s q u irte d in to te n volumes o f c o ld c h lo ro i
I »
iform . A fte r a thorough e x tra c tio n th e aqueous phase was
; d ecan ted , and th e chloroform e x tr a c t was c o n c e n tra te d u n der n i t r o -1
g en . The re s id u e s were d is s o lv e d in m ethanol and a p p lie d to a
Z a ffa ro n i chrom atography system (23) • P aper chrom atographs were
s a tu r a te d w ith m ethanol-form am ide ( 3 :2 ) ; th e sample was a p p lie d ,
and th e chrom atographs were developed in fo rm am id e -sa tu ra ted
c h lo ro fo rm . The p ercen tag e of ^ C - la b e lle d 1 1 -d e o x y c o rtis o l
h y d ro x y lated was determ ined w ith a T ra c e rla b radiochrom atogram
s t r i p c o u n te r. C o rtis o l was by f a r th e m ajor p ro d u c t form ed.
However, when h ig h c o n c e n tra tio n s of c o r t i s o l had been a tta in e d
d u rin g th e re a c tio n , a second more p o la r 14C -la b e lle d p ro d u ct
appeared in sm all am ounts. T his was n o t i d e n t i f i e d .
i
P y rid in e N u cleo tid es
Assay f o r p y rid in e n u c le o tid e c o n c e n tra tio n s were d e te r
mined e s s e n t ia ll y a cco rd in g t o th e method of E stab ro o k and
M a itra ( 2 4 ). A liq u o ts of 2 ml were withdrawn from th e in c u b a tin g
M ito ch o n d rial suspension and quenched in e i t h e r 3 N K O H a t 100° C
1
o r 15% TCA a t 40° C. The K O H sam ples were cooled in ic e immedi
a t e l y , b u t th e TCA sam ples were k ep t a t 40° C f o r a p p ro x im ate ly
2 m inutes b e fo re c o o lin g in i c e .
I
13 I
! F o r m easurem ent o f red u ced p y r id in e n u c le o tid e , K O H e x -
' |
't r a c t s w ere n e u tr a li z e d by d ro p w ise a d d itio n s o f 0 .7 ml 1 M t r i - '
e th a n o la m in e b u f f e r fo llo w e d by 0 .5 6 ml 3 N HC1 t o sam ples b e in g
I
r a p id ly s t i r r e d in a s m a ll b e a k e r c o n tin u a lly c o o le d by an e x te r
n a l i c e b a th . F o llo w in g n e u t r a l i z a t i o n t h e sam ples w ere spun a t
'20,000 g x 15 m in u te s . A liq u o ts o f 3 ml were used f o r su b seq u en t
a ssa y o f N A D H (18 m M a c e ta ld e h y d e p lu s a lc o h o l dehydrogenase) and
NADPH ( 3 .3 m M NH 4.CI and 5 m M a - k e t o g lu t a r a t e p lu s g lu ta m ic
d e h y d ro g e n a se ). The enzym es w ere d i l u t e d and added so t h a t th e
r e a c tio n was com pleted in a b o u t 2 m in u te s . The flu o re s c e n c e o f
red u ced p y r id in e n u c le o tid e in th e e x t r a c t s was s ta n d a rd iz e d by
ad d in g a known amount o f red u ced p y r id in e n u c le o tid e t o a sam ple
b e fo re a d d itio n o f enzym es.
To d e te rm in e o x id iz e d p y rid in e n u c le o tid e s , th e p r e c i p i
t a t e s form ed by th e i n i t i a l a c id tr e a tm e n t w ere packed as s e d i
m ents by (1000 g x 25 m in u te s) c e n t r i f u g a t i o n . The s u p e rn a ta n t
was d e c a n te d . Two e x tr a c t i o n s w ith 5 ml e th y l e th e r removed m ost
o f th e a c id from th e aqueous p h a s e . The sam ples were d iv id e d and
[tre a te d w ith 1 M T r is t o m a in ta in th e pH optimum o f t h e r e s p e c tiv e
I 1
enzyme a s s a y s , f o r NAD+ w ith 0 .0 3 ml e th a n o l p lu s a lc o h o l dehy-
i
jdrogenase, and f o r NADP+ w ith 0 .0 6 ml 0 .1 M g lu c o s e - 6-P p lu s
g lu c o s e - 6-P d e h y d ro g e n ase. The flu o re s c e n c e o f th e s e sam ples was
14
sta n d a rd iz e d by adding 0*01 ml o f sta n d a rd (1 m M ) N A D + o r NADP+
to th e c u v e tte a t th e end of th e re a c tio n and m easuring th e
i
i
|in c re a s e d flu o re s c e n c e . A flu o re s c in g p in k -c o lo re d su b sta n c e
jappeared in th e s e e x tr a c t s . I t was found t h a t th e a d d itio n of
i
1 0.02 ml o f 0 .2 M a sc o rb ic a c id m inim ized i t s fo rm a tio n .
j
i
P re s e n ta tio n o f D ata
I F ig u re 12, showing th e k in e tic s of p y rid in e n u c le o tid e
I
redox changes and oxygen u t i l i z a t i o n , i s th e only k i n e t i c stu d y
I
i
t o be i l l u s t r a t e d by a photograph of th e o r ig in a l c h a r t . A ll
o th e rs w i l l be p re se n te d as tr a c in g s o f s im ila r e x p e rim e n ta l
i
d a ta . The tr a c e d f ig u re s reduce th e space re q u ire d t o show th e
|Same amount of d a ta and in some c a se s allo w d i f f e r e n t experim ents
i
to be p re s e n te d in a s in g le f ig u r e . F urtherm ore s c a le changes,
seen in th e oxygen tr a c e of F ig u re 12 p r io r t o m alate a d d itio n ,
jand s t i r r i n g a r t i f a c t s te n d to u n n e c e s s a rily co m p lica te th e
'fig u re and have been e lim in a te d in th e tr a c in g s .
j 15
| D e f in itio n o f M e tab o lic S ta te a
Chance has d e s c rib e d th e m e ta b o lic s t a t e s of m ito c h o n d ria
i
jas numbered S ta te s ( 2 5 ) . The c h a r a c t e r i s t i c s o f th e s e S ta te s
I a r e :
i
C h a r a c te r is ti c s
S ta te O2 E le c tro n Donor ADP
! 1 +
i
! 2 +
' 3 +
4 +
i
D -
| U sing Chance*s te rm in o lo g y , th e S ta te s in w hich th e m ito
c h o n d ria w i l l m ost f r e q u e n tly be seen in th e s tu d ie s t o be
d e s c rib e d a re 1 , 4 , and 5 — th e l a t t e r d i f f e r i n g somewhat from
C hance’s d e f i n i ti o n in t h a t ADP i s n o t p re s e n t in e x c e s s . When
Chance r e f e r s t o a change o f ste a d y s t a t e a s a p e rc e n t change,
i
t h i s i s in r e fe re n c e t o th e change from S ta te 2 t o S ta te 5 as
>100%. In th e s e s tu d ie s a n a e ro b io s is was n o t u s u a lly a c o n d itio n
s tu d ie d . S in c e th e amount o f red u ced p y r id in e n u c le o tid e does
g r e a tly in c r e a s e upon a n a e ro b io s is o v e r S ta te 4 l e v e l s , th e t o t a l
used t o d e term in e p e rc e n t changes w i l l be S ta te 1 t o S ta te 4
u n le s s in d ic a te d t o th e c o n tr a r y .
+
+
+
+
+
16 I
j
Approach to S tead y S ta te Method o f j
i
|
C a lc u la tin g R ate C o n sta n ts
Broman e t a l . (26) d e s c rib e a method th e y used t o o b ta in
in d iv id u a l r a t e c o n s ta n ts in a c o n se c u tiv e r e a c tio n from ste a d y
s t a t e c o n c e n tra tio n s and th e tim e re q u ire d t o re a c h h a lf th e
ste a d y s t a t e c o n c e n tra tio n . Use o f t h e i r method re q u ir e s th e
assum ption t h a t e n z y m e -su b stra te com plexes a re r a p id ly form ed.
T h e ir method has been used t o c a lc u la te r a t e c o n s ta n ts o f p y rid in e
n u c le o tid e re d u c tio n from flu o re s c e n c e m easurem ents. The f o l
low ing e q u a tio n was used
M = k . c°
T l / 2 r c o " c s
w here:
CG = t o t a l r e a c tiv e o x id iz e d p y rid in e n u c le o tid e
( S ta te 5 - S ta te 1)
Cs = o x id iz e d p y rid in e n u c le o tid e a t a ste a d y s t a t e
k r = r a t e c o n s ta n t ( s ) le a d in g t o re d u c tio n
T l/ 2 = tim e re q u ire d f o r p y rid in e n u c le o tid e t o become
h a lf as reduced a s th e y a re a t S ta te 5.
CHAPTER I I I
PROPERTIES OF THE STEROID lip-HYDROXYLASE SYSTEM
H i s t o r i c a l B ackground
W ith th e d is c o v e ry o f th e a b i l i t y o f c o r tis o n e t o r e l i e v e
symptoms o f rh eu m a to id a r t h r i t i s in 1949 ( 2 7 ) , th e r e was a
s tim u lu s t o f in d m eans o f p ro d u c in g la r g e q u a n ti t ie s o f t h i s
s t e r o i d . The i s o l a t i o n o f c o r tic o s te r o n e when bovine a d re n a ls
were p e rfu s e d w ith 1 1 -d e o x y c o rtic o s te fo n e (DOC) (2 8 , 29) was
fo llo w e d by s i m i l a r d e m o n s tra tio n s t h a t a d re n a ls p o sse sse d s y s
tem s f o r th e s te p w is e in tr o d u c tio n o f h y d ro x y l groups a t C-17 and
C -21, a s w e ll a s a t C - l l o f th e p ro g e s te ro n e m o lecu le ( 3 0 ) .
S u b se q u e n tly s tu d ie s were begun o f th e enzymology o f th e
s t e r o i d h y d ro x y la tin g sy s te m s . Hayano and Dorfman found th e
a c t i v i t y f o r th e 1 1 0 -h y d ro x y la tio n of DOC to be a s s o c ia te d w ith
th e p r e c i p i t a t e o f a 5 ,000 g x 25 m in u te s c e n tr if u g a tio n o f a
hom ogenate p re p a re d in a s a lin e -p h o s p h a te b u f f e r in a W aring
iblender ( 3 1 ) . They found t h a t fu m ara te in c re a s e d th e a c t i v i t y ,
puid even th o u g h o t h e r K rebs C ycle in te r m e d ia te s a ls o s tim u la te d
h y d ro x y la tio n ( 3 2 ) , fu m a ra te was assum ed to p la y some s p e c ia l
17
r o l e , p o s s ib ly t h a t o f o x id a n t. I f t h i s r e s id u e was washed in j
| • I
th e s a lin e -p h o s p h a te medium, fu m arate and magnesium w ere re q u ire d j
I
f o r a c t i v i t y ( 3 3 ) . M alate c o u ld re p la c e fu m a ra te , and s u c c in a te ;
! I
;was ap p ro x im ate ly 75% a s e f f e c t i v e , b u t numerous d i - and t r i
c a rb o x y lic a c id s vtfiich were a c tiv e in cru d e hom ogenates w ere no
ilo n g e r a c t i v e . Washed re s id u e s p re p a re d from g lan d s s to r e d a t
0° f o r s e v e r a l weeks p r i o r t o u se re q u ir e d NAD+ and ATP as w e ll
a s fu m ara te and magnesium f o r f u l l a c t i v i t y . S ubsequent s tu d ie s
showed t h a t th e N A D + , ATP and magnesium re q u ire m e n t c o u ld be
re p la c e d w ith NADP+ ( 3 4 ) ,
Sw eat, u s in g 0 ,8 8 M su c ro se a s a hom ogenizing medium,
w hich p re s e rv e s th e i n t e g r i t y o f th e c e l l u l a r c o n s titu e n ts b e t t e r
th a n s a lin e s o lu tio n s , found th e lip -h y d ro x y la s e a c t i v i t y t o be
'c o n c e n tra te ^ in th e f r a c t io n se d im e n tin g betw een 2,000 g x
10 m in u tes and 19,000 g x 30 m in u tes — th e m ito c h o n d ria l f r a c t io n
•(1 8 ). F o llo w in g th e le a d o f Hayano and Dorfm an, he used a
i
s a lin e -p h o s p h a te in c u b a tio n medium c o n ta in in g magnesium and
fu m arate t o stu d y th e lip -h y d ro x y la s e a c t i v i t y o f i n t a c t
m ito c h o n d ria .
Brownie and G rant (35) rec o g n ize d th e im p o rtan ce o f
o b ta in in g c le a n s u b - c e l l u la r f r a c t io n s b e fo re a co m p artm en talized
i *
enzym atic fu n c tio n co u ld be a d e q u a te ly c h a r a c te r iz e d . They f e l t
I
! 19 !
(
i !
t h a t th e h a rsh methods such a s th o se used by Hayano and Dorfman ;
i
j
in p re p a rin g 110-hydroxylas e p re p a ra tio n s could r e s u l t in a r t i - j
jf a c titio u s c o fa c to r re q u ire m e n ts . They o b ta in e d from a 0.25 M j
|sucrose homogenate o f ox a d re n a l c o rte x a m ito c h o n d ria l f r a c tio n J
jsedim ented between 700 g x 10 m inutes and 5,000 g x 10 m in u te s. j
! j
|In th e s e p a r t i c l e s th e 110-hydroxylase a c t i v i t y su p p o rted by j
i i
| su c c in a te and a - k e to g lu ta r a te was e q u iv a le n t t o t h a t su p p o rted by ;
jfum arate. S u c c in a te when supplem ented w ith ATP o r A DP was even
[ :
imore e f f e c t i v e •
F u rth e r o b se rv a tio n s o f Brownie and G ra n t, which were
[la rg e ly ig n o red by in v e s tig a to r s in t h i s c o u n try , c le a r ly showed
!a c o r r e la tio n o f 110-hy droxylase a c t i v i t y w ith th e o x id a tiv e and j
!
Iphosphorylative a c t i v i t i e s o f th e m ito c h o n d ria . They m easured
’ th e amount o f oxygen u t i l i z e d d u rin g th e h y d ro x y la tio n o f D O C in
|
t h e p resen ce of d i f f e r e n t K rebs C ycle in te rm e d ia te s and n o ted t h a t
jthose in te rm e d ia te s which u t i l i z e d th e most oxygen a ls o hydrox-
jy lated th e most DOC. M alonate, which i n h ib i t s s u c c in a te o x id a-
i
jtio n , was shown t o i n h i b i t h y d ro x y la tio n su p p o rted by su c c in a te
jbut not by a - k e to g lu ta r a te . S ince fu m arate could n o t be formed j
[from a -k e to g lu ta ra te in th e p resen c e o f m alo n ate, i t could n o t |
have a s p e c if ic r o le in th e h y d ro x y la tio n r e a c tio n . They demon
s tr a te d t h a t fum arate co u ld n o t re p la c e oxygen in th e r e a c tio n ,
; I
20 |
i i
t
r u l in g o u t th e su g g e stio n t h a t i t was s e rv in g a s a hydrogen j
Ja c c e p to r. T h e ir d e m o n stra tio n o f 2 ,4 - d in itr o p h e n o l in h ib i t io n o f j
jfu m a ra te -su p p o rte d h y d ro x y la tio n le d them t o su g g e st a r o le o f |
jATP in th e h y d ro x y la tin g p r o c e s s . They p o in te d o u t th e n e c e s s ity |
!
t o d i s t in g u i s h betw een c o fa c to r s in v o lv e d in en erg y p ro d u c tio n !
I
jand th o s e in v o lv e d more d i r e c t l y in th e h y d ro x y la tio n p ro c e ss in !
! !
[stu d ie s o f 1 1 ^ -h y d ro x y la tin g a c t i v i t y . i
| In su b seq u e n t s tu d ie s Brownie and G rant p u rsu ed th e •
l 1
! i
problem o f th e r o le o f fu m a ra te . They found t h a t th e re q u ire m e n t ;
i
f o r fu m ara te and NADP+ a ro s e when m ito c h o n d ria were t r e a t e d w ith !
j ;
[hypotonic m edia o r when a c e to n e d r ie d ( 3 6 ) . M alate o r D L -iso-
c i t r a t e c o u ld e f f e c t i v e l y r e p la c e fu m a ra te , b u t a - k e to g lu ta r a te
! !
was i n a c t i v e . They o b serv ed t h a t fu m ara te in c re a s e d th e amount
o f 340 nil a b s o rp tio n vdien added t o an in c u b a tin g m ix tu re con
t a i n i n g NADP+ and a KC1 e x t r a c t o f a c e to n e d r ie d m ito c h o n d ria
1(37, 3 8 ). When DOC was a ls o p r e s e n t, th e amount o f 340 m (x a b -
i
i
s o rp tio n w hich c o u ld be form ed was l e s s . When th e KC1 e x tr a c t
jwas d ia ly z e d o r when 10 m M v e rse n e was added, DOC h y d ro x y la tio n
j i
[supported by fu m ara te p lu s NADP+ was i n h i b i t e d . The a c t i v i t y j
: j
jcould be r e s to r e d by ad d in g m e ta l io n s ; Co++, M n++, and Ni++ being;
; 1
m ost e f f e c t i v e . I f N A D H in s te a d o f fu m arate and NADP+ was added,
i
t h e r e was no e f f e c t o f M n * H' on th e d ia ly z e d enzyme p r e p a r a tio n . ;
21 j
|E v id e n tly th e m e ta l was re q u ire d f o r NADP+ re d u c tio n by fu m a ra te . j
! ;
:The p resen c e o f p y ru v ate in m ix tu res c o n ta in in g fu m arate and th e
d em o n stra tio n t h a t b ic a rb o n a te and a -k e to g lu ta r a te i n h ib ite d
is o c itr a te - s u p p o r te d h y d ro x y la tio n im p lic a te d th e m alic enzyme
(39) and D - i s o c i tr a te d ecarb o x y lase ( 4 0 ) . Both enzymes a r e M n++
o r Mg** r e q u ir in g and a re p re s e n t in th e m ito c h o n d ria .
The s p e c i f i c i t y o f th e llp -h y d ro x y la s e system w ith
r e s p e c t t o s t e r o id s u b s tr a te s was s tu d ie d by Hayano and Dorfman
( 3 3 ) . The b e s t s u b s tr a te s were found t o be D O C and 11-deoxy-
c o r t i s o l . R eduction o f th e 4 ,5 -d o u b le bond t o e i t h e r th e 5a o r
5£ c o n fig u ra tio n ap p ro x im ately h alv ed th e r a t e o f h y d ro x y la tio n .
A ^-A ndrostene-3 ,1 7 -d io n e , which la c k s th e tw o-carbon s id e c h ain
b e a rin g th e C -20,21 k e t a l group, was h y d ro x y lated a t ab o u t o n e-
t h i r d th e r a t e as was DOC. S te ro id s la c k in g th e C-21 h y d ro x y l o r
,ones w ith C-3 o r C-20 hy d ro x y l in s te a d o f k e to groups were hydrox-
y la te d o n ly a t v ery slow r a t e s .
K in e tic s tu d ie s by Sharma e t a l . d em o n strated t h a t
1 1 -d e o x y c o rtis o l and D O C are) hydroxy la te d by th e same enzyme (41) •
The K ^ s determ in ed f o r th e two s u b s tr a te s were 8 .4 7 pH (DOC) and
2 0 .84 pH (1 1 -d e o x y c o rtis o l). These corresponded t o th e K-j/s th e y
e x h ib ite d a s a c o m p e titiv e i n h i b i t o r o f th e o t h e r ’s h y d ro x y la tio n .
The h y d ro x y lated p ro d u c ts d id n o t i n h i b i t th e r e a c tio n . The same
22
w orkers re p o rte d t h a t A ^ -a n d ro s te n e -3 ,1 7 -d io n e a ls o c o m p e titiv e ly
i
i in h ib ite d th e lip - h y d ro x y la tio n o f D O C ( 4 2 ) . The K j_ d eterm in ed
was 18 t o 19 |xM. They found t e s to s te r o n e t o a ls o s i g n i f i c a n t l y
i n h i b i t th e r e a c tio n , b u t t h e k i n e t i c s o f in h ib i t io n were n o t
d e te rm in e d . llO -H y d ro x y -A ^-an d ro sten e-3 ,1 7 -* d io n e, p ro g e s te ro n e
and 1 7 a-h y d ro x y p ro g estero n e showed no i n h ib i t o r y e f f e c t .
The mechanism o f t h e lip - h y d r o x y la tio n r e a c tio n h as been
th e s u b je c t of much i n v e s ti g a t i o n . I t was o f p a r t i c u l a r i n t e r e s t
t o s te r o id c h e m ists, as th e lla -h y d ro g e n i s r e a c t i v e , w hereas th e
lip -h y d ro g e n i s in a r a t h e r s t e r i c a l l y h in d e re d p o s itio n and i s
c h em ic ally i n e r t (4 3 )• An e a r ly su g g e s tio n was made by
R e ic h s te in (44) o f a A^C^O -d e h y d ra tio n fo llo w ed by h y d ra tio n in
an alo g y t o th e s u c c in a te -fu m a ra te -m a la te se q u en c e. Hayano and
Dorfman t e s te d A9C 11)-dehydro-D 0C w ith a d re n a l hom ogenates and
found no co n v ersio n t o c o r tic o s te r o n e ( 3 4 ) . F u rth e rm o re , th e y
showed t h a t when D2O was p r e s e n t in th e medium d u rin g DOC h ydrox-
jy la tio n , no d eu teriu m was in c o rp o ra te d i n t o a s ta b le p o s itio n on
!
t h e m o le cu le . A lthough th e re q u ire m e n ts f o r oxygen had been
re c o g n ize d very e a r ly (3 1 , 4 5 ), i t was n o t u n t i l advances in
i
iiso to p e te c h n iq u e s e n ab led Mason e t a l . (4 6 ) t o d e m o n stra te th e
jin c o rp o ra tio n o f 180 2 in to th e d ip h e n o l p ro d u c ts by p h e n o la se ,
t h a t a s im ila r stu d y by Hayano e t a l . e s ta b lis h e d t h a t th e oxygen
23
o f th e 110-hydroxyl group was d e riv e d from m o le cu la r oxygen
r a t h e r th a n from w a ter ( 4 7 ) . A nother s i g n i f ic a n t c o n trib u tio n
was made by Hayano and co-w orkers tow ards e s ta b lis h in g th e
mechanism o f th e r e a c tio n when th e y dem o n strated t h a t 11(3-
i
h y d ro x y la tio n o c c u rre d w ith a r e te n tio n o f c o n fig u ra tio n a t C - l l 1
l(4 8 ). Analogy w ith o rg a n ic ch em istry would su g g e st t h a t th e |
; i
* j
a tta c k in g oxygen was an e le c t r o p h i li c s p e c ie s ( 4 9 ). G rant (38)
I ;
jruled o u t f r e e hydrogen p e ro x id e as th e a c tiv e oxygen s p e c ie s by j
j j
(dem onstrating th e la c k o f DOC h y d ro x y latio n in th e p re se n c e o f
H2O2 and th e f a i l u r e o f c a ta la s e t o i n h i b i t th e r e a c tio n ,
j E a r l i e r s tu d ie s o f Tomkins e t a l . (50) showed t h a t t h e r e j
I |
w ere a t l e a s t th r e e h e a t - l a b i l e components in th e 1 1 0 -h y d ro x y lase '
j I
sy stem . F o llo w in g th e i d e n t i f i c a t i o n o f P-450 as. th e oxygen j
a c t iv a t in g enzyme in th e C-21 h y d ro x y lase system (3 ) and r e p o r ts
o f i t s s im ila r r o le in 1 1 0 -h y d ro x y latio n (1 0 ), Omura e t a l . s o lu
b i l i z e d and c h a r a c te r iz e d two a d d itio n a l components o f th e 1 1 0 -
i i
i
jhydroxylase system (5 1 ). T hese were d e s c rib e d as a F A D -containing
Ifla v o p ro te in and a non-heme iro n p r o te in . Cytochrome P-450
rem ained bound to very sm a ll su b -m ito c h o n d ria l p a r t i c l e s . In d e - j
1 I
, j
p e n d e n tly Suzuki and Kimura is o la te d a non-heme iro n p r o te in (5 2 , I
53) and Nakamura e t a l . d e s c rib e d a non-heme iro n and f la v o p r o te in
( 5 4 ) . The 1 1 0 -hydroxylase system has th u s been d e s c rib e d :
i
(
NADPH -------- > Fp (FAD) --------* NHI -------- ► P-450
The r e p o r t o f H ard in g e t a l . o f th e p resen c e o f P-450 in
r a t a d re n a l m ito c h o n d ria w hich was reduced o p tim a lly w ith K rebs
C ycle in te rm e d ia te s and o n ly slo w ly in th e p re se n c e o f N A D PH (55)
renewed i n t e r e s t in th e pathw ays o f e le c tr o n tr a n s p o r t which
co u ld be u t i l i z e d t o su p p o rt m ito c h o n d ria l h y d ro x y la tio n s . A
com parison o f th e r e l a t i v e r a t e s o f DOC h y d ro x y la tio n and P-450
re d u c tio n in r a t a d re n a l m ito c h o n d ria su p p o rte d by v a rio u s e le c
tr o n donors showed good c o r r e l a t io n ( 5 5 ) . More e x te n s iv e s tu d ie s
em ploying v a rio u s r e s p i r a t o r y c h ain i n h i b i t o r s and ton c o u p le rs
w ith bovine a d r e n o c o r tic a l m ito c h o n d ria in d ic a te d e le c tr o n s
d e riv e d from v a rio u s so u rc e s co u ld be u t i l i z e d t o g e n e ra te i n t r a -
m ito c h o n d ria l NADPH t o be used by th e h y d ro x y lase system ( 5 7 ) . I t
can a p p a re n tly be form ed d i r e c t l y from m alate o r i s o c i t r a t e o x id a
t io n by NADP-linked d eh y d ro g en ases o r i n d i r e c t l y by tra n s h y d ro -
1
g e n a tio n o f N A D H red u c ed by NAD-linked dehydrogenases o r by an
e n e rg y -re q u irin g r e v e r s a l o f e le c tr o n flo w in th e r e s p ir a to r y
c h a in . The fo llo w in g s tu d ie s were d e sig n e d t o d eterm in e i f th e
a c t i v i t y o f th e h y d ro x y la se system was r a p id enough w ith r e s p e c t
t o r e s p i r a t io n t h a t th e su p p ly o f re d u c in g e q u iv a le n ts (NADPH)
!
inight c o n tr o l th e r a t e o f h y d ro x y la tio n .
25
i
A ssessm ent o f M agnitude o f A c tiv ity
1
A p y rid in e n u c le o tid e tra n s h y d ro g e n a s e l i n k betw een th e
h y d ro x y la se system and th e r e s p i r a t o r y c h ain would a llo w th e two
t o compete f o r e le c tr o n d o n o rs. P ro v id e d th e r a t e s o f o x id a tio n
w ere o f com parable m agnitude, th e two system s m ight m u tu a lly con
t r o l each o th e r *s a c t i v i t y . I n o rd e r t o a s s e s s th e m agnitudes o f
th e s e two o x id a se a c t i v i t i e s , th e r e l a t i v e r a t e s o f oxygen u t i
l i z e d by th e two system s were d e te rm in e d . The r a t e o f oxygen
u p ta k e by a m ito c h o n d ria l su sp en sio n o x id iz in g a g iv en e le c tr o n
donor was m easured. Cyanide o r a n o th e r r e s p i r a t o r y c h a in i n h i b i
t o r (ro te n o n e , an tim y cin A) was added t o i n h i b i t r e s p i r a t i o n .
Then s te r o id s u b s tr a te was added, and th e su b seq u en t r a t e o f
oxygen u t i l i z a t i o n by th e h y d ro x y lase system was d e te rm in e d .
KCN a t c o n c e n tra tio n s in e x c e ss o f t h a t a d eq u a te f o r
com plete in h ib i t io n o f s u c c in a te o x id a tio n f r e q u e n tly d id n o t
i n h i b i t m a la te o x id a tio n more th a n a p p ro x im ate ly 70%. T h is
cy an id e i n s e n s i ti v e oxygen u p tak e makes q u a n tita tio n o f th e oxygen
a c tu a lly u t i l i z e d by th e h y d ro x y lase system f o r 1 1 0 -h y d ro x y la tio n
im p o s s ib le . Thus th e amount o f h y d ro x y la te d p ro d u c t was d e te r
mined in a liq u o ts from sam ples in w hich th e amount o f oxygen used
was b e in g m easured. F ig u re 1 shows such an e x p e rim e n t. The
r e s u l t s a re in d ic a te d by th e c lo s e d c i r c l e s co n n ected by th e
M .la t*
tm M
KCN
I.OmM
0.020
O .I75 mM OOC
ooc
r C K ■
0.050 MlU d m rtfto, 0-S m | N
FftatphtM, »h re itmM
M fC Lg, • mN
KCLi 6 4 mm
(t
DOC
V 0.0S0
J-126
0 -0 5 6 0 » . OOC
m l C M
F ig . 1 . —C om parison o f oxygen u t i l i z e d by
r e s p i r a t i o n and lip - h y d r o x y la tio n . M ito ch o n d ria
e q u iv a le n t t o 0 .2 5 mg N/ml in is o t o n ic b u f f e r ,
370 c . A d d itio n s a re in d ic a te d in th e f i g u r e .
Numbers above th e l i n e s i n d ic a t e r a t e s in
ixm oles/m inute •
dashed l i n e . The r a te o f c o rtic o s te r o n e (Cpd. B) fo rm a tio n ,
; 50 mjimoles/min/mg N i s q u ite s im ila r t o the> in c re a s e in th e r a t e
o f oxygen uptake induced by th e a d d itio n o f s t e r o i d . The average
m olar r a t i o o f oxygen u t i l i z e d to h y d ro x y la te d p ro d u c t form ed was
c a lc u la te d u sin g th e r e s u l t s o f 30 such e x p e rim e n ts. I f i t was
assumed t h a t th e cyanide i n s e n s i ti v e m alate o x id ase was independ
e n t o f th e llp -h y d ro x y la s e system , and t h i s background r a t e of
oxygen u p tak e was s u b tra c te d from th e r a t e o bserved in th e
p re se n c e of s te r o id , th e r e s u l ti n g m olar r a t i o was 0 .944 + 0 .1 5 .
I f th e background r a te was n o t s u b tr a c te d , th e m olar r a t i o was
1.20 + 0 .2 2 . The sto ic h io m e try f o r 1 1 0 -h y d ro x y la tio n , a t l e a s t
in term s o f oxygen and h y d ro x y la te d p ro d u c t, a p p ea rs to be th e
same a s t h a t found by Cooper e t a l . (58) f o r th e C-21 h y d ro x y lase
system .
A group o f s te r o id s werq t e s t e d a s t o t h e i r a b i l i t y t o
induce cyanide in s e n s itiv e oxygen u t i l i z a t i o n in th e p resen c e o f
m a la te . The r e s u l ts a re shown in T able I . The v a lu e s o f oxygen
u p tak e a re e x p re ssed r e l a t i v e t o th e cy an id e i n s e n s itiv e oxygen
u p tak e o b ta in e d p r io r t o th e a d d itio n o f s t e r o i d . I t was n o t un
ex p ected t h a t th o se s te r o id s which a re known to be good s u b s tr a te s
f o r th e lip -h y d ro x y la s e system : A4-a n d ro s te n e -3 ,1 7 -d io n e ,
1 1 -d e o x y c o rtis o l, and 1 1 -d e o x y c o rtic o ste ro n e — a l l induced a
28
TABLE I
STEROID EFFECTS ON CYANIDE INSENSITIVE
OXYGEN UTILIZATION
!
j
R e la tiv e R a te s o f
O2 U t i l i z a t i o n
' M alate 3 .0
| M alate + K CN 1 .0 *
+ 1 1 -D e o x y c o rtiso l 4 .5
+ C o r tis o l 2 .7
+ C o rtis o n e 0 .9
+ 1 1 -D e o x y c o rtic o ste ro n e 5 .8
+ C o rtic o s te ro n e 2 .9
+ A ^ -A n d ro ste n e-3 , 1 7 -d io n e .5 .4
+ lip -H y d ro x y -A ^ -a n d ro ste n e -3 ,1 7 -d io n e 1 .9
+ A5-P reg n en o lo n e 0 .9
+ P ro g e ste ro n e 1 .6
i
^A verage a b s o lu te r a t e = 15 mp,moles 0 2 /m in u te .
Oxygen te n s io n s w ere m easured w ith a G ilso n O xygraph.
M ito c h o n d ria e q u iv a le n t t o 0 .4 5 mg N/ml in is o to n ic b u f f e r , 37° C,
m a la te (5 mM), KCN (1 mM), and e ac h s t e r o id 150 |1 M •
29
is ig n if ic a n t in c re a s e in oxygen u t i l i z a t i o n . 14C -L abelled
111-deoxycortisol and A4-a n d ro s te n e -3 ,17-dione were in cu b ated in
i
experim ents s im ila r t o t h a t p re v io u s ly d e sc rib e d in F ig u re 1 . As
w ith DOC, th e r e was good c o r r e la tio n between th e in c re a se d oxygen
u t il i z e d and th e amount o f H P -h y d ro x y la te d p ro d u ct form ed. How
e v e r, th r e e o th e r s te r o id s — a l l p ro d u cts of 1 1 0 -h y d ro x y latio n :
c o r t i s o l , c o rtic o s te r o n e , and lip -h y d ro x y -A ^ -a n d ro sten e -3 ,17-
d io n e , a ls o s ig n i f ic a n t l y s tim u la te d cyanide in s e n s itiv e oxygen
u t i l i z a t i o n .
In an a tte m p t to determ ine i f 110-h y d ro x y latio n o f endog
enous s te r o id was th e so u rce o f th e cyanide in s e n s itiv e oxygen
u t i l i z a t i o n , m ethopyrapone, a p o te n t i n h ib i t o r o f lip -h y d ro x y l-
a tio n (4 1 , 59) was added. In s te a d o f d e c re a sin g th e r a t e of
oxygen u p tak e , i t s tim u la te d th e r a t e 1 .5 to 2 .0 - f o ld . However,
subsequent a d d itio n o f th e 1 1 0 -h y d ro x y latab le s u b s tra te s o r t h e i r
llP -h y d ro x y la te d p ro d u c ts f a i l e d to f u r th e r in c re a s e th e r a te o f
oxygen u t i l i z a t i o n .
I t was r o u tin e ly found t h a t th e r a t e o f h y d ro x y latio n of
1 1 -d e o x y c o rtiso l o r 1 1 -d e o x y c o rtic o ste ro n e which was su pported by
i
m alate was e q u al t o o r g r e a te r th a n th e r a t e of m alate o x id a tio n
v ia th e r e s p ir a to r y c h a in . I f th e m alate dehydrogenases were not
s u f f i c i e n tl y a c tiv e t o a llo w b o th system s to o p e ra te a t a maximal
I
r a t e , th e i n i t i a t i o n o f h y d ro x y la tio n by a d d itio n of exogenous
js te ro id s u b s tr a te should r e s u l t in a p e rtu r b a tio n o f th e s te a d y - j
js ta te re d u c tio n l e v e l o f th e e le c tr o n c a r r i e r s o f th e r e s p ir a to r y '!
’ i
c h a in . An a tte m p t t o exam ine such a phenomenon was made by meas
u r in g th e absorbancy d if f e r e n c e s fo llo w in g th e a d d itio n o f s te r o id
l
i
{ su b strate betw een w avelength p a ir s a p p ro p ria te t o d e te c t changes
iin th e redox s t a t e s of cytochrom es b and c ( 2 0 ). The a bands of
th e cytochrom es were s tu d ie d in o rd e r t o av o id in te r f e r e n c e by
j
th e s te r o id induced s p e c t r a l changes in th e S o re t re g io n re p o rte d j
i i
i
b y Cooper e t a l . (6 0 ). I t was o b serv ed t h a t in d u c tio n of
h y d ro x y la tio n d id r e s u l t in s i g n i f i c a n t absorbancy changes a t
{these w a v ele n g th s. These changes were c y c l ic , and t h e i r re tu r n
: I
to S ta te 4 v a lu e s appeared c o n c u rre n t w ith th e r e tu r n o f oxygen j
{uptake t o th e p r e - s te r o id r a t e . However, when a s e r i e s of wave-
j
{length p a ir s were used t o p lo t a spectrum o f absorbancy changes,
I
{there appeared two d i s t i n c t tro u g h s , one a t 530 my and th e o th e r I
j
a t 575 my, w hich were n o t in te r p r e t a b l e as changes in e i t h e r
{cytochromes c o r b . In a d d itio n , t h i s p a tte r n of absorbancy
j
{change was th e same in th e p re se n c e o f c y a n id e , w hich should keep
j |
{the r e s p ir a to r y enzymes in a m axim ally reduced s t a t e . The i n i t i a l ;
i i
phase o f th e c y c le could a ls o be ob serv ed in th e absence o f exog
enous e le c tr o n d o n o r. I t was concluded t h a t th e absorbancy
! !
i 3 i
t
i
!changes were p rim a r ily due t o com ponents o f th e h y d ro x y lase s y s -
!
Item r a t h e r th a n th e r e s p ir a to r y c h a in . The stu d y o f th e s p e c t r a l
p r o p e r tie s o f th e h y d ro x y lase system w hich ensued com prises
C h a p te r IV .
CHAPTER IV
i
! PROPERTIES OF C Y TO C H R O M E P-450
| H i s t o r i c a l Background
i
i
j A pigm ent com bining w ith carbon monoxide which was
I
i
[rep o rted t o be p re s e n t in l i v e r m icrosom es by K lin g en b erg (4) and
b a r f in k le (5 ) was i d e n t i f i e d by Omura and S a to as a hem oprotein
i
Iwith r a t h e r u n u su al s p e c t r a l p r o p e r tie s , la c k in g an i d e n t i f i a b le
[reduced spectrum ( 6 ) . The carbon monoxide complex form ing w ith
i
th e reduced hem oprotein had a c h a r a c t e r i s t i c s in g le band a t
I
|450 nit. Omura and S ato s o lu b iliz e d th e pigm ent from th e m icro
tom es ( 7 ) , b u t in b e in g s o lu b iliz e d th e pigm ent (P-450) was
filte re d t o a form in w hich th e CO-complex absorbed a t 420 m p ,
I
j(P -420). C onvincing e v id en ce t h a t P-450 i s th e oxygen a c tiv a tin g
enzyme f o r dru g m e ta b o liz in g a c t i v i t i e s o f l i v e r m icrosom es,
s te r o id C-21 h y d ro x y la tio n by a d re n o c o r tic a l m icrosom es, and C-18
and lip -h y d ro x y la tio n by a d re n o c o r tic a l m ito c h o n d ria has been
o b tain ed (3 , 8 , 10, 15, 6 1 ).
A c h a r a c t e r i s t i c EPR sp ectru m , f i r s t o bserved by
Hashim oto e t a l » (62) in l i v e r m icrosom es, h as been found to be
! 32
I 3 3
i
jp re s e n t in c e l l f r a c t io n s c o n ta in in g cytochrom e P -4 5 0 , in c lu d in g
i
iliv e r m icrosom es, a d re n o c o r tic a l m icrosom es and a d r e n o c o r tic a l
-m itochondria (6 3 , 6 4 ). S ig n if ic a n tly , l i v e r m ito c h o n d ria , w hich
f a i l t o g iv e s p e c t r a l ev id en ce o f P -450, la c k th e EPR spectrum
a s w e ll ( 6 5 ) . The spectrum i s c h a r a c te r iz e d by s ig n a ls a t
g -2 .4 1 , 2 .2 5 , and 1 .9 1 .
H ashim oto e t a l . (62) o b serv ed t h a t carbon monoxide
e n a b le d b o th N A D H and N ADPH t o c o m p le te ly red u ce t h i s component
w hich th e y c a lle d FeX a s ev id en ced by th e d is a p p e a ra n c e o f th e
s i g n a l s . In th e absence o f 00 th e p y rid in e n u c le o tid e s could
o n ly reduce th e s ig n a ls 29% and 71% r e s p e c tiv e ly u n d er a n a e ro b ic
c o n d itio n s . The a p p a re n t b in d in g of CO t o th e component g iv in g
r i s e t o EPR s ig n a ls le d th e s e w orkers t o exam ine th e r e l a ti o n s h i p
o f FeX and cytochrom e P-450 ( 6 5 ) . I t a p p eared t h a t p r e p a r a tio n s
in which th e CO complex absorbed a t 450 m p, a ls o e x h ib ite d th e
EPR s i g n a ls . However, th e y found t h a t by t r e a t i n g m icrosom es
w ith d i f f e r e n t a g e n ts th e y co u ld a l t e r th e r a t i o o f P -450 p lu s
P-420 to FeX. Some p re p a ra tio n s of P-420 had no EPR c h a r a c t e r i s
t i c s . They concluded t h a t th e EPR s ig n a ls a r e d e riv e d from a
l
i
s t r u c t u r a l o rg a n iz a tio n o f th e a c tiv e enzyme system w hich i s
R equired f o r a c t i v i t y b u t does n o t n e c e s s a r ily d i c t a t e i t s
S p e c tr a l c h a r a c t e r i s t i c s •
| 34 I
! '
O b serv a tio n s o f s p e c tr a l changes a s s o c ia te d w ith a d d itio n
i I
| ;
|o f s u b s tr a te to th e a p p ro p ria te P-450 c o n ta in in g system was f i r s t
I
: re p o rte d by N arasim hulu e t a l . , who d e sc rib e d a peak a t about
i
, 385 m ( jL and a tro u g h a t 420 m p, ap p earin g as a d if fe re n c e spectrum
when 17o-hydroxyprogesterone was added t o a d re n o c o rtic a l m icro-
!somes ( 6 6 ) . In an accompanying p ap er Cooper e t a l . (60)
re p o rte d fo rm atio n o f a s im ila r spectrum upon a d d itio n o f D O C to
!a d re n o c o rtic a l m itochondria* Subsequent s tu d ie s o f o th e r
w orkers have shown s u b s tr a te induced s p e c tra in l i v e r microsomes
(6 7 , 68, 69)* A lthough th e r e appears t o be some d isagreem ent as
t o th e d e t a i l s o f s p e c if ic s p e c tra , th e r e seem to be two d i s t i n c t
i
js u b s tra te induced s p e c tra which a re observed in l i v e r m icrosom es;
one s im ila r t o t h a t re p o rte d f o r a d re n o c o rtic a l p r e p a ra tio n s , th e
o th e r c o n s is tin g of a peak a t about 430 m p, and a tro u g h a t 390 mp,.
The su g g e stio n has been made t h a t th e s e s p e c tra r e f l e c t an i n t e r
a c tio n of th e s u b s tr a te s w ith th e heme o f cytochrom e P-450 e i t h e r
d i r e c t l y as a lig a n d i t s e l f o r i n d ir e c t l y th ro u g h a p ro te in con
fo rm a tio n a l change ( 6 7 ) . Cammer e t a l . in an EPR stu d y o f th e s e
mixed fu n c tio n o x id ase system s show sm a ll s h i f t s in th e g -v alu e s
o f th e g -2 .4 2 and 1 .9 1 s ig n a ls upon a d d itio n of s u b s tr a te (6 3 ).
They i n t e r p r e t th e s e s h i f t s a s su p p o rt f o r th e h y p o th e sis of
J
su b stra te -h e m e i n t e r a c t i o n .
I A ttem p ts t o s tu d y s p e c t r a l p r o p e r tie s o f cytochrom e P-450
i
have n o t been v e ry s u c c e s s f u l. The g r e a t e s t problem has been th e
j
e ase w ith w hich i t i s a l t e r e d t o P -4 2 0 . I t a p p e a rs in l i v e r j
m icrosom es, a t l e a s t , t o be e x tre m e ly a u to x id iz a b le which makes j
I
d if f e r e n c e s p e c tr a s tu d ie s d i f f i c u l t . Mason e t a l . re p o rte d a j
i
reduced minus o x id iz e d d if f e r e n c e spectrum o f cytochrom e P-450
!
|(6 5 ). The sp ectru m w hich th e y showed h a s maxima a t 424 my and I
| i
557 my, w hich i s s im ila r t o t h a t re p o r te d by Omura and S ato f o r
p u r if ie d P-420 ( 7 ) . The bands a t t r i b u t e d t o cytochrom e P-450
c o u ld be d is tin g u is h e d from cytochrom e b s , w hich has a very
i ;
(sim ila r a b s o rp tio n sp e ctru m , by th e la c k o f s p l i t t i n g o f th e j
! !
a band a t l iq u i d n itro g e n te m p e ra tu re . !
In a more r e c e n t s tu d y K in o s h ita e t a l . (70) have p re
p a r t i c l e s c o n ta in in g cytochrom e P-450 from a d re n o c o r tic a l
! j
(m itochondria. T hese p a r t i c l e s , w hich la c k la r g e c o n c e n tra tio n s of'
j ;
c o n ta m in a tin g cy to ch ro m es, show S o re t bands in a b s o lu te s p e c tr a atl
j j
^• 15 my ( o x id iz e d ) , 421 my ( d i t h i o n i t e re d u c e d ), and 426 my and
'447 my ( d i t h i o n i t e red u ced p lu s CO) ( 7 1 ) . U sing th e s e p a r t i c l e s
i i
M orie e t a l . a tte m p te d t o a s s e s s th e c o n tr ib u tio n o f P-450 t o
i
(these s p e c tr a by p r e f e r e n t i a l l y decom posing cytochrom e P-450 (71) .j
T h e ir r e s u l ti n g P-450 s p e c tr a have S o re t a b s o rp tio n a t 427 my
j(o x id iz e d ), 400 my (re d u c e d ), and 447 my ( d i t h i o n i t e reduced (
36
p lu s CO). As t h e i r method re q u ire s unproven assum ptions as t o th e
s e l e c t i v i t y o f th e d e g re d a tiv e p ro c e s s , th e s e v a lu e s can be
a c c ep ted o n ly t e n t a t i v e l y .
S p e c tra l and EPR E ffe c ts of S u b s tra te s
and E le c tro n Donors
The d iffe r e n c e spectrum o f a e ro b ic m ito ch o n d ria w ith
1 1 -d e o x y c o rtis o l added t o th e sample c u v e tte i s shown in F ig u re 2 .
I t i s a p p a re n t t h a t th e tro u g h s o c c u rrin g a t th e a and P re g io n s
j
' ' i
a re f u r t h e r m a n ife s ta tio n s o f th e s te r o id induced peak a t 385 m p ,
i
i (
and tro u g h a t 420 m p , re p o rte d by Cooper e t a l . ( 8 ) . These a u th o rs
j
had su g g ested t h a t th e s p e c tr a l changes in th e S o re t reg io n were !
|
(the r e s u l t s of an o x id a tio n o f a n o n -a u to x id iz a b le component o f i
!
t
th e hy d ro x y lase system . T his e n tir e d if fe r e n c e spectrum w ith i t s
S o r e t, P, and a bands g r e a tly resem bles an o x id iz e d minus reduced
i :
|spectrum o f a hem oprotein.
! j
S e v e ra l o th e r s te r o id s have been surveyed to d e term in e thej
!
s p e c i f i c i t y req u irem en ts f o r in d u cin g t h i s d iffe re n c e sp ectru m . j
j
jll-D e o x y c o rtic o ste ro n e (DOC) and A ^ -an d ro ste n e-3 ,1 7 -d io n e, b o th
I !
jof which a re liP -h y d ro x y la te d by t h i s p re p a ra tio n , produce d i f f e r - |
I j
'ence s p e c tr a i d e n t i c a l to th a t produced by 1 1 -d e o x y c o rtis o l. ;
C o rtic o s te ro n e , p ro g e ste ro n e , 17a-hydroxyprogesterone, c o r t i s o l
! |
land 5 a -a n d ro stan e d io n e had no s ig n if ic a n t e f f e c t on th e s p e c tr a l '
37
fe iW ir* .
ffc " fiw * ' ’
* " i
J-IBS
4 0 0 BOO « 0 0
WAVE LENBTH(mpl
F ig . 2 .—The d if f e r e n c e spectrum
produced by 1 1 -d e o x y c o rtis o l in a e ro b ic
m ito c h o n d ria . M ito ch o n d ria e q u iv a le n t
t o 0 .5 5 mg N/ml in is o to n ic b u f f e r .
1 1 -D e o x y c o rtiso l (140 jjM) was added t o
th e sam ple c u v e tte .
jp ro p e rtie s o f th e s e m ito c h o n d ria . B oth A5, 3 P - o l- s te r o id s te s te d
(A ^-pregnenolone and d e h y d ro iso an d ro ste ro n e ) p roduced s p e c tr a
showing a s e r i e s o f peaks r a th e r than tr o u g h s .
The pregnenolone induced spectrum i s shown in F ig u re 3 .
iThe maxima o f what ap p ears to be th e S o r e t, P and a peaks o f a
i
t
jhem oprotein o ccu r a t 425, 540 and 575 mp,, r e s p e c t iv e ly . T hese
{findings were i n i t i a l l y in te r p r e te d a s an o p p o site e f f e c t on
I
I
cytochrom e P-450 from t h a t e x e rte d by t h e lip - h y d r o x y la ta b le sub
s t r a t e s . A c o m p e titiv e in h ib itio n of p regnenolone on lip -h y d ro x -
y la tio n would th e n be p r e d ic te d . Such an i n h ib i t io n was n o t
ob serv ed u sin g c o n c e n tra tio n s o f pregnenolone s u f f i c i e n t t o m axi
m a lly form th e sp ectru m . I f 1 1 -d e o x y c o rtis o l was added t o th e
i
p reg n en o lo n e t r e a te d sam ple, th e r e s u ltin g sp ectru m was t h a t
j c h a r a c te r is tic o f 1 1 -d e o x y c o rtis o l, c o n s is tin g o f a tro u g h a t
i
420 mpi. P o s s ib ly th e p resen ce o f s u b s tr a te i n h i b i t s th e b in d in g
j
jof pregnenolone by in c re a s in g th e s y s te m ^ K m f o r t h i s s t e r o i d .
i
A n o th er p o s s i b i l i t y i s t h a t pregnenolone r e a c ts w ith th e
i
{ c h o le ste ro l h y d ro x y la tin g sy stem s, as i t does i n h i b i t th e co n v er-
jsion o f c h o le s te r o l to p ro g e ste ro n e ( 7 2 ) .
! When m ethopyrapone ( 2 - m e t h y l- l ,2 - b i s ( 3 - p y r id y l ) - l -
propanone) w hich i s re p o rte d as a c o m p e titiv e i n h i b i t o r o f
ll(3 -h y d ro x y la tio n (59) was added to a e ro b ic m ito c h o n d ria , a
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WAVE LCNtTH (mp)
F ig . 3 .—The d if f e r e n c e spectrum
produced by A5-p reg n e n o lo n e in a e ro b ic
m ito c h o n d ria . M ito ch o n d ria e q u iv a le n t
t o 1 .1 mg N/ml in is o to n ic b u f f e r .
A5-P reg n en o lo n e (350 |iM) was added to
t h e sam ple c u v e tte .
I spectrum b e a rin g some lik e n e s s t o t h a t produced by pregnenolone
j
jappeared. T here a re how ever, s i g n i f ic a n t d if f e r e n c e s in th e
I
v i s i b l e re g io n . Whereas in th e spectrum produced by p reg n e
n o lo n e , th e 575 ny peak has a g r e a te r i n t e n s i t y th an th e 540 my
jband, th e spectrum produced by m ethopyrapone shows th e g r e a te r
|a b so rp tio n a t 545 my. I t was a ls o o bserved t h a t a d d itio n of
jll- d e o x y c o r tis o l to p re p a ra tio n s p r e - t r e a t e d w ith m ethopyrapone
i
had no e f f e c t on th e m ethopyrapone induced sp ectru m .
i
l
I t was found t h a t th e 1 1 -d e o x y c o rtis o l-in d u c e d spectrum
co u ld o fte n be i n t e n s i f i e d by ad d in g m alate t o b o th c u v e tte s .
I i
iThe i n te n s i f i c a ti o n was tem porary as th e 1 1 -d e o x y c o rtis o l i s
h y d ro x y la te d under th e s e c o n d itio n s . T h is e f f e c t o f e le c tr o n
j i
! donor in d ic a te d t h a t o x id iz e d m inus reduced hem oprotein absorption)
l
m ight indeed be re s p o n s ib le f o r th e o b serv ed sp ectru m . A ccord-
i
jin g ly , c o n d itio n s were sought w hich c o u ld s e l e c ti v e ly reduce th e
|h y d ro x y la tin g pathway w ith a m inim al c o n trib u tio n of th e r e s p i r a - i
jto ry ch ain t o th e d if f e r e n c e sp ectru m . P re v io u s s tu d ie s had
; e s ta b lis h e d t h a t antim ycin A co m p letely b lo ck s llfi-h y d ro x y la tio n j
j i
su p p o rted by s u c c in a te b u t h as l i t t l e e f f e c t on m a la te -su p p o rte d j
i i
h y d ro x y la tio n (5 6 , 5 7 ). A p p a ren tly , m alate can su p p ly red u c in g j
1
! t
e q u iv a le n ts to th e h y d ro x y la tin g pathw ay in th e p resen c e of a n t i - !
jmycin A, b u t s u c c in a te c a n n o t. Cytochrome b should be e q u a lly L
I 41 :
!
jreduced by s u c c in a te o r m a la te , and th e cytochrom es on th e oxygen ;
js id e o f th e a n tim y c in A b lo c k sh o u ld be o x id iz e d in b o th sa m p le s. :
T h e re fo re a d if f e r e n c e spectrum o f a e ro b ic m ito c h o n d ria t r e a t e d
;w ith m a la te and a n tim y cin A, re a d a g a in s t i d e n t i c a l m ito c h o n d ria
t r e a t e d w ith s u c c in a te and a n tim y c in A sh o u ld show e v id e n c e o f
any r e d u c ib le com ponents in th e liP - h y d ro x y la tio n sy ste m . Such a
d i f f e r e n c e sp ectru m i s g iv en in F ig u re 4 and shows S o r e t, (3, and
'a p eak s a t 420, 530 and 570 mp,, r e s p e c t iv e ly . T h is sp ectru m h as
f e a t u r e s o f a red u c ed m inus o x id iz e d hem oprotein d if f e r e n c e
i
sp e c tru m . I t sh o u ld be em phasized t h a t no added s t e r o i d s u b s tr a t e
i s u sed t o o b ta in t h i s sp e ctru m .
The d e g re e t o w hich t h i s sp ectru m o f red u ced h y d ro x y la se
sy stem resem b led th e in v e r s e o f th e 1 1 -d e o x y c o rtis o l-in d u c e d
sp e ctru m d i f f e r e d somewhat w ith d i f f e r e n t m ito c h o n d ria l p re p a ra
t i o n s . Such a sp ectru m c an n o t be c o n s id e re d t o be produced by a
s i n g l e com ponent. Such f a c t o r s a s th e e f f e c tiv e n e s s o f th e a n t i -
m ycin A b lo c k , th e endogenous c o n c e n tr a tio n s o f e le c tr o n d o n o rs
and s t e r o i d s u b s t r a t e s , and th e r e l a t i v e am ounts o f pigm ents
a b s o rb in g in t h e s p e c t r a l re g io n c o n tr ib u te in v a r ia b le ways t o
jth is d i f f e r e n c e s p e c t r a . However, th e p o s itio n s o f th e a b s o rp tio n
i
maxima and minim a in th e s p e c tr a shown in F ig u re s 4 and 2 do
c o rre s p o n d , and o f te n th e s e two s p e c tr a have a p p ea re d t o be
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F ig . 4 .—The d if f e r e n c e spectrum
produced by s e l e c ti v e re d u c tio n o f th e
h y d ro x y lase sy ste m . M ito ch o n d ria e q u iv a
l e n t t o 0 .5 5 mg N/ml t r e a t e d w ith 32 pig
an tim y cin A/ml in i s o to n ic b u f f e r .
M alate (10 m M ) was added t o th e sam ple
c u v e tte ; S u c c in a te (10 iriM ) was added t o
th e re fe re n c e c u v e tte .
i
: 43 I
i i
alm ost m irro r images of each o th e r . I t i s p o s tu la te d t h a t l i p -
h y d ro x y la ta b le s te r o id s p erm it (o r cause) th e o x id a tio n of t h a t
hem oprotein which i s reduced by m alate (b u t n o t su c c in a te ) in th e i
ipresence o f antim ycin A. The spectrum of F ig u re 2 would in d ic a te ‘
t h a t a s ig n if ic a n t p o rtio n o f th e hem oprotein in th e h y d ro x y la tin g
pathw ay i s in a reduced s t a t e , even in th e absence o f exogenous
e le c tr o n so u rc e s , and can be o x id iz ed in th e presence o f s te r o id . :
The spectrum o f th e reduced hydroxylase system was d e te r
mined a t liq u id n itro g e n te m p e ra tu re . Two sam ples c o n ta in in g
id e n t i c a l c o n c e n tra tio n s of m itochondria were p rep ared in
50 p e rc e n t g ly c e r o l. M alate and rotenone were added t o one and
1 1 -d e o x y c o rtis o l was added to th e second. The sam ples were
oxygenated and th en tr a n s f e r r e d to th e co ld f in g e r where th e y
were ra p id ly fr o z e n . The a b so lu te spectrum o f each sam ple was
re c o rd e d . The spectrum o b tain ed from th e s te r o id t r e a te d sample
was s u b tra c te d from t h a t o b ta in e d from th e m alate tr e a te d sam ple.
The r e s u l ti n g d iffe re n c e spectrum i s shown in F ig u re 5 . T his
Spectrum shows th e appearance o f th re e bands s im ila r t o t h a t
shown in F ig u re 3 w ith no d e te c ta b le s p l i t t i n g . The S o re t peak i s
g r e a tly i n t e n s i f i e d r e l a t i v e to th e a and 0 bands and a l l th e
M axim a'appear to be s h if te d s l i g h t l y to lo n g e r w avelengths a t
t h i s te m p e ra tu re . The s ig n ific a n c e of th e s e changes i s n o t known.
44 ]
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S-177 _ (
400 490 500 990 600
W A V E L E N G T H (m ji)
F ig . 5 . —L iq u id n itr o g e n te m p e ra tu re d i f f e r
en ce sp ectru m o f red u ced m inus o x id iz e d h y d ro x y la se
sy ste m . The sp ectru m p r e s e n te d i s th e r e p l o t te d
d if f e r e n c e betw een th e tw o ’’a b s o lu te " s p e c tr a ob
ta in e d a s d e s c rib e d in C h a p te r I I . C u v e tte c o n te n ts :
B oth sam ples — m ito c h o n d ria e q u iv a le n t t o 0 .6 mg N /
m l, 0 .2 5 M s u c r o s e - g ly c e r o l ( 1 : 1 ) ;
Reduced — 12 m M m a la te , 50 |xM ro te n o n e ;
O x id ized — 200 pM 1 1 -d e o x y c o rtis o l.
The absorbancy changes produced by th e v a rio u s s u b s tr a te s '
I •
i «
!u t i l i z e d to produce th e re d u c ed -o x id ize d d iffe re n c e spectrum o f
I i
ith e h y d ro x y lase system a re shown in a step w ise fa sh io n in
F ig u re 6 . T h is stu d y shows th e re c o rd in g o f th e absorbancy d i f
fe re n c e betw een 430 and 420 m p, m easured w ith th e d u a l w avelength :
i
sp e c tro p h o to m e te r. The low er tr a c e in d ic a te s th e oxygen te n s io n
m easured sim u lta n e o u sly w ith th e v ib r a tin g p latin u m e le c tr o d e .
The w avelengths s e le c te d a re th e a b so rp tio n maxima o f reduced
cytochrom e b (430 mp,) and reduced P-450 (420 mp,). A ddition o f
antim ycin A causes a ra p id in c re a s e in a b so rp tio n a t 430 m p. r e l a
t i v e t o 420 m p, as th e r e s p ir a to r y ch ain i s blocked a t th e oxygen
s id e of cytochrom e b . A ddition of su c c in a te produces a sm all
s tim u la tio n in oxygen uptake and causes a f u r th e r re d u c tio n o f
cytochrom e b as shown by th e p o s itiv e d e fle c tio n o f th e t r a c e .
A d d itio n of m alate s ig n i f ic a n t l y in c re a s e s oxygen u t i l i z a t i o n and
ra p id ly red u ces cytochrom e P-450 r e s u ltin g in a n e g a tiv e d e fle c
t i o n o f th e tr a c e as th e a b so rp tio n a t 420 m p. in c re a s e s r e l a t i v e
t o t h a t a t 430 mp,. F in a lly , th e a d d itio n o f 1 1 -d e o x y c o rtiso l
re v e rs e s th e d e f le c tio n caused by m alate as cytochrom e P-450
becomes o x id iz e d and f u r t h e r in c re a s e s th e r a t e of oxygen u p ta k e .
jThis c y c le o f re d u c tio n and o x id a tio n of cytochrom e P-450 can be
rep ro d u ced re p e a te d ly in th e p resen ce o f a s u ita b le source of
C Y T O C H R O M E b.
R E D U C T IO N 1
C Y T O C H R O M E P-490
R E D U C T IO N |
430-420mp
L og ^ ■ O .O O frp
F ig . 6 .--A k i n e t i c stu d y o f re d u c tio n o f
cytochrom es b and P-450 and th e 1 1 -d e o x y c o rtis o l-
in d u ced o x id a tio n o f cytochrom e P -4 5 0 . The d u a l
w av elen g th sp e c tro p h o to m e te r and oxygen e le c tr o d e
t r a c in g s a r e shown. A d d itio n s o f re a g e n ts a re
in d ic a te d in th e f i g u r e . M ito ch o n d ria e q u iv a le n t
t o 0 .8 mg N/ml in is o t o n ic b u f f e r , 25° C.
1 4 7
I
e le c tr o n s , oxygen, and h y d ro x y la ta b le s u b s tra te *
i
The r e la tio n s h ip o f th e s te ro id -in d u c e d oxygen uptake and
Is p e c tra l changes t o 1 1 0 -h y d ro x y latio n i s i l l u s t r a t e d in F ig u re 7. !
A ppearance o f th e 110-hydroxylated p ro d u c t, c o r t i s o l , i s in d ic a te d '
by th e dashed l i n e . Im m ediately a f t e r adding 1 1 -d e o x y c o rtis o l to
th e m ito c h o n d ria l suspension a lre a d y c o n ta in in g m a la te , oxygen
u ptake in c re a s e s m arkedly and th e p re v io u s ly d e sc rib e d o x id a tio n
o f cytochrome P-450 o c c u rs. W hile cytochrom e P-450 i s m axim ally
■ i
o x id iz e d , h y d ro x y latio n i s ra p id and l i n e a r as i s oxygen u t i l i z a
t i o n . As th e c o n c e n tra tio n of 1 1 -d e o x y c o rtis o l d e c re a s e s ,
h y d ro x y latio n and oxygen uptake d e c re a se in r a t e ; and cytochrom e
P-450 g ra d u a lly becomes le s s o x id iz e d . F in a lly , h y d ro x y la tio n -- •
s to p s a s th e s te r o id s u b s tr a te i s q u a n tita tiv e ly co n v erted t o
c o r t i s o l , and th e absorbancy and oxygen t r a c e s re tu rn tow ard th e
p r e - s te r o id l e v e ls . I t has been c o n s is te n tly found, a s i s shown
in t h i s and th e p rec ed in g f ig u r e , t h a t th e sp e c tro p h o to m e tric
tr a c e never r e tu rn s f u l l y to th e p r e - s te r o id p o s itio n . In
a d d itio n , th e r a t e of oxygen u t i l i z a t i o n a f t e r a c y c le of hydrox-
y la tio n i s o fte n s l i g h t l y g r e a te r th an b e fo re . These e f f e c t s may
be due to th e p ro d u ct o f th e h y d ro x y la tio n r e a c tio n , c o r t i s o l ,
jwhich does s tim u la te a low r a t e of oxygen u t i l i z a t i o n in th e s e
m ito c h o n d ria .
48
II- OESOXYCORTISOL
132 mjtmolM
ISO
!
10 0
so
- 0.2
S-120-2
Imin
F ig . 7 .—C o rre la tio n o f h y d ro x y la tio n o f
1 1 -d e o x y c o rtis o l w ith th e o x id a tio n s t a t e o f c y to
chrome P -4 5 0 . The r a t e s of oxygen u t i l i z a t i o n a r e
e x p re ss e d a s |j.moles/min • To m ito c h o n d ria (0 .2 mg N/
m l) in is o to n ic b u f f e r c o n ta in in g m a la te (25 m M ) a t
25° C was added 4 - 1 4 c - ll- d e o x y c o r tis o l (132 nvunoles).
The d e f le c tio n s on th e o r d in a te in d ic a te th e tim e
a t w hich 0 .1 5 ml a liq u o ts were removed f o r c o r t i s o l
d e te rm in a tio n s . The r e s u l t s o f th e s e d e te rm in a tio n s
a r e shown by th e s o l i d c i r c l e s .
49
I f th e s te ro id -in d u c e d spectrum i s th e r e s u l t o f an oxida-
I
j !
jtio n o f a component o f th e h y d ro x y la se system o c c u rrin g d u rin g thej
h y d ro x y la tio n p ro c e s s , th e p re se n c e o f oxygen should be e s s e n tia l*
Such a req u ire m e n t f o r oxygen i s i l l u s t r a t e d in F ig u re 8 . A
su sp en sio n o f m ito c h o n d ria supplem ented w ith s u c c in a te and m alate
was d iv id e d e q u a lly * One p o rtio n was p la c e d in a sto p p e re d
c u v e tte w h ile th e o th e r was p la c e d in a Thunberg c u v e tte c o n ta in
i n g 0 .5 ftmoles 1 1 -d e o x y c o rtis o l in 0 .0 5 ml 50% e th a n o l-p ro p y le n e
g ly c o l in th e s id e arm . High p u r ity argon was blown o v e r th e
re fe re n c e sam ple, and t h i s c u v e tte was cap p ed . The Thunberg
c u v e tte was re p e a te d ly e v ac u ated and flu s h e d w ith argon t o e n su re
a n a e ro b io s is . A f te r o b ta in in g a b a s e lin e d if f e r e n c e spectrum
w hich in d ic a te d t h a t b o th c u v e tte s were e q u a lly a n a e ro b ic , th e
1 1 -d e o x y c o rtis o l was tip p e d in to th e sam ple. T here was no s i g n i f
ic a n t a l t e r a t i o n in th e r e s u l t i n g spectrum from th e b a s e lin e as
shown by th e s o lid l in e in th e f i g u r e . The re fe re n c e sam ple was
th e n th o ro u g h ly oxygenated; and th e r e s u l t i n g spectrum in d ic a te d
w ith th e dashed l i n e shows t h a t th e sam ple was indeed a n a e ro b ic .
F ollow ing t h i s , th e sam ple c u v e tte was oxygenated and th e r e f e r
ence c u v e tte reo x y g e n ate d . The r e s u l ti n g spectrum shown by th e
jio tte d l in e s i s t y p i c a l o f t h a t produced by 1 1 -d e o x y c o rtis o l in
a e ro b ic m ito c h o n d ria . S ubsequent a d d itio n o f 1 1 -d e o x y c o rtis o l
50
0.00
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-0.04
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• 0.00
- 0.12
0 0 0 0 0 0
WAVE LIN0TH (up)
400
F ig . 8 .— D em onstration o f oxygen
re q u ire m e n t f o r 1 1 -d e o x y c o rtis o l-in d u c e d
d if f e r e n c e sp ectru m . C u v e tte c o n te n ts :
M ito c h o n d ria , 0 .5 5 mg N /m l; 0 .2 5 M s u c ro s e -
is o to n ic b u f f e r ( 1 : 1 ) ; m a la te and s u c c in a te ,
each 15 nM. D iffe re n c e s p e c tr a : B a se lin e
(n o t shown) — u n tre a te d a n a e ro b ic
c u v e tte s ; S o lid lin e — 1 1 -d e o x y c o rtis o l in
a n a e ro b ic sam ple; Dashed l i n e — oxygen in
r e f e r e n c e ; D o tted l i n e — oxygen in b o th
sa m p les.
I 5 1
f a i l e d t o s i g n i f ic a n t l y in c re a s e th e i n t e n s i t y o f th e sp ectru m .
P re v io u s s tu d ie s (6 2 -6 5 ) have in d ic a te d t h a t a c h a r a c te r - I
i
I :
i s t i c spectrum w ith s ig n a ls a t g -v a lu e s o f 2 .4 2 , 2 .2 6 , and 1 .9 1
i s a s s o c ia te d w ith o x id iz e d cytochrom e P-450 in m icrosom es. In
m ito c h o n d ria l sam ples th e g -1 .9 1 s ig n a l i s u s u a lly masked by th e
s ig n a l a t g -1 .9 4 a s s o c ia te d w ith non-heme iro n p r o t e i n s . To
d eterm in e i f redox changes o f th e cytochrom e o c c u r under c o n d i
t i o n s in w hich s te r o id s u b s tr a te s in d u ce th e d e s c rib e d s p e c t r a l
ch an g es, an EPR stu d y o f a d re n o c o r tic a l m ito c h o n d ria was begun.
F ig u re 9 shows th e r e s u l t s o f one such e x p e rim e n t. M ito c h o n d ria l
su sp en sio n s were su b je c te d t o 30 seconds o f vigorous, oxygenation
b e fo re b e in g tr a n s f e r r e d t o EPR tu b e s and fro z e n in liq u id
n itr o g e n . L ine A i s th e spectrum o f a e ro b ic m ito c h o n d ria and
shows th e a n tic ip a te d s ig n a ls a t g -2 .4 2 and 2 .2 6 due to o x id iz e d
cytochrom e P -450, a s ig n a l a t 1 .9 4 a s c rib e d to non-heme iro n
p r o te in s , and a n o th e r a t 2 .0 0 . The e f f e c t o f m a la te and a n t i
mycin A on th e s e a e ro b ic m ito c h o n d ria i s shown in spectrum C. The
m ost prom inent change i s th e i n t e n s i f i c a t i o n o f th e g -1 .9 4 s ig n a l
p f th e non-heme ir o n . Spectrum B was o b ta in e d a f t e r adding
j
1 1 -d e o x y c o rtis o l t o m alate and an tim y cin A -tre a te d a e ro b ic m ito
c h o n d ria . In th e p resen ce o f 1 1 -d e o x y c o rtis o l th e r e i s a s t r i k i n g
in c re a s e in th e h e ig h ts o f th e g -2 .4 2 and 2.26 s ig n a ls .
52
2.26
2.42
B
|* 5 0 0g h |
F ig , 9 , —The EPR s p e c tr a ( f i r s t d e r iv a tiv e ) o f bovine
a d r e n o c o r tic a l m ito c h o n d ria a t 770 X, L ine A: M ito ch o n d ria
( 6 .1 mg N/m l) a s i s o l a t e d ; L in e B: M ito ch o n d ria t r e a t e d w ith
a n tim y c in A (100 |i g ) , m a la te (40 mM), NADP ( 2 ,1 m M ) and 11-
d e o x y c o r tis o l ( 1 .7 mM); L in e C: As l i n e B, w ith o u t 11-deoxy
c o r t i s o l . G -v a lu e s w ere c a lc u la te d from s ig n a ls o f sta n d a rd
p i t c h . R ecorded on V a ria n Model 4502 EPR sp e c tro p h o to m e te r.
I 53
i 1
I T his r e s u l t i s most sim ply in te r p r e te d as a s te r o id -
induced f u r th e r o x id a tio n o f cytochrom e P -450. The sm a lle r
idecrease in th e non-heme iro n s ig n a l may be a t t r i b u t a b l e to th e
o x id a tio n o f t h a t p o rtio n o f th e t o t a l m ito c h o n d ria l non-heme
iro n which i s p re s e n t in th e h y d ro x y la tin g pathw ay (5 1 -5 4 ). The
s h i f t s in g -v a lu e s re p o rte d by Cammer e t a l . (63) were n o t
observed in th e s e s tu d ie s . A lthough th e r a te of scan n in g was not
s u f f i c i e n t l y slow t o a ssig n s ig n a l p o s itio n s w ith th e h ig h e s t
a cc u ra c y , s im ila r s tu d ie s a t low er scan speeds a ls o f a i l e d to
show s ig n if ic a n t s h i f t s of s ig n a l p o s itio n s (7 3 ). I t i s concluded
t h a t o f th e s e v e ra l hypotheses b e in g c o n sid ere d in th e c u rre n t
l i t e r a t u r e to e x p la in su b s tra te -in d u c e d d if fe r e n c e s p e c tr a (66-
6 9 ), an o x id a tio n s t a t e change o f cytochrom e P-450 i s most
s tro n g ly su pported by th e s e s tu d ie s on th e a d re n a l m ito c h o n d ria l
system .
CHAPTER V
HYDRQXYLATION AS A MITOCHONDRIAL FUNCTION
H is to ry o f E n erg y -L in k ed F u n c tio n s o f M ito ch o n d ria
R e s p ira to ry C o n tro l
The e a r l i e s t q u a n ti t a t iv e s tu d ie s o f o x id a tiv e phos
p h o ry la tio n w ith th e c o n ce p t o f c o n s e rv a tio n o f r e d u c tiv e f r e e
e n erg y w ere made by B e l i t z e r and T sibakow a, who m easured th e
am ounts o f p h o sp h a te e s t e r i f i e d i n t o c r e a tin e p h o sp h ate and oxygen
ta k e n up d u rin g th e o x id a tio n o f K rebs C ycle in te rm e d ia te s by
m inced m uscle p r e p a r a tio n s ( 7 4 ) . The r o le o f p h o sp h a te and phos
p h a te a c c e p to r in th e c o n tr o l o f r e s p i r a t i o n by i s o l a t e d m ito
c h o n d ria was d e m o n stra te d by L ardy and Wellman ( 7 5 ) . U sing
g lu c o s e and h e x o k in a se a s p h o sp h a te a c c e p to r, th e y showed t h a t th e
I
r a t e s o f o x id a tio n o f v a rio u s s u b s tr a t e s w ere g r e a t ly s tim u la te d
by p h o sp h ate and ATP. S l a t e r and H olton l a t e r d e m o n stra te d t h a t
ADP was th e p rim a ry p h o sp h ate a c c e p to r ( 7 6 ) .
j
I The ap p ro x im ate s i t e s in th e r e s p i r a t o r y c h ain where phos
p h o ry la tio n o c c u rs were e s ta b lis h e d by d e te rm in in g th e m aximal P /0
I
o r P /2 e . r a t i o s o b ta in e d when v a rio u s s u b s tr a te s were o x id iz e d by
54
jth e r e s p ir a to r y c h a in . I n i t i a l s tu d ie s o f Ochoa ( 7 7 ) , which
showed no p h o sp h o ry la tio n accom panying N A D H o x id a tio n by m ito -
! :
Ich o n d ria , c a s t c o n s id e ra b le doubt on th e o x id a tiv e p h o a p h o ry la -
i ;
Itio n h y p o th e s is . However, sub seq u en t s tu d ie s o f L eh n in g er
d e m o n stra te d t h a t when th e m ito c h o n d ria were made perm eable t o
NADH, i t s o x id a tio n was a s s o c ia te d w ith p h o s p h o ry la tio n r e s u l t i n g
in a P /0 r a t i o o f about 1 .8 ( 7 8 ). More im p o rta n tly , a number o f
w orkers found P /0 r a t i o s o f ap p ro x im ate ly 3 a s s o c ia te d w ith th e
i ,
o x id a tio n o f NAD-linked s u b s tr a te s (7 9 , 8 0 ). I t was a ls o shown
t h a t th e o x id a tio n o f NAD-linked s u b s tr a te s by f e r r ic y a n id e
y ie ld e d P/O r a t i o s ap p ro ach in g 2 (81) a s d id o x id a tio n o f
s u c c in a te by oxygen ( 8 0 ) . L eh n in g er d e m o n stra te d p h o s p h o ry la tio n
a t th e cytochrom e le v e l when he found th e o x id a tio n o f f e r r o -
cytochrom e c by sw ollen m ito c h o n d ria r e s u l t i n g in P/O r a t i o s
a p p ro a ch in g 1 (8 2 , 8 3 ). T h is confirm ed fin d in g s o f o th e r w orkers
who had used cytochrom e c-m ed iated o x id a tio n o f re d u c in g a g e n ts
su ch a s a s c o rb a te t o o b ta in s im ila r P/O r a t i o s (8 4 , 8 5 ).
Chance and W illiam s s p e c tro p h o to m e tric a lly i d e n t i f i e d
b e r ta in components o f th e r e s p ir a to r y ch ain o f i n t a c t m ito c h o n d ria
( 2 0 ) . By stu d y in g th e k i n e t i c s of th e changes in o x id a tio n s t a t e
i
o f th e r e s p ir a to r y enzym es, th e y e s ta b lis h e d th e sequence o f
c a r r i e r s t o be (8 6 ):
| S u b s tra te —* N A D —* Fp' — * b — ► c —* a — t a 3 — * O2
By a p p ly in g t h e i r " c ro s so v e r theorem ” , which p r e d ic ts an o x id a-
Itio n o r re d u c tio n o f a given c a r r i e r depending on i t s p o s itio n
!r e l a t i v e t o a s i t e o f in h ib itio n o f e le c tr o n flo w , t o th e
jsp e c tro p h o to m e tric a lly observed response o f th e r e s p ir a to r y c a r - .
T ie rs t o ADP, th e y i d e n t i f i e d th e s i t e s of p h o sp h o ry la tio n as
cytochrom es c and b and N A D H (8 7 ).
i
I n i t i a l l y , ADP c o n c e n tra tio n was th o u g h t t o be re s p o n s ib le
f o r c o n tr o llin g r e s p i r a t io n . Then K lingenberg and Schollm eyer
dem onstrated t h a t ATP co u ld i n h i b i t r e s p ir a tio n o f a p a r t i a l l y
(Uncoupled p a r t i c l e ( 8 8 ). The ATP in h ib itio n could be re le a s e d by
adding p h o sp h a te. The c ro s so v e r p o in ts e s ta b lis h e d by them f o r
ATP in h ib itio n and i t s r e le a s e by phosphate were th e same as th o se
d e sc rib e d by Chance f o r ADP ( 8 7 ). K lingenberg and a s s o c ia te s con
clu d ed t h a t th e (ATP)/(ADP)(P0 4 ) r a t i o was th e s i g n i f ic a n t f a c to r
c o n tr o llin g r e s p ir a tio n o f tig h tly - c o u p le d m ito c h o n d ria . They
f u r th e r su g g ested t h a t m ito ch o n d ria d em o n stratin g r e s p ir a to r y
c o n tr o l a re in an e q u ilib riu m s t a t e which in c lu d e s a redox e q u i-
i
lib riu m between r e s p ir a to r y components and a p h o sp h o ry la tio n
i
e q u ilib riu m in th e ad en in e n u c le o tid e system (8 9 ).
'R everse E le c tro n T ra n sp o rt
|
! The co n cep t o f r e v e r s i b l e e le c tr o n t r a n s p o r t in th e
r e s p i r a t o r y c h a in , w hich th e e q u ilib riu m c o n d itio n p ro p o se d by
i
K lin g e n b e rg would im p ly , was in tro d u c e d by Chance and H o llu n g e r
!
i(90) t o a c c o u n t f o r th e o b s e rv a tio n s t h a t s u c c in a te c o u ld red u ce
more p y r id in e n u c le o tid e th a n c o u ld NA D-linked s u b s tr a t e s ( 2 5 ) .
T h e ir r a t h e r e x te n s iv e i n v e s tig a tio n o f t h i s phenomenon e s t a b
l i s h e d t h a t th e re d u c tio n o f p y rid in e n u c le o tid e by s u c c in a te
r
re q u ire d e n erg y w hich c o u ld be g e n e ra te d by s u c c in a te o x id a tio n
(9 1 ) o r o b ta in e d from ATP in a p h o s p h a te -fre e medium ( 9 2 ) .
Chance e s tim a te d th e m inim al r a t e o f p y r id in e n u c le o tid e re d u c
t i o n t o be 10% o f th e m axim al r a t e of s u c c in a te o x id a tio n .
P y rid in e n u c le o tid e re d u c tio n was i n h ib ite d by m a lo n a te , a com
p e t i t i v e i n h i b i t o r o f su c c in o x id a s e and s i g n i f i c a n t l y slow ed in
th e p re s e n c e o f a m y ta l, an tim y cin A, o r q u in o lin e -N -o x id e ( 9 3 ) .
They p ro p o sed t h i s was b u t an example o f a g e n e r a l phenomenon o f
r e v e r s i b l e e le c tr o n t r a n s p o r t in th e r e s p i r a t o r y c h a in . U sing
s u l f i d e t o i n h i b i t cytochrom e o x id a s e , th e y d e m o n stra te d t h a t ATP
paused a re d u c tio n o f p y rid in e n u c le o tid e and an o x id a tio n o f
t
C ytochrom es a and c and f la v o p r o te in ( 9 4 ) . S in c e t h e oxygen
te r m in a l end o f th e r e s p i r a t o r y chain was b lo c k e d , o x id a tio n o f
i
th e s e com ponents, w hich o c c u rre d in th e p re s e n c e o r a b se n c e o f
i
58 I
j
s u c c in a te , would re q u ire flow o f e le c tr o n s tow ard p y rid in e i
'n u c le o tid e * K lin g en b erg confirm ed Chance*s fin d in g s using
s e v e r a l n o n -p y rid in e n u c le o tid e -lin k e d s u b s tr a te s (8 8 ).
A lthough r e v e r s a l o f e le c tro n tr a n s p o r t was co n sid ered a ;
; p o s s i b i l i t y , i t s a c tu a l o ccu rren ce to cause p y rid in e n u c le o tid e
re d u c tio n d u rin g s u c c in a te o x id a tio n was s u b je c t t o sc e p tic is m .
Krebs f e l t t h a t th e p y rid in e n u c le o tid e could be g e ttin g reduced
i
by m alate g e n e ra te d from s u c c in a te o x id a tio n ( 9 5 ). He argued
t h a t s a tu r a tio n o f th e r e s p ir a to r y ch ain w ith e le c tro n s from
s u c c in a te would i n h i b i t N A D H o x id a tio n , and th u s reduced p y rid in e
n u c le o tid e would accum ulate*
To d em o n strate t h a t e le c tro n r e v e r s a l was ta k in g p la c e a t
a s i g n i f ic a n t r a t e d u rin g s u c c in a te o x id a tio n , K lingenberg and
von H aefen q u a n tita te d th e amount of oxygen u t i l i z e d , su c c in a te
o x id iz e d , a c e to a c e ta te reduced, and m alate p lu s fum arate formed
d u rin g su c c in a te o x id a tio n by l i v e r m ito ch o n d ria in th e presen ce
Of a c e to a c e ta te ( 9 6 ). They found t h a t more s u c c in a te was o x id iz e d
th an could be accounted f o r by oxygen u t i l i z a t i o n . The d iffe re n c e
was found in reduced a c e to a c e ta te (0 -h y d ro x y b u ty ra te ). When
joxygen was o m itte d from th e system , th e s u c c in a te -lin k e d red u c tio n :
j
o c cu rred only in th e p resen c e o f added ATP. They found th e r a te
o f fo rm atio n o f P -h y d ro x y b u ty rate to be on th e same o rd e r o f
; 59
{magnitude as Chance had e stim a te d to be th e r a t e of re d u c tio n o f
N A D ( 9 1 ) , Amytal and 2 ,4 - d in itr o p h e n o l i n h ib i t e d i t s fo rm a tio n .
An experim ent somewhat s im ila r t o K lin g en b erg and von
H aefen*s was re p o rte d by S l a t e r e t a l . ( 9 7 ) . They m easured th e
g lu ta m ate and a s p a r ta te form ed d u rin g th e o x id a tio n o f s u c c in a te
i
iin th e p resen ce o f a - k e to g lu ta r a te and ammonia. They found more
g lu ta m ate th an a s p a r ta te , which th e y assum ed r e s u lte d from t r a n s -
lam ination o f th e o x a la c e ta te r e s u l t i n g from m a la te o x id a tio n .
G lutam ate s y n th e s is re q u ire d p h o sp h ate in th e medium and was
in c re a s e d by oligom ycin b u t i n h ib ite d by 2 ,4 - d in itr o p h e n o l, a n t i
mycin A, am ytal and m alo n ate . They a ls o n o te d t h a t a lth o u g h th e
r a t e o f oxygen u t i l i z a t i o n was g r e a te r in th e p re se n c e o f
a - k e to g lu ta r a te , th e P/O r a t i o was re d u c e d .
P y rid in e n u c le o tid e can be red u ced by e le c tr o n s e n te r in g
th e r e s p ir a to r y c h ain a t cytochrom e c from a s c o rb a te in th e
p re se n c e o f c a t a l y t i c amounts o f TM PD (N,N,Nl ,N * -te tra m e th y l-p -
phenylenediam ine) (9 8 , 9 9 ). Ascorbate-TMPD o x id a tio n can a ls o
g e n e ra te energy which can be u t i l i z e d to re v e rs e e le c tr o n s from
s u c c in a te in an an tim y cin A -blocked c h ain (9 9 , 1 0 0 ). Many s tu d ie s
showing p y rid in e n u c le o tid e re d u c tio n by s u c c in a te in su b -
m ito c h o n d ria l p a r t i c l e s la c k in g fum arase a c t i v i t y have s u b s ta n
t i a t e d e le c tro n r e v e r s a l as a r e a l phenomenon (1 0 1 ).
| 60
C alcium A ccum ulation
i
| A r e s p ir a tio n - lin k e d accum ulation o f calcium by i s o l a t e d
im ito ch o n d ria has been s tu d ie d under two ty p e s of e x p e rim e n ta l
j
c o n d itio n s . When th e c o n c e n tra tio n o f calcium in th e medium i s
h ig h (a b o u t 3 .0 mM), la r g e amounts of calcium a re tak e n up by th e
m ito c h o n d ria . When phosphate i s p r e s e n t, i t i s a ls o accum ulated
I
(with a s to ic h io m e tric r e la tio n s h ip w ith calciu m , and th e two a re
.p r e c ip ita te d a s an in s o lu b le s a l t in th e i n t e r i o r of th e m ito -
jch o n d rio n . Under th e s e c o n d itio n s th e e x cess calcium c au ses
i r r e v e r s i b l e u n co u p lin g of r e s p ir a tio n and s w e llin g o f th e m ito
c h o n d ria (1 0 2 ). When low c o n c e n tra tio n s (ab o u t 0 .1 m M ) a re added
i
j i t s a ccu m u latio n i s accom panied by an a c c e le r a te d r a t e o f r e s p i r a
itio n . However when th e medium becomes d e p le te d o f calciu m ,
r e s p i r a t io n r e tu r n s to a " r e s ti n g ’ 1 r a t e w ith o u t im pairm ent o f
o x id a tiv e p h o sp h o ry la tio n o r o f r e s p ir a to r y c o n tro l (1 0 3 ).
i
Chance h as th o ro u g h ly d e s c rib e d th e phenomenon of r e v e r s -
I
i
jib le calciu m accu m u latio n (1 0 4 ). The resp o n se o f th e components
o f th e r e s p ir a to r y c h ain to calcium a re in m ost r e s p e c ts q u a li t a -
i
jtiv e ly s im ila r t o th o s e caused by ADP. In a phosphate c o n ta in in g
jmedium, th e r e i s a c y c lic o x id a tio n o f p y rid in e n u c le o tid e ,
!
f la v o p r o te in , and cytochrom e b and a re d u c tio n o f cytochrom es c
and a . The r e s p ir a to r y r a t e and Ca++/0 r a t i o o fte n exceeds t h a t
61
i
I
i
o b ta in e d in th e p re s e n c e o f ADP. A c c o rd in g ly , th e s te a d y s t a t e
changes o f r e s p i r a t o r y com ponents a re more e x a g g e ra te d . In th e
jcase o f p y r id in e n u c le o tid e s , t h e p e rc e n ta g e o x id a tio n r e p o r te d
jby Chance was 55% f o r calciu m com pared t o 40% f o r ADP.
I f th e m ito c h o n d ria a r e in c u b a te d in a p h o s p h a te -fre e
medium, th e c a lc iu m s tim u la tio n o f r e s p i r a t io n i s t r a n s i e n t , b u t
[the re s p o n se o f r e s p i r a t o r y c h a in com ponents i s m onophasic. The
p y r id in e n u c le o tid e s , f o r exam ple, become more o x id iz e d and
Iremain in a more o x id iz e d s t a t e even a f t e r r e s p i r a t io n h as j
re tu rn e d t o a c o n tr o lle d r a t e . Upon th e su b seq u en t a d d itio n o f
i
p h o sp h a te , r e s p i r a t i o n i s a g a in s tim u la te d f o r a s h o r t tim e ; b u t 1
i ;
^hen i t slo w s , th e e le c tr o n c a r r i e r s a ls o r e tu r n t o t h e i r j
'"norm al” S t a te 4 l e v e l . Chance p o s tu la te d t h a t calciu m i n t e r a c t s j
jwith a h ig h e n erg y in te r m e d ia te o f o x id a tiv e p h o sp h o ry la tio n as
I t i s tr a n s p o r te d t o an in tr a - m ito c h o n d r ia l s i t e . The olig o m y cin
i n s e n s i t i v i t y o f c a lc iu m s tim u la tio n o f r e s p i r a t io n (105) im p li- j
{cates a n o n -p h o sp h a te c o n ta in in g h ig h en erg y in te rm e d ia te
(X I ) • The e f f e c t o f p h o sp h a te m aking t h i s a r e v e r s i b l e process;
can be b ro u g h t a b o u t a ls o by a c e ta te and i s presum ed t o be a
!
calciu m s e q u e s te r in g r o l e .
I P y rid in e N u cleo tid e T ranshvdrocrenation
I !
Kaplan and co-w orkers f i r s t d e sc rib e d an enzymic a c tiv -
j i t y , a s s o c ia te d w ith th e p a r t i c u l a t e f r a c tio n (20 m inutes x
i
16,000 RPM) of hom ogenates p re p a re d from v a rio u s mammalian
t i s s u e s , which c a ta ly z e d th e r e v e r s ib le t r a n s f e r o f hydrogen
i
between N A D and NADP (1 0 6 ). The r a t e o f N AD PH re d u c tio n o f N A D +
has been found by many w orkers to be s ig n i f ic a n t l y f a s t e r th an
'th e re v e rse r e a c tio n . The e q u ilib riu m of th e re a c tio n was found
by Lee and E m s te r (107) to be ap p ro x im ately th e same (0 .8 )
re g a rd le s s o f th e d ir e c tio n from which i t was rea ch e d . T his
corresponded q u ite w e ll w ith th e e q u ilib riu m e stim a te d by Kaplan
(1 0 6 ). Magnesium was found t o i n h i b i t th e r a t e o f t h i s non-energy
lin k e d tra n sh y d ro g e n a se ; in i t s p resen c e e i t h e r ATP o r energy
g e n erate d a e ro b ic a lly by th e r e s p ir a to r y ch ain p r e f e r e n t i a l l y
s tim u la te d th e r a t e of NADP+ re d u c tio n by N A D H (108) • In th e
p resen ce o f energy th e e q u ilib riu m v alu e was a ls o a f f e c te d , being
in c re a s e d to alm ost 500 (1 0 7 )•
The oligom ycin i n s e n s i t i v i t y o f th e e n e rg y -lin k e d re a c tio n
When th e energy i s g e n erate d a e r o b ic a lly , in c o n tr a s t t o i t s sen -
I
p i t i v i t y when ATP i s su p p o rtin g a c t i v i t y , le a d D anielson and
fern ste r t o conclude t h a t th e h ig h energy in te rm e d ia te i s th e same
as t h a t u t il i z e d f o r r e v e r s a l of e le c tr o n tr a n s p o r t (1 0 8 ).
S e v e ra l m ethods have been em ployed t o e s tim a te th e amount o f
e n erg y re q u ire d t o red u ce N ADPH (1 0 7 -1 1 0 ) • I t h a s been co n clu d ed
t h a t one h ig h -e n e rg y e q u iv a le n t i s expended p e r m o le cu le o f NADP+
red u ced by N A D H (1 1 0 ).
T here h as been some c o n tro v e rs y a s t o t h e m u tu al i d e n t i t y
o f th e en erg y c o n tr o lle d and t h e n o n -e n e rg y lin k e d a c t i v i t i e s .
On th e b a s is o f s i m il a r h ig h a c t i v a t i o n e n e r g ie s and i n h i b i t i o n
by t r ii o d o t h y r o n in e , Hommes and E sta b ro o k (111) have c o n clu d ed
t h a t th e y a re i d e n t i c a l . T h is was s u p p o rte d by th e more r e c e n t
f in d in g s o f K aw asaki e t a l . (112) t h a t a n tib o d ie s p re p a re d
a g a in s t th e p u r i f i e d n o n -en erg y c o n tr o l le d enzyme i n h i b i t s th e
e n e rg y -lin k e d a c t i v i t y in s u b m ito c h o n d ria l p a r t i c l e s .
T h is r e a c tio n a p p e a rs t o e x p la in e a r l i e r o b s e rv a tio n s by
K lin g e n b e rg and S le n c z k a (113) and E sta b ro o k and N is s le y (114) o f
th e h ig h s te a d y s t a t e l e v e l o f red u c ed NADP in i n t a c t m ito
c h o n d ria when N A D was c o n s id e ra b ly more o x id iz e d . S e v e ra l
w orkers have d e s c rib e d a p r o g re s s iv e d e c re a s e o f th e s te a d y s t a t e
r a t i o o f endogenous p y r id in e n u c le o tid e (NADPH)(NAD+)/(NADP+)
(NADH) w ith in c r e a s in g c o n c e n tr a tio n s o f u n c o u p lin g a g e n ts
(1 1 5 , 1 1 6 ).
U sing p a r t i c l e s from s o n ic a te d r a t l i v e r m ito c h o n d ria
w hich r e t a i n p h o s p h o ry la tin g a b i l i t y , van Dam and t e r W elle (115)
64
found t h a t th e r a t e o f tra n s h y d ro g e n a tio n su p p o rte d by e n erg y
i
g e n e ra te d by s u c c in a te o x id a tio n was much l e s s s e n s i t i v e t o
m alo n ate (w hich d e c re a s e s th e r a t e o f s u c c in a te o x id a tio n ) th a n
was ATP fo rm a tio n . F u rth e rm o re , th e y found t h a t th e ATP/O and
NADPH/0 r a t i o s were in d e p e n d e n t. T h at i s , th e p re s e n c e of NADP+ j
I
and th e o th e r com ponents n e c e ssa ry t o run th e tra n s h y d ro g e n a s e '
r e a c tio n d id n o t a l t e r th e P/O r a t i o found d u rin g s u c c in a te
o x id a tio n •
Lee and E m s te r re s tu d ie d th e e f f e c t o f tra n s h y d ro g e n a
t io n on t h e P/O r a t i o u sin g su b -m ito c h o n d ria l p a r t i c l e s o b ta in e d
from b e e f h e a r t (117) • They found t h a t when a r e l a t i v e l y la r g e
amount o f enzyme was used and th e r a t e of s u c c in a te o x id a tio n was
su p p re sse d w ith m alo n ate , th e r e was a d e f i n i t e d e c re a s e in th e
P/O r a t i o r e s u l t i n g from tra n s h y d ro g e n a tio n . L i t t l e o r no e f f e c t
was o b serv ed w ith e i t h e r N A D H o r NADP+ a lo n e . A b o litio n o f phos
p h o ry la tio n e i t h e r by o m ittin g p hosphate o r a d d in g o lig o m y c in was
found t o in c r e a s e th e NADPH/O r a t i o . An a d d itio n a l i n t e r e s t i n g
f in d in g was t h a t th e c o n c e n tra tio n o f N A D H re q u ire d t o s a t u r a t e
th e tra n sh y d ro g e n a se system was h ig h e r in th e p re se n c e o f an
a c t iv e p h o sp h o ry la tin g system , and th e c o n c e n tra tio n o f p h o sp h a te
re q u ire d t o s a t u r a t e th e p h o sp h o ry la tin g system was in c r e a s e d by
th e p re se n c e o f an a c tiv e tran sh y d ro g e n a se sy ste m . T hey f e l t
65 !
t h i s could p o s s ib ly e x p la in th e fin d in g s o f van Dam and t e r W ellej,
w hich had le d them t o h y p o th e siz e t h a t d i f f e r e n t so u rc e s o f h ig h ‘
energy in te rm e d ia te s a re used f o r tra n sh y d ro g e n a tio n and e le c tr o n ;
i
i
j
r e v e r s a l. !
i
F u n c tio n s o f A d re n o c o rtic a l M ito ch o n d ria and
T h e ir E f f e c ts on H v d ro x v latio n
i
I t became obvious from th e s tu d ie s le a d in g t o th e exam i-
i
jnation o f th e s p e c t r a l p r o p e r tie s o f th e hy d ro x y lase system t h a t
a tte m p ts t o stu d y th e e f f e c t s o f h y d ro x y la tio n on th e r e s p ir a to r y •
c h ain by s p e c tro p h o to m e tric m ethods would be e x ce ed in g ly com plex.
Oxygen u p tak e would a ls o be an u n s a tis f a c to r y in d ic a to r o f th e
r e la tio n s h ip as one co u ld n o t d is tin g u is h betw een th e oxygen
u t i l i z e d by th e r e s p i r a t o r y c h ain and t h a t u t i l i z e d by th e
h y d ro x y lase system w ith o u t in d e p e n d e n tly d e te rm in in g h y d ro x y lase
a c t i v i t y . The d i r e c t m easurem ent o f p y rid in e n u c le o tid e f lu o r e s
cence coupled w ith enzym atic a ssa y s o f th e s e c o -f a c to r s seemed t o
be th e b e s t approach t o t h i s problem . The p y rid in e n u c le o tid e s
respond s e n s i t iv e l y t o r e s p ir a to r y a c t i v i t y . In a d d itio n , th e
most l i k e l y lin k betw een th e two o x id ase system s was e x p ec te d to
be th e p y rid in e n u c le o tid e tra n s h y d ro g e n a s e .
T here w as, how ever, one problem w hich appeared e a r ly in
th e s tu d ie s — t h a t o f s ta n d a rd iz in g th e flu o re s c e n c e . I t i s
66 !
known t h a t e n z y m e -a sso c ia te d p y r id in e n u c le o tid e h as d i f f e r e n t j
flu o re s c e n c e p r o p e r t i e s th a n d o es th e f r e e compound (1 1 8 , 1 1 9 ). '
The c o n c e n tra tio n o f p y r id in e n u c le o tid e in th e m ito c h o n d ria
e s tim a te d from th e flu o re s c e n c e o f a c l e a r s o lu tio n c o rre sp o n d e d '
p o o rly w ith th e c o n c e n tr a tio n d e te rm in e d by e n zy m atic a s s a y . i
I
A d d itio n o f low c o n c e n tr a tio n s o f s ta n d a rd n u c le o tid e to m ito
c h o n d ria l su s p e n s io n s was a ls o u n s u c c e s s fu l a s th e r e was a v ery
r a p i d , c y a n id e - in s e n s itiv e lo s s o f f lu o r e s c e n c e . In a d d itio n , a s !
i
l
w i l l be d is c u s s e d l a t e r , i t a p p ea re d t h a t more th a n one p o o l o f
m ito c h o n d ria l p y r id in e n u c le o tid e was b e in g red u ced in th e s e
s t u d i e s . In l i e u o f g iv in g a m is le a d in g c a l i b r a t i o n v a lu e on th e
f i g u r e s , in e ac h s tu d y t o be d is c u s s e d th e r e w i l l be an exam ple
o f p y rid in e n u c le o tid e red u ced by m a la te o r s u c c in a te t o be used
f o r co m p ariso n .
F ig u re 10 shows th e r e l a t i v e amount o f flu o re s c e n c e
produced by s u c c in a te and m a la te . As th e t r a c i n g s i n d ic a t e ,
a lth o u g h th e r a t e a t w hich th e flu o re s c e n c e in c r e a s e s i s l e s s in
th e p re s e n c e o f s u c c in a te , a b i t more p y r id in e n u c le o tid e i s
a p p a r e n tly red u ced by t h i s e le c tr o n d o n o r th a n by m a la te . The
a d d itio n o f 1 1 -d e o x y c o rtis o l r e s u l t s in a p a r t i a l o x id a tio n o f
th e p y rid in e n u c le o tid e w hich c o n s t i t u t e s a s te a d y s t a t e change
o f a p p ro x im a te ly 45% in th e c a s e o f s u c c in a te and 30% in th e case
SUCCINATE
S f f lM
ll-D E O X Y C O R T IS O L
S IpM malate
S m M
ll-D E O X Y C O R T IS O L
3l|iM
R O T E N O N E
| IS jiM
|>l mln-l S-173
F ig . 1 0 .—Com parison o f f lu o r e s
cence produced by s u c c in a te and m a la te .
The e f f e c t o f 1 1 -d e o x y c o rtis o l i s a ls o
shown. M ito ch o n d ria (0 .5 mg N/ml) in
is o to n ic b u f f e r , 25° C. A d d itio n s a re
in d ic a te d in th e f i g u r e .
68
;o f m a la te . The p y rid in e n u c le o tid e rem ains a t a more o x id iz e d
i
i
| ste a d y s t a t e w hile th e h y d ro x y la tin g system i s s a tu r a te d w ith
|s te r o id s u b s tr a te . The sm all amount o f s te r o id added in t h i s
i
jstudy i s ra p id ly c o n v erted to c o r t i s o l , and th e p y rid in e n u c le o -
itid e ag ain becomes more reduced. As seen in th e m alate t r a c e ,
I
!
jth is ste a d y s t a t e le v e l of re d u c tio n i s s l i g h t l y l e s s th a n t h a t
iobserved p r io r to th e o x id a tio n cy cle o c c u rrin g d u rin g th e
i
I
jh y d ro x y latio n o f 1 1 -d e o x y c o rtis o l. In p re v io u s s tu d ie s , i t was
ishown t h a t c o r t i s o l in d u ces a c y a n id e -in s e n s itiv e oxygen uptake
, (T ab le I ) and t h a t cytochrome P-450 a p p ea rs t o rem ain more o x i-
jdized a f t e r a p e rio d o f h y d ro x y latio n (F ig u re 7 ) . The a p p a re n t
l
o x id a tio n o f p y rid in e n u c le o tid e , as w e ll as th e s e o th e r f in d -
jin g s, i s com patible w ith an in te r a c tio n of c o r t i s o l w ith a
{hydroxylase system e i t h e r as a s u b s tr a te undergoing f u r t h e r
i
|
h y d ro x y la tio n o r in some o th e r manner in c re a s in g th e a u to x id iz a -
i *
j b i l i t y o f cytochrom e P -450. The a d d itio n of ro ten o n e r e s u l t s in
I
;only a sm a ll in c re a s e in th e amount o f p y rid in e n u c le o tid e
{reducible by m a la te . A n aero b io sis a ls o f a i l s t o g r e a tly in c re a s e
I
i
jthe re d u c tio n of p y rid in e n u c le o tid e by m alate o r s u c c in a te .
i
| F ig u re 11 compares th e a b i l i t i e s of m alate and a -k e to -
g lu ta r a te p lu s m alonate t o reduce p y rid in e n u c le o tid e . M alonate
lis added w ith a -k e to g lu ta ra te t o i n h i b i t th e o x id a tio n o f
I
!
R O T E N O N E -KET08LUTARATE
'* > •!< M A L O N A T E
•c-K E T O G L U T A R A T E (7.8m M )
M A L O N A T E (7.8 m M )
ll-D E O X Y C O R T IS O L
8 S 1 1M _ _ _ _
M A L A T E
S-194
F ig . 1 1 .—Com parison o f flu o re s c e n c e p ro
duced by m a la te and a - k e to g lu ta r a te p lu s m a lo n a te .
The e f f e c t s o f ro te n o n e and 1 1 -d e o x y c o rtis o l a re
a ls o shown. M ito ch o n d ria (0 .2 6 mg N/ml) in
i s o t o n i c b u f f e r , 25° C.
i I
s u c c in a te g e n e ra te d d u rin g a - k e to g lu ta r a te o x id a tio n . In S ta te 4
a -k e to g lu ta r a te a p p e a rs to red u c e h a lf th e p y rid in e n u c le o tid e
i
i
jreduced by m a la te . Comparison o f th e p y rid in e n u c le o tid e reduced
|in th e p resen c e and absence o f ro te n o n e shows t h a t in th e c a se of
i
m alate 84% i s reduced in S ta te 4 w hereas in th e c a se o f a -k e to - j
I
i
g lu ta r a te o n ly 55% i s red u c ed . A d d itio n o f 1 1 -d e o x y c o rtis o l j
i 1
i I
^ in d ic a te d by u n la b e lle d arrow s in th r e e o f th e t r a c e s ) r e s u l t s
i
jin th e o x id a tio n o f ab o u t 34% and 50% o f th e p y rid in e n u c le o tid e
i :
Reduced by m alate and a - k e t o g lu t a r a t e , r e s p e c tiv e ly . In th e j
i • !
I
p resen c e o f ro ten o n e th e p e rc e n t o x id a tio n s change t o 21% and
|57%. The f i r s t o rd e r r a t e c o n s ta n ts c a lc u la te d by th e approach ;
1 i
i
t o ste a d y s t a t e method d e s c rib e d in th e Methods s e c tio n from th e
'in c re a s in g flu o re s c e n c e in d ic a te s th e r a t e of re d u c tio n by m alate
(3 .9 m in"1) i s about th r e e tim e s th e r a t e o f re d u c tio n by a -k e to -
I
ig lu ta ra te (1 .2 m in " l).
i !
i 1
R e s p ira to ry C o n tro l
i
t
A d re n o c o rtic a l m ito c h o n d ria , when f r e s h ly p re p a re d as
^ e s c rib e d in M ethods, d e m o n stra te r e s p i r a t o r y c o n tr o l d e fin e d by j
1 ;
J :
th e ADP s tim u la tio n o f r e s p i r a t io n ( 2 1 ) . As was re p o rte d by
Chance f o r o th e r m ito c h o n d ria ( 2 5 ) , th e s tim u la tio n o f r e s p ir a tio n
j 1
t o S ta te 3 i s accom panied by an o x id a tio n o f p y rid in e n u c le o tid e i
i ■
iand cytochrom e b ( 4 6 ) . The o th e r r e s p ir a to r y components were n o t !
71
s tu d ie d . D e p le tio n of ADP r e s u l t s in th e r e tu r n o f r e s p i r a t i o n
and o x id a tio n s t a t e o f c a r r i e r s t o t h e i r p re v io u s S ta te 4 l e v e l s .
The s tim u la tio n o f m a la te -su p p o rte d r e s p i r a t io n was c o n s i s t e n t l y
a p p ro x im a te ly 2 - f o ld ; and ADP/O r a t i o s , a lth o u g h n o t e x te n s iv e ly
d e te rm in e d , f e l l betw een 2 and 3 .
F ig u re 12 i s a stu d y show ing th e k i n e t i c s o f changes in
i
jp y rid in e n u c le o tid e re d u c tio n r a t e s and oxygen u t i l i z a t i o n in
I
jresponse t o ADP and 1 1 -d e o x y c o rtis o l. A d d itio n o f m a la te r e s u l t s
i
in a ra p id re d u c tio n o f p y rid in e n u c le o tid e — T ^/2 t o ste a d y
s t a t e ( S ta te 4) i s betw een 6 and 10 se c o n d s. A m o d erate r a t e of
i
r e s p i r a t i o n (26 |iM/min) i s a ls o o b ta in e d . A d d itio n o f ADP
jr e s u lts in a 2 5 % d e c re a se in flu o re s c e n c e and a 1 .7 - f o l d s tim u la
t i o n o f r e s p i r a t i o n . The ADP/O r a t i o c a lc u la te d from th e
in c re a s e d oxygen u t i l i z e d i s 2 .8 . When th e added ADP i s d e p le te d ,
jthe p y rid in e n u c le o tid e becomes more reduced (n o t q u ite r e tu r n in g
to th e o r i g i n a l l e v e l ) , and r e s p i r a t io n d e c re a se s t o a p p ro x i
m ately th e o r i g i n a l r a t e .
| The second c y c le o f in c re a s e d oxygen u t i l i z a t i o n and
i
jp y rid in e n u c le o tid e o x id a tio n r e s u l t s from th e a d d itio n o f 11-
p e o x y c o r tis o l. The p e rc e n t o x id a tio n i s a b i t g r e a t e r (30%) and
jthe r a t e o f oxygen u t i l i z a t i o n i s s tim u la te d ab o u t 2 - f o l d . As
i
w ith ADP, when th e s te r o id s u b s tr a te i s d e p le te d , th e p y rid in e
F ig . 1 2 .--T h e e f f e c t o f ADP on p y rid in e
n u c le o tid e flu o re s c e n c e and r e s p i r a t i o n . T h is i s a
p h o to g rap h o f th e o r i g i n a l flu o ro m e te r (u p p er) and
oxygen e le c tr o d e (lo w e r) t r a c i n g s . M ito ch o n d ria
e q u iv a le n t t o 0 .6 6 mg N/ml in is o to n ic b u f f e r ,
250 C, in 3 .2 ml t o t a l volum e. C o n c e n tra tio n o f
re a g e n ts in c u v e tte : M a la te , 7 .7 mM ; ADP, 61 |iM;
1 1 -d e o x y c o rtis o l, 30 pM. C h art speed = 1 in /m in .
73
n u c le o tid e becomes more red u ced and th e r a t e o f oxygen u t i l i z a t i o n
r e tu r n s to t h e S t a te 4 l e v e l . As i s r o u t in e ly t h e c a s e , a sm a ll
p e rc e n ta g e (a p p ro x im a te ly 7%) o f t h e p y r id in e n u c le o tid e rem ains ;
more o x id iz e d a f t e r a c y c le o f hydroxy l a t i o n . When t h e medium i s j
d e p le te d o f oxygen, t h e r e i s a f u r t h e r s m a ll r e d u c tio n o f i
1 I
■ i
p y r id in e n u c le o tid e ( S t a te 5 ) .
The p e rc e n ta g e change in p y r id in e n u c le o tid e in th e
S ta te 4 — * 3 t r a n s i t i o n i s a p p ro x im a te ly h a l f t h a t r e p o r te d by
i
Chance f o r r a t l i v e r m ito c h o n d ria . As th e d e g re e o f c o u p lin g as
a s s e s s e d by r e s p i r a t i o n re sp o n se t o ADP i s a ls o low , t h i s may
i n d ic a t e t h a t th e a d r e n o c o r tic a l m ito c h o n d ria a r e l e s s t i g h t l y
c o u p le d th a n th o s e i s o l a t e d from o th e r t i s s u e s .
P y rid in e N u c le o tid e R ed u ctio n bv S u c c in a te
A lthough s u c c in a te d o e s , in m ost c a s e s , i n i t i a t e a f a i r l y
ra p id re d u c tio n o f p y r id in e n u c le o ti d e , ATP c o u ld n e v e r be shown
t o s u b s t i t u t e f o r a e r o b i c a ll y g e n e ra te d en erg y in i n t a c t ad ren o
c o r t i c a l m ito c h o n d ria . T h is i s in c o n tr a s t t o f in d in g s o f
Chance (92) and K lin g e n b e rg (8 9 ) in o th e r t i s s u e s . O fte n th e r e
was an o b se rv a b le la g in re d u c tio n o f p y rid in e n u c le o tid e beh in d
s tim u la tio n o f oxygen u p tak e upon a d d itio n o f s u c c in a te . A c o rr e
l a t i o n betw een th e tim e o f p r e - in c u b a tio n o f th e m ito c h o n d ria and
th e le n g th o f th e la g tim e was n o tic e d . The c o ld m ito c h o n d ria
74
seem t o be a n a e ro b ic and t o c o n ta in s i g n i f ic a n t endogenous su b
s t r a t e , as i s shown by a la rg e i n i t i a l flu o re s c e n c e and r a t e o f
oxygen u t i l i z a t i o n , b o th o f which d e c re a se f o r ab o u t 3 m in u te s .
A d d itio n of s u c c in a te a t th e tim e where p y rid in e n u c le o tid e h a s
j u s t re a c h e d a m aximal o x id a tio n r e s u l t s in a ra p id in c re a s e in
flu o re s c e n c e . The d e c re a se in r a te and e x te n t o f p y rid in e
n u c le o tid e re d u c tio n which alo n g w ith an in c re a s e d la g tim e seems
to fo llo w p re -in c u b a tio n can p ro b ab ly be a t t r i b u t e d to d e p le tio n
o f endogenous s u b s tr a te s and th e subsequent la c k of h ig h en erg y
in te rm e d ia te s to be u t i l i z e d in th e r e v e r s a l of e le c tr o n flo w .
i
Chance re p o rte d s tu d ie s u sin g m ito c h o n d ria d e p le te d o f
s u b s tr a te where s u c c in a te f a i l s t o reduce p y rid in e n u c le o tid e .
!
jwhen h e added ATP t o th e s e a e ro b ic p re p a ra tio n s in a p h o sp h a te -
i
f r e e medium, th e r e was a ra p id re d u c tio n o f p y rid in e n u c le o tid e
'(9 2 ). A lthough numerous a tte m p ts were made to show t h i s phenome
non in th e s e m ito c h o n d ria , s i g n i f ic a n t e f f e c t s o f ATP were n e v e r
|observed on th e r a t e o r th e e x te n t o f p y rid in e n u c le o tid e re d u c -
!
jtio n . ATP r e v e r s a l o f e le c tro n t r a n s p o r t , e s p e c ia lly under
a e ro b ic c o n d itio n s , re q u ire s t i g h t l y coupled m ito c h o n d ria w hich
la c k s i g n i f i c a n t ATPase a c t i v i t y . S in ce th e s e m ito c h o n d ria
ap p ea r to be le s s coupled th an th e pigeon h e a r t p re p a r a tio n s used
by C hance, i t i s n o t to o s u r p r is in g t h a t ATP e f f e c t s co u ld n o t be
|
d em o n strated .
The e f f e c t s o f ro ten o n e and am ytal on th e re d u c tio n o f
p y rid in e n u c le o tid e by s u c c in a te and i t s p a r t i a l re o x id a tio n by
1 1 -d e o x y c o rtis o l i s seen in F ig u re 13. L ine A shows a ty p ic a l
tr a c in g of th e changes in p y rid in e n u c le o tid e d u rin g s u c c in a te -
su p p o rted h y d ro x y la tio n • I n i t i a t i o n o f h y d ro x y latio n by th e
a d d itio n o f 1 1 -d e o x y c o rtis o l r e s u l t s in th e o x id a tio n o f a p p ro x i
m ately 50% o f th e reduced p y rid in e n u c le o tid e . As observed in
L in e B, a d d itio n o f ro ten o n e r e s u l t s in an i n i t i a l g ra d u a l
i
1
'in c re a s e in flu o re s c e n c e as p y rid in e n u c le o tid e i s reduced by
endogenous s u b s tr a te s and cannot be o x id iz e d by th e in h ib ite d
r e s p ir a to r y c h a in . The subsequent a d d itio n o f su c c in a te c au ses a
f u r t h e r re d u c tio n of p y rid in e n u c le o tid e . The r a te and e x te n t of
re d u c tio n i s somewhat le s s th a n t h a t seen in th e c o n tro l stu d y .
A dd itio n o f 1 1 -d e o x y c o rtis o l cau ses an o x id a tio n of p y rid in e
n u c le o tid e t o ap p ro x im ately th e same e x te n t seen in th e upper
t r a c in g . The r a t e o f o x id a tio n may be s l i g h t l y g r e a t e r . The
e f f e c t o f am ytal i s a b i t more pronounced. Not only does i t
i n h i b i t th e i n i t i a l r a t e and e x te n t o f re d u c tio n , i t a ls o
i n h i b i t s th e o x id a tio n o f th e reduced p y rid in e n u c le o tid e in
resp o n se t o 1 1 -d e o x y c o rtis o l. T h is l a t t e r fin d in g i s perhaps
76
S U C C IN A T E
7.8mM
R O T E N O N E
I« J IM
S U C C IN A T E
7.8hiM
ll-D E O X Y C O R T IS O L
SI 11M /
S-173
F ig . 1 3 .—E f f e c ts o f ro ten o n e and am ytal on
s u c c in a te re d u c tio n o f p y rid in e n u c le o tid e . M ito
c h o n d ria (0 .4 mg N/ml) in is o to n ic b u f f e r , 25° C.
e x p la in e d by th e in h ib i t io n o f h y d ro x y la tio n by am y tal w hich was
o b serv ed in th e p re se n c e o f m alate o r N A D FH in c a lc iu m -tre a te d
m ito c h o n d ria (5 7 ). B oth o f th e s e e le c tr o n donors a p p e a r t o p a ss !
e le c tr o n s d i r e c t l y t o th e h y d ro x y lase system which would in d ic a te ,
t h a t am y tal i s i n h ib i t in g e le c tr o n tr a n s p o r t in th e h y d ro x y lase
' > 1
sy ste m . The e f f e c t o f ro ten o n e and am ytal on th e r a t e s o f ;
h y d ro x y la tio n in t h i s ex perim ent w i l l be shown below .
The i n i t i a l la g tim e f o r p y rid in e n u c le o tid e re d u c tio n by !
s u c c in a te i s g r e a tly in c re a s e d in th e T ris -m a n n ito l-s u c ro s e
medium (See F ig u re 1 7 ). T h is la g i s r e f le c te d in th e i n i t i a l
r a t e s o f s u c c in a te -s u p p o rte d h y d ro x y la tio n . The i n i t i a l tim e
c o u rse o f h y d ro x y la tio n i n i t i a t e d by s u c c in a te i s shown in
F ig u re 14. In th e experim ent in d ic a te d by s o lid t r i a n g l e s ,
s u c c in a te was added t o m ito c h o n d ria p re -in c u b a te d f o r 3 m inutes
in is o to n ic phosphate b u f f e r . 1 1 -D e o x y c o rtiso l had been added
p r i o r t o s u c c in a te . As in c u b a tio n o f 1 1 -d e o x y c o rtis o l w ith m ito
c h o n d ria in th e ab sen ce o f exogenous e le c tr o n donor fo llo w in g a
few m inutes of p re -in c u b a tio n r e s u lte d in no h y d ro x y la tio n , z e ro
tim e i s c o n sid e re d t o be when su c c in a te was added. T ris -m a n n ito l-
su c ro se b u f f e r was used in th e experim ent in d ic a te d by th e s o lid
c i r c l e s . A ll o th e r c o n d itio n s were th e sam e. In th e m a n n ito l
b u f f e r th e i n i t i a l la g in h y d ro x y la tio n a c t i v i t y i s s i g n i f i c a n t l y
78
F ig . 1 4 .—I n i t i a l tim e c o u rse o f s u c c in a te -
su p p o rte d h y d ro x y la tio n . M ito ch o n d ria e q u iv a le n t to
0 .6 3 mg N/ml were in cu b a te d a t room tem p era tu re w ith
ra p id s t i r r i n g in sm a ll b e a k e rs . In cu b a tio n m ix tu re:
A — is o to n ic b u f f e r ; s u c c in a te , 7 .7 rriM ; 4 - 1 4 c - ll-
d e o x y c o rtis o l, 151 m^moles. x — a s A p lu s ADP,
2 .5 [xmoles. • — T ris -m a n n ito l-s u c ro s e b u f fe r ;
o th e r a d d itio n s a s in A.
g r e a t e r , and th e r a t e o f h y d ro x y la tio n which i s o b ta in e d a f t e r j
i ]
t h e la g i s l e s s th a n in th e is o to n ic medium.
The e f f e c t o f p h o sp h o ry la tin g c o n d itio n s on. s u c c in a te -
su p p o rted h y d ro x y la tio n was exam ined in th e experim ent in d ic a te d |
I
by x in F ig u re 14. C o n d itio n s were i d e n t i c a l t o t h a t in th e
experim ent in d ic a te d by A ex cep t e x c e ss ADP was in c lu d e d in th e
medium — more th an cou ld be p h o sp h o ry la te d by th e c o n c e n tra tio n
i
o f m ito ch o n d ria used w ith in th e tim e o f th e e x p e rim e n t. Appar
e n tl y under th e s e c o n d itio n s , ADP does n o t i n h i b i t th e r a t e of
h y d ro x y la tio n n o r does i t ap p ear t o e x ten d th e i n i t i a l la g tim e .
The a p p a re n t dependence o f h y d ro x y la tio n su p p o rte d by
su c c in a te on g e n e ra tio n of reduced p y rid in e n u c le o tid e c e r t a i n l y
su g g e sts t h a t p y rid in e n u c le o tid e m ed iate s e le c tr o n t r a n s f e r
betw een s u c c in a te and th e h y d ro x y lase sy stem . However, th e
f a i l u r e o f ro ten o n e t o s i g n i f ic a n t l y i n h i b i t th e r a t e o f hydrox
y l a t i o n , d id n o t su p p o rt th e h y p o th e s is t h a t re v e rs e e le c tr o n
tr a n s p o r t was r e q u ire d . I t was c o n sid e re d p o s s ib le t h a t a m ajor
c o n trib u tio n t o th e r a t e o f s u c c in a te -s u p p o rte d a c t i v i t y came
from th e o x id a tio n o f m alate form ed v ia fu m arase. To t e s t t h i s
id e a , th e r a t e s o f h y d ro x y la tio n su p p o rte d by s e v e r a l low con
c e n tr a tio n s o f m alate were d eterm in ed in th e p resen c e and absence
o f ro te n o n e . The r e s u l t s o f one such experim ent i s shown in
80 >
jFigure 1 5 . The p re se n c e o f ro te n o n e , in d ic a te d by s o lid t r i -
i
a n g le s , h as alm ost no e f f e c t on th e r a t e of h y d ro x y la tio n even !
i
t j
when th e c o n c e n tra tio n o f m alate i s c le a r ly su b -o p tim a l. The K m |
I
f o r m alate as determ in ed from an in v e rs e p lo t i s ap p ro x im ate ly
t i
0 .2 mM . The r a t e o f oxygen uptake by t h i s p re p a ra tio n in th e
p resen c e o f s u c c in a te was 132 m m oles /m in u te . The maximum amount
o f m alate form ed from s u c c in a te in a m inute would be 264 m m o le s.
Assuming t h i s i s in e q u ilib riu m w ith th e medium, th e c o n c e n tra - '
t i o n o f m alate would be 0.082 m M . The r a te o f h y d ro x y la tio n
which t h i s c o n c e n tra tio n o f m alate can su p p o rt, even in a
ro ten o n e -b lo c k ed c h ain (ap p ro x im ately 20 m ^ o le s /m in u te ) , i s
s i g n i f i c a n t l y le s s th a n th e r a te o f h y d ro x y la tio n su p p o rte d by
s u c c in a te which was 53 m JU ^es/roinut® • E ith e r th e m alate g e n e r
a te d i s c o n c e n tra te d a t th e s i t e o f i t s dehydrogenase, o r a
m ajor component o f su c c in a te -s u p p o rte d a c t i v i t y i s n o t v ia m alate
and presum ably in v o lv e s re v e rse d e le c tr o n t r a n s p o r t .
P y rid in e N u c leo tid e R eduction by S u lfid e p lu s ATP
W hile a tte m p tin g t o f in d e x p e rim e n ta l c o n d itio n s where ATP
would s tim u la te s u c c in a te -s u p p o rte d p y rid in e n u c le o tid e re d u c tio n ,
i t was found t h a t s u l f i d e , an a g en t u t i l i z e d t o i n h i b i t cytochrom e
o x id a s e , caused an in c re a s e in flu o re sc e n c e — th e d u ra tio n and
i n te n s i t y o f which c o u ld be in c re a s e d w ith ATP. F ig u re 16 shows
81
c
E
80
i
E
z
o
2
K
S
•o
£ 40
«
t
0
u
& 20
1
F ig . 1 5 .—Dependence o f h y d ro x y la tio n on
m a la te c o n c e n tr a tio n . M ito ch o n d ria e q u iv a le n t to
0 .5 mg N/ml were in c u b a te d in c u v e tte s . 4 - l^C -1 1 -
d e o x y c o rtis o l (226 nvunoles) was added, and th e n
th e r e a c tio n was begun w ith th e a d d itio n o f th e
c o n c e n tra tio n o f m a la te in d ic a te d above. R ates
were d eterm in ed from 4 -p o in t p ro g re s s c u rv e s .
82
N o 2S
A T P
A T P
20%! M A L A T E -F L U O R E S C E N C E
A T P N o 2S
B
(H min*l
F ig . 1 6 .—E f fe c t of s u lf id e and ATP
on flu o re sc e n c e (A, B) and oxygen e le c tr o d e
(A1)* M itochondria e q u iv a le n t t o 0 .6 mg N/ml
in T ris -m a n n ito l-s u c ro s e medium. An e stim a
tio n o f th e m agnitude of flu o re sc e n c e i s
in d ic a te d by th e amount of flu o re sc e n c e
produced by m alate in an i d e n t i c a l p re p a ra
t i o n . A d d itio n s: Na2S , 1 m M ; ATP, 0 .3 m M .
;exam ples o f t h i s e f f e c t . The i n s t a b i l i t y o f th e e le c tro d e j
! i
t r a c i n g , A», in d ic a te s s u lf id e in te r a c tio n w ith e i t h e r th e s i l v e r '
o r p la tin u m c o u p le . A d d itio n o f ATP f u r t h e r in c re a s e s f l u o r e s
cence and th e e le c tro d e tr a c e r a p id ly f a l l s . As th e e le c tro d e
t r a c e a p p ea rs t o c ea se f a l l i n g , th e r e i s a r a th e r a b ru p t red u c
t io n in flu o re s c e n c e . T rac in g B shows a s im ila r c y c le of
flu o re s c e n c e when ATP i s added p r i o r t o s u l f i d e . The medium used
in th e s e e x p erim en ts was th e T ris -m a n n ito l-s u c ro s e medium
d e s c rib e d e a r l i e r . S im ila r resp o n ses t o s u lf id e w ith or w ith o u t
ATP co u ld a ls o be ob serv ed in a T ris-K C l o r a g ly c y l-g ly c in e
b u f f e r . However, p hosphate in r e l a t i v e l y low c o n c e n tra tio n s
in h ib ite d b o th th e s u lf id e and ATP e f f e c t s .
When 1 1 -d e o x y c o rtis o l was added to a p re p a ra tio n t r e a t e d
t o g iv e t h i s flu o re s c e n c e , th e flu o re sc e n c e d e c re a se d . A r e a
so n ab le assum ption was t h a t th e flu o re sc e n c e was due to p y rid in e
n u c le o tid e re d u c tio n . I t was somewhat s u r p r is in g t o o b serv e t h a t
s u c c in a te , a lth o u g h n o t as e f f e c t i v e l y as 1 1 -d e o x y c o rtis o l, a ls o
seemed to remove o r p re v e n t th e fo rm atio n o f t h i s flu o re s c e n c e .
S in c e th e 1 1 -d e o x y c o rtis o l appeared to o x id iz e th e p y rid in e
n u c le o tid e , i t was o f i n t e r e s t t o see i f h y d ro x y la tio n c o u ld be
su p p o rte d by th e sulfide-A T P sy stem .
I The r e s u l t o f one h y d ro x y la tio n e x p erim e n t i s shown in
j
T ab le I I . T h is i s t h e m ost ,co m p lete e x p e rim e n t in r e s p e c t t o
a d eq u a te c o n tr o l s , b u t in th e s e v e r a l e x p e rim e n ts o f t h i s ty p e
p erfo rm ed , i t was found t h a t h y d ro x y la tio n c o u ld be w e ll su p
p o rte d by t h i s sy ste m . As can be s e e n , in th e T r is - m a n n ito l-
su c ro se medium, th e su lfid e-A T P sy stem s u p p o rts h y d ro x y la tio n a t
a r a t e even f a s t e r th a n t h a t su p p o rte d by s u c c in a te and 7 t o 8
tim e s f a s t e r th a n e i t h e r s u l f i d e o r ATP does a lo n e . As was p re
d ic te d from th e flu o r e s c e n c e t r a c i n g s , th e s u l f i d e o r su lfid e-A T P
system does n o t s u p p o rt h y d ro x y la tio n in a p h o sp h a te b u f f e r . The
i n h ib i t io n o f th e su lfid e-A T P sy stem by s u c c in a te i s r e p e a ta b le
and can be shown t o be an im m ediate e f f e c t by a d d in g s u c c in a te in
th e m iddle o f an in c u b a tio n , ta k in g s e v e r a l a li q u o t s b e fo re and
a f t e r i t s a d d itio n .
T hese o b s e rv a tio n s a r e p re s e n te d a s an i n t e r e s t i n g b u t
u n e x p la in e d phenomenon. I t may i n d ic a t e a u se o f s u lf id e a s a
re d u c in g a g e n t, a s c y a n id e and a z id e y ie ld e d l i t t l e , i f an y ,
flu o re s c e n c e u n d er th e same c o n d itio n s . I t c o u ld in d ic a te t h a t
s u lf id e i s a much b e t t e r i n h i b i t o r o f th e r e s p i r a t o r y c h a in and
ATP can a c t i v a t e re d u c in g s u b s tr a t e s g iv in g r i s e t o red u ced
p y rid in e n u c le o tid e . In any c a s e , t o my know ledge such a
TABLE II
110-HYDROXYLATION SUPPORTED BY SULFIDE A N D ATP
R ate o f C o r tis o l P ro d u ctio n (mpjnole/min/mg N)
Medium
None S u c c in a te S u lfid e ATP S u lfid e + ATP
S u lfid e +
ATP +
S u c c in a te
T ris-M an n ito l-S u cro se 0 16 3.2 2 .4 20 12
I s o to n ic Phosphate
-----
39 0
-----
0
-----
A d d itio n s o f re a g e n ts were made a f t e r 2 to 3 m inutes of p re -in c u b a tio n in a b eak er
s t i r r e d t o m ain tain a e ro b ic c o n d itio n s a t room tem perature* C o n c e n tra tio n s: M ito ch o n d ria,
0 .6 3 mg N/m l; s u c c in a te , 7 .7 m M ; s u l f i d e , 0 .3 m M ; ATP, 0 .7 6 m M ; 4 - ^ -^ -1 1 -d e o x y c o rtis o l,
151 mumoles; t o t a l volume o f 3 .3 m l.
00
cn
8 6
phenomenon h a s n o t been p re v io u s ly o b se rv e d , o r a t l e a s t re p o rte d ^
; f
in m ito c h o n d ria .
i
Calcium A ccum ulation
A d re n o c o rtic a l m ito c h o n d ria respond t o low c o n c e n tra tio n s
o f calciu m in a m anner s im ila r t o t h a t d e s c rib e d by Chance f o r
l i v e r and h e a r t m ito c h o n d ria (1 0 4 ). F ig u re 17 shows th e o x id a -
i
t i o n o f p y rid in e n u c le o tid e fo llo w in g a d d itio n o f calciu m t o
m ito c h o n d ria o x id iz in g s u c c in a te . The ex p erim en t shown in th e
u p p er t r a c i n g was c a r r ie d o u t in p h o s p h a te -c o n ta in in g is o to n ic
b u f f e r . A d d itio n s o f 150 |iM CaCl2 r e s u l t in ra p id c y c le s o f
o x id a tio n -r e d u c tio n o f p y rid in e n u c le o tid e . I t a p p ea rs t o
become 45% more o x id iz e d . The low er t r a c i n g shows an i d e n t i c a l
ex p erim en t in th e T ris -m a n n ito l-s u c ro s e medium. The la g tim e
b e fo re p y r id in e n u c le o tid e i s red u ced i s g r e a t ly e x te n d e d , and
th e r a t e a t w hich re d u c tio n o c c u rs i s much slo w er in t h i s medium.
A d d itio n o f 150 |iM calciu m o x id iz e s ap p ro x im ate ly 60% o f th e
reduced p y r id in e n u c le o tid e . I t rem ains in th e more o x id iz e d
s t a t e u n t i l p h o sp h ate i s added.
The e f f e c t o f calciu m on s t e r o id h y d ro x y la tio n s has
re c e iv e d c o n s id e ra b le a tt e n t i o n fo llo w in g th e su g g e stio n by
X o ritz and Perone (120) t h a t calciu m may have a r o le in th e
87
i
SUCCINATE
7.7mM
CaCl2
ISOpM
SUCCINATE
I 7.7mM
PHOSPHATE
S-183
|-l m in— |
F ig . 1 7 .- - E f f e c t o f calciu m on s u c c in a te -
produced flu o re s c e n c e in th e p re se n c e and absence
o f p h o sp h a te . M ito ch o n d ria (0 .4 3 mg N/ml) in
is o to n ic b u f f e r (u p p e r t r a c e ) and in T r is -
m a n n ito l-s u c ro se (lo w er t r a c e ) , 25° C.
mechanism of a c tio n o f AC1H. From subsequent s tu d ie s Perone (
| i
l e t a l . (121) re p o rte d th e locus o f calcium a c tio n t o be m ito - |
I |
jch o n d ria l. They found 8 to 11 m M calcium s tim u la te d C-18 and j
i ;
lip -h y d ro x y la tio n of D O C by r a t a d re n a l m ito c h o n d ria in th e !
! _ I
p resen ce o f NADPH. '
I The stim u la tio n of NADPH-supported lip -h y d ro x y la tio n by |
i \
[a d re n o c o rtic a l m ito ch o n d ria was confirm ed by s tu d ie s in t h i s
[lab o rato ry (5 7 ). T h is e f f e c t has been a t t r i b u t e d t o an in c re a s e d i
|
|p e rm e a b ility of th e m itochondria t o NADPH. NADPH, a s w e ll a s , j
i
N A D H su p p o rts a very low r a te of r e s p ir a tio n in i n t a c t adreno
c o r t i c a l m ito c h o n d ria . A ddition o f 2.0 m M calcium g r e a tly stim u - I
i ' !
jla te s N A D H o x id a tio n and a ls o NADPH o x id a tio n when exogenous NAD+
i j
jis added in c a t a l y t ic am ounts.
G u erra, P ero n e, and McCarthy more r e c e n tly re p o rte d an
i ’ !
in h ib i t io n of lip -h y d ro x y la tio n su p p o rte d by K rebs C ycle i n t e r
m ediates by r e l a t i v e l y low c o n c e n tra tio n s (0 .1 to 1 .0 m M ) of
| ;
jcalcium (1 2 2 ). S im ila r fin d in g s had been o b ta in e d in t h i s la b o - !
jrato ry ( 5 7 ). The experim ents d e sc rib e d by th e s e w orkers were
j30-minute in c u b a tio n s w ith o u t p ro g re s s iv e d e te rm in a tio n s . A slow
b u t s ig n if ic a n t leakage o f p y rid in e n u c le o tid e caused by calcium
could account f o r t h e i r fin d in g s . The in c u b a tio n s done in t h i s
[lab o ra to ry to o k 5 to 10 m inutes. The r a t e s o f h y d ro x y latio n
i
| 89
d e te rm in e d by 3 o r 4 -p o in t p ro g re s s cu rv es were o fte n n o n - lin e a r
jin th e p re se n c e o f c a lc iu m . The r a p id f a l l - o f f o f a c t i v i t y would
!
■support th e e x p la n a tio n o f p y rid in e n u c le o tid e le a k a g e . However,
!
jthe c o n tr o l s tu d ie s in th e T ris-X C l b u f f e r used were o fte n non-
i
l i n e a r to o .
In l i g h t o f th e r e v e r s ib le un co u p lin g e f f e c t s of calcium
jdem onstrated in th e s e m ito c h o n d ria , i t was of i n t e r e s t t o d e te r
m ine th e im m ediate e f f e c t s o f calcium on th e r a t e of 110-hydrox-
j y l a t i o n . Two ex p erim en ts a re i l l u s t r a t e d in F ig u re 18. The
tr a c in g s show p y rid in e n u c le o tid e flu o re s c e n c e in th e p resen c e of
i
Im alate and s u c c in a te . T hese were d i f f e r e n t e x p e rim e n ts; so th e
ia b so lu te v a lu e s sh o u ld n o t be com pared. A d d itio n of 11-deoxy
c o r t i s o l r e s u l t s in a p a r t i a l o x id a tio n o f th e reduced p y rid in e
n u c le o tid e w ith a g r e a te r p e rc e n ta g e change in th e case of
s u c c in a te . The r a t e s o f h y d ro x y la tio n can be e s tim a te d from th e
s lo p e s o f th e l in e s c o n n e c tin g th e d o ts in d ic a tin g th e amounts of
jc o r tis o l form ed. The a d d itio n o f calcium r e s u l t s in a f u r t h e r
c y c lic o x id a tio n of p y rid in e n u c le o tid e . In b o th e x p e rim e n ts,
t h e r e a p p ea rs to be a t r a n s i e n t c e s s a tio n o f h y d ro x y la tio n which
joccurs a f t e r th e maxim al o x id a tio n of p y rid in e n u c le o tid e has
i
been re a c h e d . The l i n e a r r a t e o f h y d ro x y la tio n which i s th en
[a tta in e d i s s l i g h t l y b u t n o tic e a b ly slo w er th a n th e p re -c a lc iu m
S U C C IN A T E
7,7m M
B O m jim olac C O R T IS O L
ll-O E O X Y C O R T IS O L
246m|imol«*
S-184
IO O m |H n o to > C O R T IS O L ll-O E O X Y C O R T IS O L
> 224mjliMlM
F ig . 1 8 .—E f f e c t o f calciu m on f lu o r e s
cence and h y d ro x y la tio n . M ito ch o n d ria e q u iv a le n t
t o 0 .6 1 mg N/ml f o r Exp. 184 and 0 .6 7 mg N/ml f o r
Exp. 172; in is o to n ic b u f f e r , 25° C.
r a t e . In n e i t h e r c a se i s t h e r e any a p p a re n t l o s s o f p y r id in e
n u c le o tid e . I t would t h e r e f o r e seem t h a t c a lc iu m does have an
'in h ib ito r y e f f e c t on h y d ro x y la tio n w hich i s im m ediate and does
n o t seem a t t r i b u t a b l e t o p y r id in e n u c le o tid e le a k a g e . The t r a n
s i e n t i n h i b i t i o n o b se rv ed d u rin g th e r e v e r s i b l e c a lc iu m u n c o u p l-
I
;ing may i n d ic a te a s e n s i t i v i t y o f t h e h y d ro x y la se system t o th e
en erg y s t a t e o f th e m ito c h o n d ria o r p e rh a p s r e f l e c t s th e co m p e ti
t i o n f o r red u ced p y rid in e n u c le o tid e betw een th e r e s p i r a t o r y
c h a in and t h e h y d ro x y la se sy ste m .
A sco rb a te O x id a tio n
The r o le o f a s c o r b ic a c id in a d r e n o c o r tic a l m etabolism
in c lu d in g i t s re sp o n se t o ACTH, h as rem ained a m y stery in s p i t e
o f much s tu d y . A sc o rb ic a c id , w hich i s accu m u lated in th e
a d re n a l g la n d i s d e p le te d in re s p o n se t o ACTH (1 2 3 ). T h is sug
g e s ts a r o le o f a s c o r b ic a c id in th e c o n tr o l o f c o r t i c o s te r o i d o -
g e n e s is . Sharma e t a l . (124) r e p o r te d th e a cc u m u latio n o f
a s c o rb a te by s l i c e s o f a d r e n a l c o r te x t o be e n erg y d ep en d en t and
t h a t b o th ACTH and 1 1 0 -h y d ro x y la ta b le s t e r o i d s u b s tr a t e s i n h i b i t
t h e a c c u m u la tio n . B oth s tim u la tio n and i n h i b i t i o n o f s t e r o i d
b io s y n th e s is by a s c o rb a te in v i t r o have been r e p o r te d (1 2 5 , 1 2 6 ).
I t was found in t h i s la b o r a to r y t h a t a s c o rb a te supplem ented w ith
TM PD (N ,N ,N l ,N , “te tra m e th y l-p -p h e n y le n e d ia m in e ) c o u ld s u p p o rt a
low r a t e o f 1 1 0 -h y d ro x y latio n ( 5 7 ) . !
j
It was o f i n t e r e s t t o determine th e p ath w ay (s) o f e le c - !
|
jtro n tr a n s p o r t u t i l i z e d when h y d ro x y la tio n was b e in g su p p o rte d by ;
I
la s c o rb a te . In a s o lu b le s t a t e , th e non-heme ir o n p r o te in o f th e j
; ' i
ihydroxylase system can fu n c tio n as a cytochrom e c re d u c ta s e . j
i
I
A lthough th e r e does n o t ap p ear t o be a c o n n e c tio n in i n t a c t m ito
c h o n d ria , i t seemed p o s s ib le t h a t TM PD c o u ld l in k cytochrom e c j
i
w ith th e h y d ro x y lase sy stem . A p ro b ab ly more l i k e l y means o f j
I I
a sc o rb a te -s u p p o rte d h y d ro x y la tio n would be th ro u g h p y rid in e
i
i
n u c le o tid e red u ced by r e v e r s a l o f e le c tr o n t r a n s p o r t . I f such
i \
{were th e c a s e , t h i s would be th e c le a r e s t e v id e n ce f o r e le c tr o n j
I !
r e v e r s a l in a d re n o c o r tic a l m ito c h o n d ria . The e f f e c t o f uncou
p l e r s and r e s p ir a to r y c h a in i n h ib i t o r s on b o th a s c o rb a te - !
S u p p o rted p y rid in e n u c le o tid e re d u c tio n and h y d ro x y la tio n was
js tu d ie d .
A sco rb a te alo n e i s u n ab le to red u ce p y rid in e n u c le o tid e .
!
However, a s i s shown in F ig u re 19, upon th e su b seq u en t a d d itio n
I
|of 0 .6 m M TMPD, a ra p id re d u c tio n of p y rid in e n u c le o tid e o c c u rs .
The a d d itio n o f cyanide r e s u l t s in an o x id a tio n o f p a r t o f th e
: I
f lu o r e s c e n c e . The flu o re s c e n c e w hich rem ains i s a t t r i b u t e d to
S ;
TM PD a s i t v a r ie s w ith i t s c o n c e n tra tio n . When m a la te i s added,
t h e r e i s a second ra p id re d u c tio n o f p y rid in e n u c le o tid e . I t can |
T M P D
0.66ml*
A S C O R B A T E
7.7 m M
—I m i n -
S-ll-2
F ig . 1 9 .--F lu o re sc e n c e produced by
ascorbate-TM PD. The e f f e c t o f cyanide and com
p a ris o n w ith m alate-produced flu o re sc e n c e i s a ls o
shown. M ito ch o n d ria e q u iv a le n t to 0 .5 1 mg N/ml
in is o to n ic b u f f e r , 250 C.
94
be seen t h a t t h e amount o f f lu o r e s c e n c e produced by a s c o r b a te -
IMPD w hich i s r e v e r s i b l e w ith c y an id e i s a p p ro x im a te ly 50% th e
amount o f flu o re s c e n c e produced by m a la te .
When t h e r e a c tio n i s fo llo w e d f o r a lo n g e r p e rio d o f
tim e , as i s shown in F ig u re 2 0 , i t a p p e a rs t h a t th e maxim al
i
re d u c tio n o f p y rid in e n u c le o tid e s l a s t s f o r o n ly ab o u t a m inute !
|
b e fo re a s l i g h t o x id a tiv e tr e n d o c c u r s . T h is i s p ro b a b ly due t o
a p ro g re s s iv e u n c o u p lin g o f th e m ito c h o n d ria by TMPD. When th e
su sp en sio n becomes d e p le te d o f oxygen, o x id a tio n of p y r id in e
!
n u c le o tid e becomes more r a p i d . As can be seen in th e low er !
tr a c in g , o x id a tio n o f p y rid in e n u c le o tid e under a e ro b ic c o n d i- j
tio n s can be made im m ediate and com plete by ad d in g an u n co u p lin g j
agent — in t h i s c a s e , p e n ta c h lo ro p h e n o l (PC P). In a d d itio n t o 1
!
cyanide and p e n ta c h lo ro p h e n o l, am y tal and ro ten o n e a ls o i n h i b i t
j
th e ascorbate-TM PD re d u c tio n o f p y rid in e n u c le o tid e . A ntim ycin A I
i 1
, \
f lu o r e s c e s i t s e l f , m aking s tu d ie s o f p y rid in e n u c le o tid e f l u o r e s - i
| !
Icence d i f f i c u l t in i t s p re s e n c e . However, i t to o seemed t o
i *
I ,
i n h i b i t an in c r e a s e in flu o re s c e n c e when TM PD was added t o j
i
a s c o r b a te - tr e a te d m ito c h o n d ria . T hese e f f e c t s o f i n h i b i t o r s a re
i
s im ila r t o th o s e r e p o rte d by P a c k er (9 8 ) and T ager e t a l . (99)
f o r ascorbate-TM PD re d u c tio n o f NAD+ in i n t a c t m ito c h o n d ria .
95 !
m M
PC P
67|lM
I-I57b
|— I m in— “ J
F ig . 2 0 .—E f fe c t o f p e n ta c h lo ro
p h en o l (PCP) on flu o re s c e n c e and oxygen
u t i l i z a t i o n in th e p resen c e of a s c o r b a te -
TMPD. C u v e ttes c o n ta in m ito c h o n d ria
e q u iv a le n t to 0.18 mg N/ml and a s c o r b a te ,
14 m M , in is o to n ic b u f f e r , 250 C .
The re s p o n se o f ascorbate-T M PD red u c ed p y r id in e n u c le o -
|tid e t o th e a d d itio n o f 1 1 - d e o x y c o r tis o l i s shown in F ig u re 21.
j
jAs can be s e e n , th e i n i t i a t i o n o f h y d ro x y la tio n i s accom panied by
t h e o x id a tio n o f a s i g n i f i c a n t p o r tio n (75%) o f t h e red u c ed
{pyridine n u c le o tid e and KCN can o x id iz e th e re m a in in g 25%. The
!
( i n a b i li t y o f 1 1 -d e o x y c o rtis o l o r PCP ( n o t shown h e re ) t o d e c re a s e
I ;
th e f lu o re s c e n c e more th a n t h a t r e s u l t i n g from th e a d d itio n o f
i
c y a n id e s u p p o rts th e c o n c lu s io n t h a t th e p y r id in e n u c le o tid e i s
j i
m axim ally o x id iz e d in t h e p re s e n c e o f c y a n id e .
Ascorbate-TM PD s u p p o rts a s i g n i f i c a n t r a t e o f h y d ro x y la
t i o n b y co u p le d m ito c h o n d ria . T a b le I I I shows r e s u l t s o f two j
• i
h y d ro x y la tio n e x p e rim e n ts in w hich ascorbate-TM PD was u sed as th e j
! i
e le c t r o n d o n o r. In b o th e x p e rim e n ts th e r e a c tio n s w ere c a r r i e d
1 i
o u t in a c u v e tte w h ile f lu o r e s c e n c e and oxygen te n s io n w ere b e in g
m o n ito re d . In e x p e rim e n t 159, a s in g le sam ple was removed j u s t
b e fo re th e c u v e tte c o n te n ts became d e p le te d o f oxygen. The r e a c - '
; i
t i o n r a t e was c a lc u la te d from th e tim e betw een th e a d d itio n of
? -4 c -la b e lle d 1 1 - d e o x y c o r tis o l and when th e sam ple was quenched.
i
(Both p e n ta c h lo ro p h e n o l and a m y ta l i n h i b i t e d ascorbate-T M PD - 1
su p p o rte d h y d ro x y la tio n a s th e y had p y r id in e n u c le o tid e re d u c
t i o n . The r a t e s shown f o r e x p e rim e n t 171 were d e te rm in e d from
4 - p o in t p ro g re s s c u r v e s . The t o t a l e x p e rim e n t tim e was l e s s th a n !
97
TMPD
0.68mM
t
KCN
0-3ltnM
1 1 -D E O X Y C O R T IS O L
3l|iM
T M PD
0.66 m M
1 1 -D E O X Y C O R T IS O L
3ljiM
K C N
0.3lmM
S-I7I
1 * 1 m in -H
F ig . 2 1 .—E ffe c t o f 1 1 -d e o x y c o rtiso l on
flu o re sc e n c e produced by ascorbate-TOCPD. M ito
c h o n d ria e q u iv a le n t t o 0.35 mg N/ml and a s c o rb a te ,
8 .0 nM, in is o to n ic b u f f e r , 25° C,
TABLE I H
ASCORBIC ACID-SUPPORTED HYDROXYLATION
1
A d d itio n s
C o r tis o l Form ation
(m|xmoles/min/mg N)
Exp. 159
None 0
Ascorbate-TMPD 28, 24
Ascorbate-TMPD + PCP 0
Ascorbate-TMPD + Amytal 2 .4
Exp. 171
Ascorbate-TMPD 1 9, 21
Ascorbate-TMPD + KCN 0 .9
M alate 32, 37
S u c c in a te
■ t
24, 26
Exp. 159: M ito c h o n d ria e q u iv a le n t t o 0 .4 3 mg N/ml in
is o to n ic b u f f e r , 250 C. A d d itio n s : A sco rb a te , 14 mM ; TMPD,
0 .6 m M ; PCP, 67 |iM; a m y ta l, 2 .7 m M ; 4 - 1 4 C - ll-d e o x y c o rtis o l,
100 m|imoles.
Exp. 171: M ito c h o n d ria e q u iv a le n t t o 0 .3 5 mg N/ml in
i s o to n ic b u f f e r , 2 5 0 C. A d d itio n s : A sc o rb a te , 8 .0 m M ; TMPD,
0 .6 8 mM ; KCN, 0 .3 mM ; 4 - H c - l l - d e o x y c o r t i s o l , 100 mjimoles;
m a la te , 8 .0 mM ; s u c c in a te , 8 .0 m M .
99
2 m in u te s , e x c e p t f o r t h e cy an id e I n h i b it e d in c u b a tio n w here th e
i
f i n a l sam ple was removed a t 5 m in u te s• T h is ex p erim e n t showed
t h a t c y a n id e , a s would b e p r e d ic te d from th e f lu o re s c e n c e t r a c e s
i f p y r id in e n u c le o tid e r e d u c tio n w ere r e q u ir e d , i s a v e ry e f f e c
t i v e i n h i b i t o r o f ascorbate-T M P D -supported h y d ro x y la tio n .
F u rth e rm o re , th e i n i t i a l r a t e o f th e u n in h ib ite d r e a c t i o n , w hich
f e l l o f f r a p i d l y , i s n o t much le s s th a n th e r a t e o f s u c c in a te -
s u p p o rte d h y d ro x y la tio n .
isupported h y d ro x y la tio n in v i t r o in r e s p e c t t o in v iv o c o n d itio n s
can a t b e s t be s p e c u la tiv e . The dy es w hich have been m ost su c
c e s s f u l l y u t i l i z e d t o m ed iate e le c tr o n t r a n s f e r from a s c o r b a te t o
cytochrom e c o f th e r e s p i r a t o r y c h a in have a phenazonium fu n c
t i o n a l group a s p a r t o f a r a t h e r hy d ro p h o b ic m o le cu le (A and B ).
The s ig n if ic a n c e o f th e d e m o n stra tio n o f a s c o r b a te -
CH3
,N
CH3
A
TM PD
B
P h e n a z in e m e th o s u lfa te
-100
c
i Adrenochrome
i A compound (C) w ith somewhat s im ila r s t r u c t u r a l f e a tu r e s
j(127) i s form ed in e x t r a c t s from a d re n a l t i s s u e s , Adrenochrom e,
jas i t i s c a lle d , i s r e d u c ib le by a s c o rb a te and a u to x id iz a b le —
a s a re th e redox dyes shown ab o v e. I n i t i a l s tu d ie s a tte m p tin g to
use adrenochrom e in a TMPD-like r o le were u n s u c c e s s fu l. The
adrenochrom e sam ple, w hich ap p eared t o c o n ta in in s o lu b le im p u ri
t i e s , was v ery r a p id ly , n o n -e n z y m a tic a lly a u to x id iz e d .
j I t i s c le a r t h a t a v a r i e ty o f s u b s tr a te s can p ro v id e
^ electro n s t o th e h y d ro x y la se system . In d o in g s o , how ever, th e
energy which would n o rm ally be g e n e ra te d by i t s o x id a tio n v ia th e
jre s p ira to ry c h ain would n o t be a v a ila b le t o th e c e l l . I t would
i
seem advantageous to t h e a d re n a l c e l l t o have a re s e rv e so u rc e of
e le c tr o n s f o r e ith e r .h y d r o x y la tio n o r ATP g e n e ra tio n a v a ila b le to
be u t i l i z e d under th e s t r e s s of ACTH s tim u la tio n of th e g la n d .
The h ig h c o n c e n tra tio n o f a s c o rb a te p re s e n t in th e g lan d co u ld be
isuch a so u rce o f e le c tr o n s s e rv in g t h i s p u rp o se . The p o s s i b i l i t y
I
jof th e e x is te n c e of a p h y s io lo g ic a l m e d ia to r o f a s c o rb a te
101
o x id a tio n by th e r e s p ir a to r y ch ain i s in tr ig u in g as i t would
p erh ap s su p p ly a reason d ’e t r e f o r a d re n a l a s c o rb ic a c id .
P v rid in e N u c leo tid e T ranshvdroqenation
I
The p a r tic ip a tio n o f a p y rid in e n u c le o tid e tr a n s h y -
!
dro genase in th e g e n e ra tio n of N ADPH f o r 1 1 0 -h y d ro x y latio n was
su g g e ste d by Sweat and Lipscomb when th e y found th e h y d ro x y la tio n
o f D O C t o be su p p o rted as e f f e c t i v e l y by N A D H p lu s N A D P+ as i t
i
was by NADPH, w hereas n e ith e r N A D H n o r NADP+ a lo n e was a c tiv e
1(128). S te in e t a l . (129) found th e r a t e of re d u c tio n o f th e
ja c e ty l-a n a lo g u e o f N A D + by N AD PH to be v ery ra p id in r a b b it
a d re n a l m ito c h o n d ria . The r a t e o f th e re v e rs e r e a c tio n , NADH-
|AcPyADP+ was, however, n e g lig ib le . U sing bovine a d re n o c o r tic a l
i
m ito c h o n d ria , S p iro and B a ll (130) found q u ite h ig h tra n s h y -
jdrogenase a c t i v i t y . The re d u c tio n o f N A D + by N ADPH was about
i
I
|3 tim e s f a s t e r th an th e re v e rs e r e a c tio n . H arding and N elson
i
found a h ig h tran sh y d ro g en ase a c t i v i t y in r a t a d re n a l hom ogenates
i
w hich a ls o showed a h ig h e r r a te in th e d ir e c tio n o f N A D +
i
re d u c tio n (1 3 1 ).
I
More r e c e n tly , B e ll w orking in t h i s la b o ra to ry i n v e s t i -
I
'gated th e p y rid in e n u c le o tid e tran sh y d ro g e n ase a c t i v i t y o f bovine
ia d re n o c o rtic a l m ito ch o n d ria u sin g p h o sp h o ry la tin g su b -m ito -
I
jch o n d rial p a r t i c l e s . She found i t t o be an energy c o n tr o lle d
|enzyme w ith s i m il a r p r o p e r t i e s a s have been d e s c r ib e d by
D a n ie lso n and E m s te r (1 0 8 ) and Hommes and E sta b ro o k (1 1 1 ).
i
U sing th e s e s u b -m ito c h o n d ria l p a r t i c l e s t o g e n e ra te NADPH, l i p -
:h y d ro x y la tio n o f 1 1- d e o x y c o r tis o l was s tu d ie d in a r e c o n s t i tu t e d
h y d ro x y la tin g sy ste m . The r a t e o f h y d ro x y la tio n su p p o rte d by
t h e ATP s tim u la te d tra m s hydrogen a s e was a b o u t tw o - th ir d s t h e r a t e
s u p p o rte d by m a la te p lu s NADP+ , th e m ost e f f e c t i v e s u b s t r a t e -
c o f a c to r co m b in atio n found (1 3 2 ) .
T hese s t u d i e s c l e a r l y p re s e n te d t h e p o s s i b i l i t y t h a t th e
p y r id in e n u c le o tid e tra n s h y d ro g e n a s e was p a r t i c i p a t i n g in NADPH
g e n e ra tio n f o r s u p p o rt o f 11(3-h y d ro x y la tio n in i n t a c t m ito
c h o n d ria . The fo llo w in g stu d y was d e sig n e d t o d e te rm in e i f th e
tra n s h y d ro g e n a s e was a s i g n i f i c a n t p a r t i c i p a n t in t h e s u p p o rt o f
h y d ro x y la tio n by K rebs C y cle in te r m e d ia te s . An a sse ssm e n t o f i t s
c o n tr o l by th e e n erg y s t a t e o f th e m ito c h o n d ria and o f i t s r a t e
u s in g endogenous p y r id in e n u c le o tid e w ere a d d i t i o n a l aim s.
T h ree e le c tr o n d o n o rs w ere u sed t o s tu d y p y rid in e n u c le o
t i d e tra n s h y d ro g e n a s e a c t i v i t y in i n t a c t m ito c h o n d ria s m a la te ,
w hich i s a p p a r e n tly o x id iz e d by b o th N A D and NADP-linked dehy
d ro g e n a se s i n th e s e p a r t i c l e s ; s u c c in a te , w hich can red u c e N A D
presum ably by e le c tr o n r e v e r s a l ; and a - k e t o g l u t a r a t e , w hich
s p e c i f i c a l l y re d u c e s NAD*. K in e tic s t u d i e s o f th e flu o r e s c e n c e
produced In th e p resen c e o f th e s e e le c tr o n do n o rs had shown t h a t
upon a d d itio n o f 1 1-d e o x y c o rtis o l t o m ito c h o n d ria l su sp en sio n s
o x id iz in g m a la te , s u c c in a te , o r a - k e to g lu ta r a te , a p p ro x im ately
30, 45, o r 50 p e rc e n t o f th e reduced p y rid in e n u c le o tid e was
o x id iz e d . I n i t i a l l y th e drop in reduced p y rid in e n u c le o tid e was
a t t r i b u t e d t o NADPH, which i s known t o be o x id iz e d s p e c i f i c a ll y
by th e h y d ro x y lase sy stem . However, th e amount o f p y rid in e
n u c le o tid e e s tim a te d t o have been o x id iz e d was g r e a t e r th a n th e
c o n c e n tra tio n o f t o t a l NADP which had been e n z y m a tic a lly d e te r
m ined by a co-w orker (5 7 ). S u b seq u e n tly , ex p erim e n ts were
d e sig n ed t o d e term in e th e o x id a tio n s t a t e o f th e endogenous N A D
and NADP w h ile in th e p resen c e and absence o f 1 1 -d e o x y c o rtis o l.
The method d e s c rib e d by E stab ro o k and M a itra (24) was
e s s e n t i a l l y used t o p re p a re and a ssa y th e m ito c h o n d ria l e x t r a c t s .
However, s in c e th e s e n s i t i v i t y o f th e flu o ro m e te r employed in
th e s e s tu d ie s i s a p p a re n tly le s s th a n th e E ppendorf in stru m e n t
employed in t h e i r s tu d ie s , m o d ific a tio n s w ere in tro d u c e d t o m in i
m ize d i lu t i o n o f th e e x tr a c ts d u rin g n e u t r a l i z a t i o n . The neu
t r a l i z a t i o n and a ssa y of th e e x tr a c ts i s d e s c rib e d in some d e t a i l
in C h ap ter I I .
M ito ch o n d ria were in c u b a te d a t room te m p e ra tu re in a
b e a k e r r a p id ly s t i r r e d to m a in ta in a e ro b ic c o n d itio n s . Volumes
104
I
^adequate t o e n a b le th e r e q u i s i t e number o f 2 ml a liq u o ts f o r
p y rid in e n u c le o tid e a ssa y and 1 ml a liq u o ts f o r c o r t i s o l d e te r
m in a tio n s t o be tak e n from a s in g le sam ple were p re p a re d con
t a i n i n g m ito c h o n d ria e q u iv a le n t t o 1 t o 2 mg N/m l. In o rd e r t o
d e te rm in e th e tim e re q u ire d t o re a c h th e d e s ire d ste a d y s t a t e
a f t e r a d d itio n o f e le c tr o n donor o r 1 1 -d e o x y c o rtis o l, a 3 ml
sam ple was sim u lta n e o u sly p rep a red and m onitored w ith th e
flu o ro m e te r and oxygen e le c tr o d e . A liq u o ts f o r d e te rm in a tio n o f
S ta te 1 v a lu e s were ta k e n when th e flu o re s c e n c e o f th e s i s t e r
sam ple in d ic a te d t h a t th e maximum o x id iz e d ste ad y s t a t e was
re a c h e d . The e le c tro n donor was added to b o th sam ples; and when
S ta te 4 had been re a c h e d , a liq u o ts were ag ain ta k e n . Then
1 1 -d e o x y c o rtis o l was added. Two a liq u o ts were ta k e n to d e te r
mine c o r t i s o l p ro d u c tio n b e fo re th o se f o r p y rid in e n u c le o tid e
a ssa y and two were removed a fte rw a rd s . In t h i s way i t could be
e s ta b lis h e d t h a t th e p y rid in e n u c le o tid e le v e l m easured in d ic a te d
a ste a d y s t a t e s u p p o rtin g a l i n e a r r a t e o f h y d ro x y la tio n .
S ta n d a rd n u c le o tid e was added t o sam ples and t r e a t e d to
th e quenching and n e u tr a liz a tio n p ro c e d u re . Recovery was q u ite
v a ria b le among e x p e rim e n ts. I t was found t h a t re c o v e rie s of th e
reduced form were o fte n h ig h e r th a n f o r th e o x id iz e d . T h is i s
presum ably due to th e more e f f e c t i v e in a c tiv a tio n o f d e g ra d a tiv e
105 |
enzymes by base th a n by a c id (1 3 3 ). The rec o v ery o f th e o x id iz e d
form s was 60 to 70% w ith th e reduced form s b e in g 70 t o 80%.
T his ty p e o f ex p erim en t, in c lu d in g th e h y d ro x y la tio n
a s s a y s , was perform ed a t l e a s t tw ic e w ith each e le c tr o n d o n o r.
I
W ithout th e a d d itio n a l s te r o id d e te rm in a tio n , th e p y rid in e
i ’
n u c le o tid e v a lu e s u sin g m alate and s u c c in a te were re p e a te d ly
m easured. The r e s u l t s o f th r e e ex p erim en ts a re shown in
F ig u re 2 2 . The v a lu e s shown f o r m alate and s u c c in a te a re q u ite
t y p i c a l . The v a r ia tio n in th e c o n c e n tra tio n of t o t a l p y rid in e
n u c le o tid e betw een experim ents was found t o be q u ite l a r g e . As
each experim ent employed a d i f f e r e n t m ito c h o n d ria l p r e p a r a tio n ,
t h i s p ro b ab ly r e f l e c t s th e v a r ia tio n in th e c o n d itio n o f th e
a d re n a ls u se d . The v a lu e s f o r S ta te 1 a re n o t shown in th e
f i g u r e . In t h i s s t a t e N A D g e n e ra lly appeared t o t a l l y o x id iz e d ,
w hereas NADP was fre q u e n tly 10 to 20% red u c ed . Each p a i r o f b a rs
in d ic a te s th e c o n c e n tra tio n o f t o t a l N A D and NADP m easured. The
shaded p o rtio n re p re s e n ts th e c o n c e n tra tio n o f th e reduced
s p e c ie s . The v a lu e s in d ic a te th e av erag e o f d u p lic a te a li q u o t s .
The low er graph shows th e average p e rc e n t re d u c tio n o f th e two
p y rid in e n u c le o tid e s . The p resen ce o f e le c tr o n d o n o r, 11-deoxy
c o r t i s o l , and u n co u p ler a re in d ic a te d a t th e b ase o f th e f i g u r e .
106
sor
to
TOTAL
n tK M K
NUCLBOTKIC
B tatliL
m N
1 0
|N M M
■ NAOMI
I .
I
0L _ J U I U
I
pcrccnt
■EDUCTION
1 0 0
•0
> K 0 « K 0 « * • M ali
ll-OEOX VCONTISOL - + - + - * - +
O IC U M A ftO L “ “ “ “ - - + +
■ O T E N O N C - - - “ - _ 4 +
F i g . 2 2 .—The e f f e c t o f h y d ro x y la tio n
on th e o x id a tio n s t a t e o f p y rid in e n u c le o tid e s
in th e p re s e n c e o f s u c c in a te , m a la te , and
a - k e t o g lu t a r a t e p lu s m a lo n a te . A ssays a r e
d e s c rib e d in C h a p te rs I I and V. The concen
t r a t i o n s o f s u b s tr a te s em ployed w ere: S u c c in a te ,
15 mM ; m a la te , 20 m M ; a - k e t o g lu t a r a t e , 10 mM .
The a - k e t o g lu t a r a t e sam ples a ls o c o n ta in e d
m a lo n a te , 10 mM. The c o n c e n tra tio n o f added
c o n s t it u e n t s w ere: 1 1- d e o x y c o r tis o l, 0 .2 t o
0 .3 mM ; d ic u m a ro l, 43 pM; and ro te n o n e , 22 pM.
The e f f e c t o f u n co u p ler was s tu d ie d o n ly in th e c a se o f a -k e to -
I
i
g l u ta r a t e and w i l l be d is c u s s e d below .
C o n sid e rin g f i r s t th e n u c le o tid e reduced by th e d i f f e r e n t
e le c tro n donors in th e absence of u n c o u p le r, i t i s seen t h a t N A D
i s reduced m ost co m p letely by s u c c in a te (70%) fo llo w ed by m alate
(45%) and a - k e to g lu ta r a te (30% ). M alate ap p ears t o more e f f e c
t i v e l y red u ce NADP (99%) th a n s u c c in a te (90%) w ith a - k e to g lu ta
r a t e b e in g somewhat le s s e f f e c tiv e (83% ). These o rd e rs o f
re d u c in g c a p a b i l i t i e s rem ain in th e p resen ce o f 11- d e o x y c o r tis o l.
When one com pares th e resp o n se o f N A D H ( s o l i d b a r) and
i
N A D PH (h a tc h e d b a r) t o th e p resen ce o f 1 1 -d e o x y c o rtis o l, i t i s
e v id e n t t h a t w ith each e le c tr o n d o nor, th e d e c re a se in N A D H i s o f
g r e a te r m agnitude th a n th e d e c re a se in NADPH. T h is su p p o rts th e
p ro p o sa l t h a t th e N AD PH su p p lie d f o r o x id a tio n by th e h y d ro x y lase
system i s g e n e ra te d a t th e expense o f N A D H v ia a tra n sh y d ro g e n a se
r e a c tio n . A lthough th e p e rc en ta g e change in N A D H i s l e s s in th e
case o f m a la te , i t i s i n te r e s ti n g t h a t i t o ccu rs a t a l l w ith a
NADP-linked m alate dehydrogenase p re s e n t (5 7 ). A p p aren tly th e
NADP-linked dehydrogenase does n o t g e n e ra te N A D PH r a p id ly enough
t o s a tu r a te th e h y d ro x y lase c h a in . Nor does th e tra n sh y d ro g e n a se
as in d ic a te d by th e drop in N A D PH in th e c a se s o f a - k e to g lu ta r a te
and s u c c in a te .
108
, F or each e le c tr o n donor th e p e rc e n ta g e change in t o t a l
i
reduced p y rid in e n u c le o tid e (NADH + NADPH) as determ ined enzy
m a tic a lly i s q u ite s im ila r t o th e p e rc e n ta g e change in f lu o r e s
c en ce. The r e l a t i v e amount o f p y rid in e n u c le o tid e reduced by
a - k e to g lu ta r a te and m a la te a ls o co rre sp o n d s q u ite w e ll t o th e
r e l a t i v e flu o re s c e n c e produced in t h e i r p re s e n c e . On th e o th e r
hand, from th e c o n c e n tra tio n o f p y rid in e n u c le o tid e reduced by
s u c c in a te , one would e x p e c t su c c in a te -p ro d u c e d flu o re s c e n c e to
jbe s i g n i f ic a n t l y g r e a te r th a n t h a t produced by m a la te .
| The ste a d y s t a t e r a t i o o f p y rid in e n u c le o tid e s ,
(NADPH)(NAD+)/(NADP+)(NADH), c a lc u la te d from th e s e a ssa y s i s
g r e a te r th a n u n ity d u rin g S ta te 4 r e s p ir a tio n o f each o f th e
e le c tr o n d o n o rs. The a c t u a l v a lu e s v ary from a p p ro x im ately 3 f o r
s u c c in a te t o 8 f o r a - k e to g lu ta r a te , th e l a r g e r v alu es r e f l e c t i n g
p rim a rily l e s s re d u c tio n o f NAD. P r e f e r e n t i a l re d u c tio n o f NADP
in d ic a te d by a la r g e p o s i t iv e r a t i o h as been c it e d as evidence
f o r an e n e rg y -c o n tro lle d asym m etric tran sh y d ro g e n ase (1 1 3 ).
Im p o sitio n o f an in c re a s e d r a t e o f o x id a tio n o f N A D PH by a d d itio n
o f 11- d e o x y c o rtis o l, w h ile c a u sin g a s i g n i f ic a n t o x id a tio n of
b o th p y rid in e n u c le o tid e s , does n o t d e c re a se t h i s r a t i o , and in
some c a se s a p p e a rs t o in c r e a s e i t . T h is in d ic a te s t h a t th e r a t e
o f th e tra n sh y d ro g e n a se i s q u ite ra p id and under th e s e c o n d itio n s
109
i s d e f i n i t e l y n o t r a t e - l i m i t i n g , o r a g r e a te r o x id a tio n o f NADPH
would be e x p e c te d . The s h i f t o f b o th p y rid in e n u c le o tid e s t o a
more o x id iz e d ste a d y s t a t e su g g e sts t h a t under c o n d itio n s where
jthe h y d ro x y lase system i s s a tu ra te d w ith s te r o id s u b s tr a t e , th e
jra te o f re d u c tio n o f p y rid in e n u c le o tid e may be d e te rm in in g th e
r a t e o f th e o v e r a ll h y d ro x y la tio n p ro c e s s .
I
| The e f f e c t o f u n co u p ler on th e o x id a tio n s t a t e o f p y r i -
I
I |
dine n u c le o tid e was s tu d ie d o n ly in th e case o f a - k e to g lu ta r a te j
as t h i s was th e o n ly e le c tro n donor a p p ea rin g t o g e n e ra te N A D H I
e x c lu s iv e ly . In th e experim ent shown on th e rig h t-h a n d s id e o f j
I
jpigure 2 2 , th e m ito c h o n d ria were in c u b a te d in th e p resen c e o f
I
b.0 m M dicum arol f o r ap p ro x im ately 1 m inute b e fo re a - k e to g lu ta r a te j
j
and m alonate were added. Rotenone was a ls o added t o p re v e n t th e j
uncoupled r e s p ir a to r y c h ain from o x id iz in g NADH. The " s te a d y
jsta te " r a t i o o f p y rid in e n u c le o tid e s c a lc u la te d from th e su b se
quent a ssa y s was ap p ro x im ately 1 a s has been found by o th e r i
i
W o r k e r s i n u n c o u p l e d m i t o c h o n d r i a (1 1 5 , 1 1 6 ). The r a t e o f h y -
i f
l
d ro x y la tio n su p p o rte d by a -k e to g lu ta r a te in t h i s experim ent was
i
d e c re a se d by ap p ro x im ately 50% when ro ten o n e and d icu m aro l were
:p re s e n t. The e f f e c t o f th e s e a g e n ts on h y d ro x y la tio n su p p o rte d
I
!)y m alate and a -k e to g lu ta r a te in a n o th e r experim ent i s shown in
stable IV . I
TABLE IV
THE EFFECT OF UNCOUPLER ON THE RATE OF 11P-HYDR0XYLATI0N
A d d itio n
C o r t is o l F orm ation
(m(xmoles/min/mg N)
Exp. 194
M alate 6 1 .4
M alate + R otenone 5 5 .7
M alate + R otenone + D icum arol 3 6 .1
a -K e to g lu ta r a te 4 2 .4
a -K e to g lu ta r a te + M alonate 4 7 .0
a -K e to g lu ta r a te + M alonate +
R otenone 39.0
a -K e to g lu ta r a te + M alonate +
D icum arol 1 2 .6
a -K e to g lu ta r a te + M alonate +
R otenone + D icum arol 3 6 .6
I
Ill
The r a t e s o f h y d ro x y la tio n show some i n t e r e s t i n g c h a ra c -
I
t e r i s t i c s . The in c re a s e o f th e a -k e to g lu ta ra te -s u p p o rte d
a c t i v i t y in th e p resen ce o f m alonate i s i n t e r e s t i n g in l i g h t o f
jth e o b se rv a tio n t h a t m alonate appeared a ls o t o in c r e a s e th e r a t e
j
a t which p y rid in e n u c le o tid e was reduced by a - k e t o g lu t a r a t e .
A lthough m alate i s g e n e ra lly th e most e f f i c i e n t e le c tr o n donor in
su p p o rt o f h y d ro x y la tio n , in th e p resen ce of ro ten o n e and
dicu m aro l th e r a t e s o f h y d ro x y la tio n in th e p resen c e o f m alate
and a - k e to g lu ta r a te and m alonate become e q u a l. The la r g e i n h ib i
tio n o f th e a -k e to g lu ta ra te -m a lo n a te r a t e caused by d icu m aro l
jprobably r e f l e c t s th e co m p etitio n o f th e uncoupled r e s p ir a to r y
c h a in f o r N A D H and th e lo s s o f th e e n e rg y -lin k e d tra n sh y d ro g e n a se
i
( a c tiv ity . The in h ib itio n by dicum arol i s reduced in th e p resen c e
'of ro te n o n e ; and w h ile th e r e s u ltin g r a t e i s n o t much d i f f e r e n t
t
jthan th e r a t e in th e p resen c e o f ro ten o n e a lo n e , i t does in d ic a te
la 20% i n h ib itio n of th e c o n tr o l h y d ro x y la tio n a c t i v i t y . The 50%
I
iin h ib itio n found in th e p y rid in e n u c le o tid e ex perim ent p ro b ab ly
i
can be a t t r i b u t e d t o th e lo n g e r tim e in which d icu m aro l was
p re s e n t p r i o r t o h y d ro x y la tio n . In t h i s experim ent d icu m aro l
was added j u s t a s h o rt tim e b e fo re th e h y d ro x y la tio n was begun,
w hereas f o r th e p y rid in e n u c le o tid e a ssa y s i t was p re s e n t f o r ap
p ro x im a te ly 5 m inutes d u rin g th e i n i t i a l p a r t o f th e ex p erim e n t.
The r e s u l t s o f th e s e ex p erim e n ts in d ic a te t h a t th e p y rid in e
n u c le o tid e tra n sh y d ro g e n a se h as an im p o rta n t r o le in g e n e ra tin g
th e n e c e ssa ry e le c tr o n s f o r 1 1 0 -h y d ro x y la tio n • S in ce th e p y r i
d in e n u c le o tid e tra n sh y d ro g e n a se a p p ea rs to be under s im ila r
c o n tr o l as r e s p i r a t io n , i . e . , (ATP)/(ADP)(PO4) , i t would seem
t h a t th e energy s t a t e o f th e a d re n a l m ito c h o n d ria may be an
|
im p o rtan t r e g u la tin g f a c t o r in c o r tic o id b io s y n th e s is by th e
I
|
a d re n a l g la n d . |
' CHAPTER VI
I
CLASSIC INHIBITORS OF CORTICOSTEROID BIOSYNTHESIS
i
H i s t o r i c a l Background
i
i
i
j The f i r s t compound t o be s tu d ie d e x te n s iv e ly a s an
{ a d re n o c o rtic a l i n h i b i t o r was D D D ( 2 , 2-b is ( p a r a - c h lo r o p h e n y l) -
i
jl, 1 - d ic h lo r o e th a n o l) . T h is r e s u lte d from th e o b s e rv a tio n s o f
i
N elson and Woodward (134) o f a d re n a l a tro p h y in dogs fe d th e
i n s e c t i c i d e . Subsequent in v iv o s tu d ie s (135) showed t h a t D D D
i
su p p re sse d th e p ro d u c tio n o f a d re n a l c o r t i c o i d s . C ueto and Brown
|(136) d e m o n stra te d t h a t th e m ost a c tiv e component o f com m ercial
!
I
DDD, which c o n ta in e d a m ix tu re o f iso m e rs, was o,p*-DDD.
A lthough t h i s a g e n t h as been used c l i n i c a l l y , i t s t o x i c i t y has
lim ite d w id esp read u s e .
i
j The second i n h i b i t o r t o be s tu d ie d was a compound c a lle d
i
jamphenone B, s y n th e s iz e d by A lle n and Corwin (1 3 7 ), w hich was
I
Shown by in v iv o studies to inhibit corticoid production (1 3 8 ).
~
jl stu d y t o f in d a compound r e t a in i n g th e a b i l i t y o f amphenone t o
I
S uppress a d re n a l s e c r e tio n b u t h a v in g s id e e f f e c t s l e s s to x ic le d
(
C h a rt and Sheppard (139) t o stu d y a s e r i e s o f amphenone an alo g u es
113
f o r a b i l i t y t o d e c re a se th e 17-hydroxy s te r o id c o n te n t in p e r-
i
f u s a te s o f i n t a c t a d re n a ls in d o g s. They a ls o stu d ie d t h e i r
e f f e c t on c o r tic o id p ro d u ctio n by r a t a d re n a l s l i c e s . One d ru g ,
2 -m e th y l-l,2 -b is (3 -p y rid y l)-l-p ro p a n o n e , known as SU-4885
m ethopyrapone, and M etopirone, was in tro d u c e d by C h art e t a l .
(59) a s a p o te n t i n h ib i t o r o f c o r tic o id p ro d u c tio n . I t h as been
shown t o i n h i b i t llfi-h y d ro x y la se in v i tr o (4 1 , 140, 141, 1 4 2 ).
R ecent s tu d ie s have dem onstrated i t t o a ls o in h ib i t C-18 (142)
and C-19 (143) h y d ro x y la tio n . E a rly s tu d ie s of Sharma e t a l .
'in d ic a te d t h a t th e d egree o f in h ib itio n of llfJ-h y d ro x y la tio n of
DOC was n o t dependent on th e c o n c e n tra tio n o f D O C (4 2 ). However
Dominquez and Samuels (140) u sin g r a t a d re n a l homogenate demon
s t r a t e d t h a t m ethopyrapone was a c o m p e titiv e i n h ib ito r of 110-
h y d ro x y la tio n of DOC. They c a lc u la te d a of 16.3 pH in a
p re p a ra tio n d e m o n stra tin g a f o r D O C o f 20 pH. . In a more
re c e n t stu d y , W illiam son and O’D onnell (141) confirm ed th e com-
s
p e t i t i v e n a tu re o f m ethopyrapone in h ib itio n in an 1 1 0-h y d ro x y lase
system e x tra c te d from an aceto n e powder o f bovine a d re n a l m ito
c h o n d ria . W hereas t h e i r e stim a te d Km f o r D O C (18 pH) was
com parable t o t h a t determ ined by Dominquez and Sam uel, th e Ki f o r
m ethopyrapone determ ined by them was s ig n if ic a n tly le s s (0 .3 2 pH)
In a d d itio n , th e y found t h a t m ethopyrapone stim u la te d th e r a te o f
115 !
I
NADPH o x id a tio n by th e s t e r o i d sy stem w ith o u t a d d itio n o f DOC,
i
0 .1 m M in c r e a s in g i t by a p p ro x im a te ly tw o - f o ld . S a n z a ri and !
P erone (142) have r e p o r te d a d e c re a s e in l e v e ls of red u ced p y r i
d in e n u c le o tid e in th e p re s e n c e o f m ethopyrapone.
S p e c tr a l and K in e tic S tu d ie s
The d if f e r e n c e sp e ctru m pro d u ced by m ethopyrapone in
a e ro b ic m ito c h o n d ria and i t s s tim u la tio n o f c y an id e i n s e n s i t i v e I
i
oxygen u t i l i z a t i o n have been p r e v io u s ly d e s c rib e d in C h a p te rs I I I :
i
and IV . More r e c e n tly f lu o r e s c e n c e s tu d ie s showed i t t o d e c re a s e ;
t h e s te a d y s t a t e l e v e l o f p y r id in e n u c le o tid e in m ito c h o n d ria
! j
'o x id iz in g m a la te , c o n firm in g th e f in d in g s o f S a n z a ri and P erone
(1 4 2 ). In l i g h t o f th e d is c re p a n c y betw een th e K ^ s re p o rte d in
t h e l i t e r a t u r e w hich were o b ta in e d from q u ite d i f f e r e n t 1 1 0-
h y d ro x y la se p r e p a r a tio n s , a k i n e t i c stu d y o f m ethopyrapone i n h i
b i t i o n was u n d e rta k e n u s in g i n t a c t m ito c h o n d ria . The r e s u l t s of
a t y p i c a l stu d y a re shown in F ig u re 23. The n e g a tiv e s lo p e o f
t h e c o n tr o l p l o t shows a Km f o r 1 1- d e o x y c o r tis o l o f 17 pM, w hich
i s s im ila r t o th e v a lu e r e p o r te d by Sharma e t a l . ( 4 1 ) . M etho
p yrapone in c o n c e n tr a tio n s in d ic a te d in th e f ig u r e in c re a s e d th e
Km b u t d id n o t a l t e r th e o r d in a te i n t e r c e p t (Vmax, 12 t o
13 pM /m in). T hese r e s u l t s c o n firm p re v io u s f in d in g s re g a rd in g
t h e n a tu r e o f th e i n h i b i t i o n (1 4 0 , 1 4 1 ). The Ki c a lc u la te d from
14.0
a- a control
0 - 0 O.MpM M CTRV .
0 - 0 O-Ttptt MCTPT.
I O O
V ELO C ITY
0.0
0.0
M O .140 J O O
F ig . 2 3 .—R e c ip ro c a l p lo ts of th e e f f e c t o f
m ethopyrapone on llp -h y d ro x y la tio n of 1 1-d e o x y c o rtis o l.
M itochondria e q u iv a le n t to 0 .1 7 mg N/ml in c u b a te d in
is o to n ic b u f f e r , 37° C. C o n c e n tra tio n s o f 11-deoxy
c o r t i s o l (pM): 167, 100, 75, 50, and 25.
I
117 ;
i
th e e x p re ss io n :
I
| Kmi = *mc <1 +
I
was 0 .1 2 p M which i s c lo s e to t h a t determ ined by W illiam son and j
' I
0*D onnell (1 4 1 ). I t was found t h a t s te r o id and i n h i b i t o r had t o j
j ;
be added sim u lta n e o u sly to th e m ito ch o n d ria in o rd e r to o b ta in j
■data c o n s is te n tly showing c o m p e titiv e i n h ib i t io n . I f m etho- |
! j
jpyrapone was p re -in c u b a te d w ith th e m ito c h o n d ria , even f o r a I
i
short time, the Vmax was often decreased which indicates a mixed
I
t
i n h i b i t i o n , and p ro b ab ly r e f l e c t s th e much s tro n g e r b in d in g of
m ethopyrapone th an s te r o id t o th e hydroxylase system .
The d if f e r e n c e spectrum produced by amphenone in a e ro b ic
m ito ch o n d ria has a b so rp tio n bands a t 4 3 0 n i p . , 5 4 5 m p . , and 5 8 5 m p , j
I I
w ith a tro u g h a t about 390 m p . , which i s s im ila r to th o se induced
by pregnenolone and m ethopyrapone. The s h i f t to s l i g h t l y lo n g e r !
w avelengths i s a c o n s is te n t fin d in g of unknown s ig n if ic a n c e . The j
I I
k i n e t i c s o f th e e f f e c t of amphenone on th e s p e c tr a l p r o p e r tie s o f j
i
th e s e m ito c h o n d ria a re shown in F ig u re 24. At a p o in t where th e
R e s p ira to ry ch ain i s blocked w ith antim ycin A and has been m axi
m ally reduced by s u c c in a te , a d d itio n of amphenone r e s u l t s in a j
i |
la rg e in c re a s e in 430 m p. a b so rp tio n r e l a t i v e to 420 mp.. T h is w ould 1
j
be ex p ected in view o f th e a b so rp tio n spectrum i t p ro d u ce s. A ddi-
: i
jtion o f m alate r e s u l t s in a v ery sm a ll, slow downward d e f le c tio n j
118
A M P H E N O N E
90pM
K -O E O X Y C O R T IS O L
I » •> •« «
SUCCINATE
r.Sm M
H Ril*|
8 -1 6 1
F ig . 2 4 .—E f fe c ts o f amphenone on r a t e s o f
re d u c tio n and o x id a tio n o f cytochrom e P -450.
M ito ch o n d ria e q u iv a le n t t o 0 .7 5 mg N/ml in is o to n ic
b u f f e r , 250 C.
iin d ic a tin g th e i n a b i l i t y o f m alate to form th e u su a l 420 m p,
i
a b so rp tio n band. Comparison w ith th e c o n tr o l stu d y shown in th e
low er tr a c e shows t h a t th e o x id a tio n produced by 1 1-d e o x y c o rtis o ls
i s a ls o , though n o t to o m arkedly, red u c ed . However, th e d u ra tio n !
{
o f th e o x id a tio n i s about th e same in th e two s tu d ie s . E v id e n tly :
amphenone a t t h i s c o n c e n tra tio n d o es n o t g r e a tly i n h i b i t th e r a t e
o f h y d ro x y latio n in s p ite o f i t s r a t h e r profound e f f e c t s on th e
s p e c tr a l p ro p e rtie s of th e m ito c h o n d ria . The e f f e c t s o f
amphenone shown in t h i s f ig u r e c o u ld o n ly s l i g h t l y be in c re a s e d
by d o u b lin g i t s c o n c e n tra tio n . At much h ig h e r c o n c e n tra tio n s o f :
amphenone, in h ib itio n o f h y d ro x y la tio n o f 11-d e o x y c o rtis o l
o c c u rs. A stu d y s im ila r t o t h a t shown f o r m ethopyrapone i n d i
c a te d t h a t i t s in h ib itio n was a ls o of a c o m p e titiv e n a tu re w ith
a of 0 .5 to 0 .6 m M .
A stu d y o f th e e f f e c t s o f o ,p ’-DDD on th e s p e c tr a l p ro p
e r t i e s and a c t iv i t y o f th e 1 1 0-h y d ro x y la se system gave q u ite
d if f e r e n t r e s u l ts th an th o se observed f o r e i t h e r m ethopyrapone
o r amphenone. I t appeared t o have no s i g n i f i c a n t e f f e c t on th e
s p e c tr a l p ro p e rtie s o f a e ro b ic m ito c h o n d ria a lth o u g h i t d id seem
t o sym m etrically d ecrease th e m agnitude o f th e reduced minus
o x id iz ed d iffe re n c e spectrum of t h e h y d ro x y lase system (F ig u re 4 ) .
As i s shown in F ig u re 25, o , p ’-DDD d e c re a se s th e r a t e and e x te n t
120
ZSmM tueeinat.
I ^ i m M Of
S -IM
F ig . 2 5 .--T he e f f e c t o f o,p*-DDD on th e
r a t e s o f re d u c tio n and o x id a tio n o f cytochrom e
P -4 5 0 . C u v e tte c o n ta in s m ito c h o n d ria , 0 .8 mg N/m l;
an tim y cin A, 6 M.g/ml; and i s o to n ic b u f f e r , 25° C.
;of re d u c tio n o f P -450. T his i s q u ite a p p a re n t in th e t r a c e s upon j
i !
th e a d d itio n o f m a la te , and may acco u n t f o r th e re ta rd e d re tu rn j
; i
o f th e tr a c in g a f t e r a p e rio d o f h y d ro x y la tio n . I t , how ever,
does n o t seem t o g r e a tly i n h i b i t th e r a te of h y d ro x y la tio n . !
F u rth e r s tu d ie s showed t h a t i t i s n o t a p a r t i c u l a r l y p o te n t
i n h i b i t o r . I t i s q u ite in s o lu b le , even in e th a n o l, and th e
la r g e s t c o n c e n tra tio n which would g iv e c o n s is te n t r e s u l t s was
100 pM. A k i n e t i c stu d y s im ila r to t h a t c a r r ie d out f o r metho
pyrapone and amphenone was made. The V v s . V/S p lo ts showed
d e f i n i te n o n -co m p e titiv e i n h i b i t i o n . The Ki was e stim a te d t o be
about 1 .0 m M .
The n o n -c o m p e titiv e n a tu re o f o ,p ’-DDD in h ib itio n o f
h y d ro x y la tio n to g e th e r w ith i t s i n h ib itio n of th e re d u c tio n of
cytochrom e P-450 su g g e ste d a s i t e of a c tio n in th e hydroxylase
system p r i o r to th e te rm in a l enzym es. S im ila r k i n e t i c r e s u l ts
would be o b ta in e d i f i t were b lo c k in g th e flow o f e le c tr o n s to
th e h y d ro x y la tin g enzymes o r i f i t were b le e d in g e le c tr o n s out o f
th e hyd ro x y lase sy stem . I t was th o u g h t t h a t th e e f f e c t o f
o ,p ’-DDD on th e le v e l o f reduced p y rid in e n u c le o tid e m ight d is
tin g u is h between th e s e two p o s s i b i l i t i e s . As i l l u s t r a t e d in
F ig u re 26, o , p ’-DDD cau ses a c o n c e n tra tio n -d e p e n d e n t o x id a tio n o f
p y rid in e n u c le o tid e . I t i s s i g n i f ic a n t t h a t in th e p resen c e of
M A L A T E
7.5mM
11-D EO X Y C O R TISO L
68.5 jiM
t
S-175
— ETOH
30*iM o,p-DDD
9ljiM o,p-DDD
|-l min-|
F ig . 2 6 .—The e f f e c t of o ,p ’-DDD
on p y rid in e n u c le o tid e flu o re s c e n c e .
A d d itio n s to m ito ch o n d ria (0 .5 5 mg N/ml)
in is o to n ic b u f f e r , 25° C, a re in d ic a te d
in th e f ig u r e .
123
o , p ’ -DDD th e a d d itio n o f 1 1 -d e o x y c o rtis o l r e s u l t s in l e s s o x id a
t i o n o f p y rid in e n u c le o tid e . As a r e s u l t , th e t o t a l d e c re a se in
flu o re s c e n c e a f t e r a d d itio n o f e i t h e r s te r o id alo n e o r s te r o id
p lu s o , p ’ -DDD i s c o n s ta n t. A lthough more s tu d ie s a re d e f i n i t e l y
re q u ire d b e fo re a s i t e o f a c tio n o f o ,p ’-DDD c o u ld be a s s ig n e d ,
th e s e r e s u l t s in d ic a te t h a t i t a c ts a t a s i t e betw een p y rid in e
n u c le o tid e and th e te rm in a l enzymes o f th e h y d ro x y lase system and
p ro v id e s an a l t e r n a t e pathw ay o f e le c tr o n flo w th a n t o th e te r m i
n a l h y d ro x y la tin g enzym es.
CHAPTER V II I
1 i
DISCUSSION
I
The s tu d ie s w hich have been p re s e n te d c le a r ly d e m o n s tra te ;
i
t h a t a d re n o c o rtic a l m ito c h o n d ria e x h ib it many p r o p e r tie s a s s o c i
a te d w ith m ito ch o n d ria i s o l a t e d from o th e r t i s s u e s • W hile in o u r ,
i
hands, th ey do n o t seem a s t i g h t l y coupled a s some p re p a ra tio n s j
. i
jof h e a rt o r l i v e r m ito c h o n d ria , th e y do show evidence f o r r e s p i r - ;
a to r y c o n tro l as w e ll a s th e c a p a c ity t o perform e n e rg y -lin k e d
fu n c tio n s such a s ion acc u m u latio n , r e v e r s a l o f e le c tro n tr a n s p o r t
and p y rid in e n u c le o tid e tra n s h y d ro g e n a tio n .
The most s ig n if ic a n t way in which a d re n o c o r tic a l m ito
ch o n d ria d i f f e r from o th e r m ito c h o n d ria i s th e p resen ce in them
o f s te r o id hydroxylase a c t i v i t i e s . These in c lu d e a d d itio n a l
e le c tr o n tr a n s p o r t pathw ays th ro u g h which e le c tr o n s a re provided
f o r th e h y d ro x y latio n r e a c t i o n . U sing th e e x tin c tio n c o e f f ic ie n t
d e riv e d f o r P-450 by Omura and S a to ( 7 ) , i t ap p ears t h a t bovine
a d re n o c o rtic a l m ito ch o n d ria c o n ta in about seven tim es a s much
P-450 as cytochrom e a j . The r a t e s a t w hich e le c tr o n s a re t r a n s
p o rte d through th e r e s p ir a to r y ch ain and th e 1 1 0-h y d ro x y lase
124
sy stem , a t l e a s t i r th e p resen c e o f m a la te , a re co m p arab le.
| In p re v io u s s tu d ie s in t h i s la b o r a to r y , i t was found t h a t
jw ith i n t a c t m ito c h o n d ria m alate su p p o rte d th e h ig h e s t r a t e of
jh y d ro x y la tio n o f th e e le c tr o n do n o rs t e s t e d ( 5 7 ) . T h is c o u ld n o t
I
jbe in c re a s e d by u sin g NADPH (5 m M ) and c a lc iu m . L a te r s tu d ie s on
i
! su b -m ito c h o n d ria l p a r t i c l e s showed t h a t 5 m M NADPH was to o h ig h a
|
jc o n c e n tra tio n t o give >maximal a c t i v i t y (1 3 2 ). T hese s tu d ie s were
jnot re p e a te d in i n t a c t m ito c h o n d ria . However th e r e l a t i v e l y
sm a ll p e rc e n ta g e o x id a tio n o f p y rid in e n u c le o tid e r e s u l t i n g from
i n i t i a t i o n o f h y d ro x y la tio n in th e p re se n c e o f m a la te , su p p o rts
ithe su g g e stio n t h a t m a la te can g e n e ra te in tr a - m ito c h o n d r ia l NADPH
I
lalm ost r a p id ly enough t o s a tu r a te th e h y d ro x y la se sy stem .
! The r e s p i r a t o r y ch ain and i t s c o n n e c tio n to th e hydrox-
j
iy la se system by th e p y rid in e n u c le o tid e tra n s h y d ro g e n a s e a s w e ll
i
a s th e co n cep t o f th e r e a c tio n o f cytochrom e P-450 w ith s te r o id s
land th e s i t e s o f a c tio n o f h y d ro x y la se i n h i b i t o r s w hich has
|
jevolved from th e d e s c rib e d s t u d i e s , a re shown in F ig u re 27. I t
jappears t h a t in m ito c h o n d ria , P-450 i s e i t h e r o n ly slo w ly a u t-
I
o x id iz a b le ( in d ic a te d by dashed a rro w ), o r t h e r e i s s u f f i c i e n t
jendogenous e le c tr o n d o n o r p re s e n t t o m a in ta in i t in a p a r t i a l l y
t ;
red u ced ste a d y s t a t e u n d er a e ro b ic c o n d itio n s . A d d itio n o f
s t e r o id s u b s tr a te g r e a t ly in c r e a s e s th e r a t e o f o x id a tio n o f th e
I
s a-Ketoglutarate f
Fumarate Succinate
•V
_ r < — >
F P
t (~)
TM PC
A scorbate
IHjO
F ig . 2 7 .—Pathw ays o f e le c tr o n tr a n s p o r t
in a d re n o c o r tic a l m ito c h o n d ria and r e a c tio n s o f
cytochrom e P-450 w ith s t e r o i d .
cytochrom e. I t s o x id a tio n i s s u f f i c i e n t l y ra p id t h a t even when
an e f f i c i e n t e le c tro n donor such a s m alate i s p r e s e n t, th e c y to
chrome i s m ain tain ed in a h ig h d eg ree o f o x id a tio n by a s a t u r a t
in g c o n c e n tra tio n o f s te r o id s u b s tr a te . Thus a s te p le a d in g t o a
re d u c tio n of th e cytochrom e ap p ears t o be lim itin g th e o v e r a ll
r a t e o f h y d ro x y la tio n . From a k i n e t i c stu d y o f th e r a t e s o f
fo rm atio n o f th e g -1 .9 4 EPR s ig n a l o f non-heme iro n p r o te in and
th e d isa p p ea ran c e o f th e g -2 .2 4 s ig n a l o f P-450 upon a d d itio n o f
NADPH t o a d re n o c o rtic a l hom ogenate, Miyake e t a l . (64) have sug
g e ste d t h a t th e t r a n s f e r of e le c tro n s from non-heme iro n to
P-450 may be r a t e lim itin g . I t would be i n t e r e s t i n g t o re p e a t
t h i s stu d y u sin g m alate a s e le c tro n donor in o rd e r t o avoid th e
p e rm e a b ility problem s a s s o c ia te d w ith NADPH.
The i n h ib i t o r s o f lip -h y d ro x y la tio n which have been
s tu d ie d can be c o n sid ere d to a f f e c t th e r a t e o f re d u c tio n o r
o x id a tio n o f cytochrom e P -450. M ethopyrapone and amphenone
produce d if f e r e n c e s p e c tr a in a e ro b ic m ito c h o n d ria which show
c o n sid e ra b le lik e n e s s t o th e reduced minus o x id iz e d d if f e r e n c e
sp ectru m . The c o m p e titiv e n a tu re o f t h e i r in h ib itio n su g g e sts
t h a t th e y a c t a t o r n e a r th e s i t e o f s te r o id b in d in g . I t i s p o s
s i b l e t h a t th e y a ls o i n h i b i t th e b in d in g o f oxygen as w e ll as
s t e r o id s u b s tr a te ; however, i n i t i a l s tu d ie s in d ic a te d t h a t
128 1
m ethopyrapone d id n o t i n h i b i t fo rm atio n o f th e CO-complex. i
!
o ,p ’-DDD, on th e o th e r hand, a p p ea rs to i n h i b i t th e e f f e c tiv e j
r a t e o f re d u c tio n o f th e cytochrom e. I t s o x id a tio n o f p y rid in e
n u c le o tid e would su g g e st i t s s i t e o f a c tio n i s lim ite d t o some
where betw een N A D PH and P-450 in th e h y d ro x y lase system . I
I t would be p r e d ic te d t h a t any p ro c e ss which would low er
th e ste a d y s t a t e l e v e l o f N ADPH would slow th e r a t e o f h y d ro x y la-
j
t io n p ro v id ed th e e f f e c tiv e c o n c e n tra tio n o f N A D PH i s lim itin g i
th e n e t r a te of h y d ro x y la tio n . Such i s a p p a re n tly th e c a s e ,
even in m ala te -su p p o rte d h y d ro x y la tio n , a s th e r e l a t i v e l y sm all
o x id a tio n o f p y rid in e n u c le o tid e induced by o,p*-DDD does cause a
sm all b u t s i g n i f ic a n t in h ib i t io n o f 1 1 0 -h y d ro x y la tio n • Since
N A D PH i s in e q u ilib riu m w ith NADH, lo w erin g th e ste a d y s t a t e
l e v e l o f N A D H by in c re a s in g th e r a t e o f i t s o x id a tio n by th e
r e s p ir a to r y ch ain sh o u ld d e c re a se th e r a t e o f h y d ro x y la tio n .
Such was observed when d icu m aro l was in c lu d e d in th e o -k e to -
g lu ta r a te -c o n ta in in g re a c tio n m ix tu re . T h is may a ls o be th e
e x p la n a tio n f o r in h ib i t io n by low c o n c e n tra tio n s o f calciu m .
The d e c re a se in h y d ro x y lase a c t i v i t y when an e le c tr o n donor
o th e r th a n m alate i s u t i l i z e d , in d ic a te s t h a t th e r a t e o f red u c
t io n o f p y rid in e n u c le o tid e i s a ls o a s i g n i f ic a n t f a c to r d e te r
m ining th e o v e r a ll r a te o f h y d ro x y la tio n . In T able V th e T y 2 t 0
129
TABLE V •
COMPARISON OF RATES OF PYRIDINE NUCLEOTIDE REDUCTION
A N D CORTISOL PRODUCTION
E le c tro n Donor
P y rid in e N u cleo tid e
R eduction
T1 /2 k r
(m in) (m in~l)
i
C o r tis o l
P ro d u ctio n
(iqim oles/m in/
mg N)
Exp. 173
S u ccin ate 0 .2 1 .8 32
S u c c in a te + Rotenone 0 .6 1 .1 30
! S u c c in a te + Amytal
----- -----
7 .8
Exps. 185 & 183
S u c c in a te in I s o to n ic 0 .4 1 .7 39
S u c c in a te in T r is -
M annitol 1 .0 0.65 16
j Exp. 194
i M alate 0 .1 5 3.8 61
a -K e to g lu ta ra te +
M alonate 0 .3 1.2 47
| Exp• 171
M alate 0 .1 5 3.8 37
| S u ccin ate 0 .3 1 .6 26
Ascorbate-TMPD 0 .2 1 .9 24
T1 /2 i s th e tim e re q u ire d f o r p y rid in e n u c le o tid e f lu o r e s
cence t o rea ch 1/2 th e v a lu e o b ta in e d in S ta te 4 r e s p i r a t io n ,
la te c o n s ta n ts , kr , and r a t e of c o r t i s o l p ro d u ctio n were d e te r
mined as d e sc rib e d in C h ap ter I I .
■ R eactio n C o n d itio n s: E xp. 173, as in F ig u re 13;
Exp. 185, as in F ig u re 14;
Exp. 183, as in F ig u re 17;
Exp. 194, as in F ig u re 11;
Exp. 171, as in T able I I I .
130 |
i
I
s te a d y s t a t e and a p p a re n t r a t e c o n s ta n ts o f p y rid in e n u c le o tid e i
l
r e d u c tio n , e s tim a te d from flu o re s c e n c e m easurem ents, can be com- |
jpared w ith th e r a t e s o f h y d ro x y la tio n found in s e v e r a l o f th e
i
e x p e rim e n ts d e s c rib e d in C h ap ter V. These r a t e c o n s ta n ts (and
i
uTi/2 *s) a re complex fu n c tio n s in c lu d in g th e r a t e and e x te n t o f
re d u c tio n o f b o th N A D and NADP as r e f l e c t e d in n e t flu o re s c e n c e
'in c r e a s e , and th e y sh o u ld be view ed o n ly as in d ic a tio n s o f r e l a
t i v e e f f ic ie n c y o f p y rid in e n u c le o tid e r e d u c tio n . T here does i
I
Iseem t o be a q u a l i t a t i v e c o r r e la tio n betw een th e s e nc o n s ta n ts M
and th e r e l a t i v e r a t e s o f h y d ro x y la tio n su p p o rte d by th e d if f e r e n t!
i ■ !
e le c tr o n donors u n d er th e in d ic a te d e x p e rim e n ta l c o n d itio n s . j
|
T hese s tu d ie s have been p a r t o f a c o n tin u in g e f f o r t in
t h i s la b o r a to r y t o u n d e rsta n d a d re n o c o r tic a l m etabolism in hopes
6 f ‘e s t a b l is h i n g th e mechanisms which c o n tr o l c o r t i c o s te r o i d b io
s y n th e s is . Of p a r t i c u l a r i n t e r e s t i s , o f c o u rs e , th e p rim ary
b f f e c t o f ACTH. The o b s e rv a tio n o f Chance e t a l . (144) o f a :
! I
p a p id d e c re a s e in p y rid in e n u c le o tid e flu o re s c e n c e in an i n t a c t
jadrenal g la n d in resp o n se t o ACTH would su g g e st t h a t i n i t i a l l y |
i
jthe h y d ro x y la tio n p ro c e ss i s a c tiv a te d . T h is co u ld be b ro u g h t j
i ' '
a b o u t by m aking a so u rc e o f s te r o id s u b s tr a te a v a ila b le t o th e j
I i
I ,
h y d ro x y la se system s o r by a c t iv a t in g th e system so t h a t i t would
; ]
U t i li z e a v a ila b le s u b s tr a te s f a s t e r . The r a t e a t w hich I
1 in tra m ito c h o n d ria l N A D PH must be g e n erate d to su p p o rt th e r a te of
i
(c o rtic o s te ro id b io s y n th e s is which occurs in vivo under th e stim u
l u s o f A CTH has been e stim a te d to be s im ila r to t h a t o b tain ed
(under maximal c o n d itio n s in th e in v i t r o s tu d ie s d e sc rib e d above
I
j(5 7 ). T h erefore a secondary, i f n o t p rim ary, response to A CTH
(would be expected t o be an in c re a s e in p o te n tia l f o r N A D PH gen-
j
je ra tio n . The p o ss ib le ro le o f a d re n a l a sc o rb ic a c id in t h i s
(resp ect has been d isc u sse d .
I
I The h y d ro x y latio n p ro cess u t i l i z e s e le c tro n s which would
o th erw ise be o x id iz ed by th e r e s p ira to r y chain t o g e n erate ATP.
(The im portance o f o x id a tiv e p h o sp h o ry latio n to th e g e n e ra l m etab
o lism of th e c e l l would make i t d e s ira b le to have a means o f con-
i
( tr o llin g th e r a te a t which e le c tro n s could be siphoned in to th e
I
(hydroxylase system . The p y rid in e n u c le o tid e tran sh y d ro g en ase in
Which th e r e l a ti v e r a te s o f tran sh y d ro g e n atio n between N A D and
I nADP a re re g u la te d by th e energy s t a t e of th e m itochondrion p ro -
j
jvides an id e a l s i t e of c o n tr o l. I t has been dem onstrated t h a t by
uncoupling th e tran sh y d ro g en ase system , h y d ro x y latio n can be
in h ib ite d , even though th e c o n c e n tra tio n of N A D H i s h ig h . In an
i
(uninhibited m ito ch o n d ria, th e N A D H le v e l would a ls o r e f l e c t th e
j
energy s t a t e , b ein g h ig h when th e re i s excess energy and low when
jre s p ira tio n i s l e s s c o n tro lle d (S ta te 3 ).
j In the above studies, ADP plus phosphate did not seem to j
i n h i b i t th e r a t e o f s u c c in a te -s u p p o rte d h y d ro x y la tio n . T h is
! i
; •
'would te n d t o arg u e a g a in s t th e e n erg y s t a t e o f th e m ito c h o n d ria j
j
b e in g an im p o rta n t r e g u la to r y f a c t o r . I t was e s p e c i a l l y s u r p r i s
in g in view o f th e f in d in g s o f Cammer and E sta b ro o k (145) t h a t
j i
h y d ro x y la tin g c o n d itio n s i n h i b i t ATP fo rm a tio n d u rin g s u c c in a te |
I
o x id a tio n in a d r e n o c o r tic a l m ito c h o n d ria . The a p p a re n t d is c r e p - j
j
ancy may be e x p la in e d by th e f in d in g s o f Lee and E r n s te r (117) j
i
t h a t a c o m p e titio n betw een tra n s h y d ro g e n a tio n and o x id a tiv e i
i
p h o s p h o ry la tio n c o u ld o n ly be seen when t h e r e was a lim ite d supply!
i !
o f h ig h e n erg y in te r m e d ia te s and s a t u r a t i n g c o n c e n tra tio n s of j
I ' i
s u b s tr a t e s f o r th e tw o e n e r g y - r e q u ir in g p r o c e s s e s . I t w ould be
1 I
1 i
e x p e c te d t h a t what e v id e n c e o f c o m p e titio n f o r e le c tr o n s o r 1
e n e rg y w hich can be d e m o n stra te d when b o th a r e b e in g s u p p lie d a t aj
m axim al r a t e would be g r e a t ly a m p lifie d when th e su p p ly o f e le c
tr o n d o n o rs became l i m i t i n g .
| _ j
! T hese s t u d i e s , w hich were i n i t i a t e d w ith th e d e te rm in a tio n
i !
I
jof th e r a t e - l i m i t i n g s te p in lip - h y d r o x y la tio n as an aim , have
i i
; l
jshown t h a t v a rio u s c o n d itio n s d i c t a t e w hich w i l l be th e r a t e - i
l i m i t i n g s t e p . C e r ta in ly a v a ila b le s t e r o i d s u b s tr a t e i s a
I ;
p r i t i c a l f a c t o r . S e c o n d ly , th e s te a d y s t a t e l e v e l o f in tr a m ito -
c h o n d ria l NADPH becomes s i g n i f i c a n t when th e r e i s s a t u r a t i n g
133
c o n c e n tra tio n s o f s t e r o id s u b s tr a t e . T h is i s d eterm in ed by th e
j r e la tiv e r a t e s o f re d u c tio n and o x id a tio n o f b o th p v rid in e
jn u c le o tid e s and can be c o n tr o lle d by th e energy s t a t e o f th e
m ito c h o n d ria . T h ird ly , when b o th N A D PH and s t e r o id s u b s tr a te a re
!
p r e s e n t in e x c e s s , th e r a t e o f e le c tr o n t r a n s p o r t t o cytochrom e
P-450 w ith in th e h y d ro x y lase system a p p ea rs to become th e slow
j
is te p . I t sh o u ld be c le a r t h a t an a l t e r a t i o n o f each o f th e s e
!
j f a c to r s c o u ld , u n der a given c o n d itio n , se rv e t o r e g u la te c o r t i -
i
c o s te r o id b io s y n th e s is . W hile a mechanism o f a c tio n o f A CTH
seems no n e a r e r , any which may be su g g e ste d in th e f u t u r e , must
I
i
'in c o rp o ra te th e re c o g n itio n o f c o r tic o s te r o id b io s y n th e s is as an
i n t e g r a l fu n c tio n o f a d r e n o c o r tic a l m ito c h o n d ria .
CHAPTER V IH
I i
; I
! S U M M A R Y i
i |
' i
F a c to rs c o n tr o llin g th e r a t e o f s te r o id lip -h y d ro x y la tio n j
I i
have been s tu d ie d in i s o la te d i n t a c t a d r e n o c o rtic a l m ito c h o n d ria .
|
jThese m ito ch o n d ria e x h ib it r e s p ir a to r y c o n tr o l and perform such i
i !
jen erg y -lin k ed fu n c tio n s as calcium accu m u latio n , r e v e r s a l o f j
; j
[ele ctro n tr a n s p o r t in th e r e s p ir a to r y c h a in , and asym m etric i
I i
tra n s h y d ro g e n a tio n . L ike r e s p i r a t io n , s te r o id h y d ro x y latio n
i u tiliz e s e le c tro n donors and oxygen, and th e a c t i v i t i e s of th e s e j
i i
tw o o x id ase system s a re of com parable m agnitude. NADPH, which i s [
i ' |
!the prim ary e le c tro n donor u t i l i z e d by th e 110-hydroxylase s y s -
l
tern, m ust be g e n erate d in tra m ito c h o n d ria lly a s th e i n t a c t m ito -
i
c h o n d ria a re imperm eable to e x te r n a l reduced p y rid in e n u c le o tid e .
! When h y d ro x y la tio n i s i n i t i a t e d by a d d itio n of s te r o id I
j !
jsu b stra te to m ito ch o n d ria supplem ented w ith an a p p ro p ria te e le c -
l
jtron do n o r, th e endogenous p y rid in e n u c le o tid e s s h i f t to a more j
I I
[oxidized ste a d y s t a t e . Enzym atic a ssa y s o f th e p y rid in e n u c le o - j
I !
jtid es in m ito c h o n d ria l e x tr a c ts showed t h a t p rim a rily N A D H r a th e r ;
th an N A D PH i s o x id iz e d . In th e p resen ce o f u n coupling a g en ts th e ;
!r e l a t i v e o x id a tio n o f NADPH in c r e a s e s . An e n e rg y -lin k e d p y r id in e j
n u c le o tid e tra n sh y d ro g e n a se w ith s u f f i c i e n t a c t i v i t y t o m a in ta in
NADP in a h ig h d eg ree o f re d u c tio n in s p i t e o f th e in c re a s e d r a t e
o f i t s o x id a tio n by th e h y d ro x y lase system a p p e a rs t o l i n k th e ;
! ' I
;h y d ro x y la se system to th e r e s p i r a t o r y c h a in .
|
j When s te r o id s u b s tr a te s u f f i c i e n t t o s a tu r a te th e hydrox-
ly la se system i s p r e s e n t, th e ste a d y s t a t e l e v e l o f NADPH a p p ea rs -
i
t o c o n tr o l th e r a t e o f h y d ro x y la tio n . S in ce N A D PH i s in e q u il i b -
|
jrium w ith NADH, lo w e rin g th e ste a d y s t a t e l e v e l o f N A D H by
in c r e a s in g th e r a t e o f i t s o x id a tio n by th e r e s p i r a t o r y c h a in , a s !
o c c u rs in th e p re se n c e o f u n c o u p lin g a g e n ts o r low c o n c e n tra tio n s |
o f c a lc iu m , d e c re a se s th e r a t e o f h y d ro x y la tio n . Comparison o f j
t h e r a t e s o f h y d ro x y la tio n su p p o rte d by v a rio u s e le c tr o n donors
w ith th e r a t e s o f t h e i r re d u c tio n o f p y rid in e n u c le o tid e s shows a
good q u a l i t a t i v e c o r r e l a t i o n . Any p ro c e ss w hich would a l t e r th e
r e l a t i v e r a t e s o f o x id a tio n and re d u c tio n o f th e s e c o fa c to rs couldj
I i
b e a mechanism f o r c o n tr o llin g c o rtic o s te r o id o g e n e s is a t th e m ito-j
i ' <
c h o n d ria l l e v e l . The en erg y s t a t e o f th e m ito ch o n d rio n would be a;
1 i
c r i t i c a l f a c t o r a s n o t o n ly does i t c o n tr o l th e r a t e o f N A D H o x i- j
i
d a tio n by th e r e s p ir a to r y c h a in , b u t i t a ls o d e te rm in e s th e r a t e
a t w hich re d u c in g e q u iv a le n ts can be s u p p lie d to th e h y d ro x y lase !
sy stem v ia th e p y rid in e n u c le o tid e tra n s h y d ro g e n a s e .
! 136
|
C e rta in s p e c t r a l and EPR p r o p e r tie s o f a d re n o c o rtic a l
jm ito c h o n d ria l P -450, th e o x y g e n -a c tiv a tin g enzyme f o r s te r o id
i
h y d ro x y la tio n s, have been exam ined. D iffe re n c e s p e c tr a th ro u g h
o u t th e v is ib le re g io n i l l u s t r a t e th e e f f e c t o f s t e r o id sub-
j s t r a t e s on th e m ito c h o n d ria l hy d ro x y lase sy stem s. The s p e c tra
| produced by s u b s tra te s o f th e lip -h y d ro x y la s e system have been
i
jin te r p r e te d to r e f l e c t an o x id a tio n o f cytochrom e P -4 5 0 . The
ifo llo w in g evidence su p p o rts t h i s i n te r p r e t a t i o n : (1 ) a spectrum
iproduced by s e le c tiv e ly reducing th e hyd ro x y lase system i s th e
in v e rs e o f th e ste ro id -in d u c e d spectrum ; (2) oxygen i s re q u ire d
i
|f o r s te r o id s u b s tr a te s t o induce t h e i r c h a r a c t e r i s t i c s p e c tra ;
i
land (3) a s e r ie s o f EPR s tu d ie s show an i n te n s i f i c a ti o n of s ig -
i
|n a ls a s s o c ia te d w ith P-450 in th e p resen ce of 1 1 -d e o x y c o rtiso l
•and oxygen. K in e tic s tu d ie s of changes in redox s t a t e of P-450
iduring h y d ro x y la tio n in d ic a te t h a t th e r a t e o f re d u c tio n o f P-450
!
jis th e slow ste p in th e hyd ro x y lase p ro c e s s .
| T hree c la s s ic in h ib ito r s o f 1 1 0 -h y d ro x y latio n have been
|
:s tu d ie d . Amphenone B [ 3 , 3 -b is(p -am in o p h en y l)-2 -b u tan o n eJ and
|SU-4885 [2 -m e th y l-l,2 -b is (3 -p y rid y l)-l-p ro p a n o h e ] produce ab so rp
t io n bands in th e d iff e re n c e s p e c tra in a e ro b ic m ito ch o n d ria and
I '
iact as c o m p e titiv e in h ib ito r s of lip -h y d ro x y la tio n , e x h ib itin g
K i*s o f 0 ,5 m M and 0,12 pM, r e s p e c tiv e ly . o,p*-DDD [ l - ( o - c h l o r o -
p h e n y l)- l-( p -c h lo ro p h e n y l) - 2 , 2 -d ic h lo ro e th a n e ], n o n -c o m p e titiv e ly
j in h ib its llp -h y d ro x y la tio n w ith a o f ap p ro x im ate ly 1 .0 mM .
i
T h is i n h i b i t o r ap p ears t o in d u ce a p a r t i a l o x id a tio n o f reduced
l
(p y rid in e n u c le o tid e , and in i t s p resen c e th e s h i f t in p y rid in e
I
in u c le o tid e t o a more o x id iz e d ste a d y s t a t e which n o rm ally o c cu rs
i
upon i n i t i a t i o n o f h y d ro x y la tio n i s d e c re a s e d . The e sta b lis h m e n t
of an a l t e r n a t e pathw ay o f e le c tr o n flow a t th e s i t e o f one o f
th e e le c tr o n c a r r i e r s w hich m ed iate e le c tr o n tr a n s p o r t betw een
^ADPH and P-450 in th e h y d ro x y lase system i s p o s tu la te d as i t s
jnechanism o f i n h ib i t io n .
L I S T OF R E F E R E N C E S
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This dissertation has been
microfilmed exactly as received
OLDHAM, Susan Banks, 1942-
INTERACTION BETWEEN 11 (3 -HYDROXYLATION
AND RESPIRATION IN BOVINE ADRENOCORTICAL
MITOCHONDRIA.
University of Southern California, PluD., 1968
Chemistry, physical
University Microfilms, Inc., Ann Arbor, Michigan

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Asset Metadata

Creator Oldham, Susan Banks, 1942- (author)

Core Title Interaction between 11β-hydroxylation and respiration In bovine adrenocortical mitochondria

School Graduate School

Degree Doctor of Philosophy

Degree Program Biochemistry

Degree Conferral Date 1968-01

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Harding, Boyd W. (committee chair), Brodie, Arnold F. (committee member), Mehl, John W. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c18-605231

Unique identifier UC11360154

Identifier 6807194.pdf (filename),usctheses-c18-605231 (legacy record id)

Legacy Identifier 6807194.pdf

Dmrecord 605231

Document Type Dissertation

Rights Oldham, Susan Banks

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

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