Studies on cholesterol esterases

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Content STUDIES O N CHOLESTEROL ESTERASES
A D is s e r ta tio n
P re se n te d to
tlie F a c u lty o f th e G raduate School
U n iv e rsity of S outhern C a lifo rn ia
In P a r t i a l F u lfillm e n t
o f th e R equirem ents f o r th e Degree
D octor o f P hilosophy
by
M arie L ouise N ie ft
June 1948
T/.D, b>o, Vg- fJ&iC
This dissertation, written by
....................MBXK..MU.ISELNXEF.I..................
under the guidance of hB.v. Faculty Committee
on Studies, and approved by all its members, has 3 - g ^
been presented to and accepted by the Council
on Graduate Study and Research, in partial ful- /
fillment of requirements for the degree of
DOCTOR OF P H ILO SO P H Y
v Dean
H.W.PATKORE
Secretary
JUKE 1948
Date.
Committee on Studies
V>OjetX>
The a u th o r has g r e a tly a p p re c ia te d th e h e lp
and guidance o f Dr, H, J . D euel, J r . , in th e p la n n in g
and e x ecu tio n o f t h i s p r o je c t.
Thanks a re a lso due th e L ife In su ran ce M edical
R esearch imnd, which has su p p o rted t h i s work thro u g h
a S tudent F ello w sh ip .
TABLE OF CONTENTS
CHAPTER PAGE
I . INTRODUCTION............................................................................. 1
II* REVIEW OF THE LITERATURE.............................................., 3
I I I . MATERIALS A N D METHODS......................................................... V
P re p a ra tio n o f C h o le s te ro l P a lm ita te . . • . . ?
P re p a ra tio n o f C h o le ste ro l C o llo id ........................ V
P re p a ra tio n of C h o le ste ro l P a lm ita te C o llo id , . 8
T issu e E x t r a c t s ............................... 8
Assay u sin g Dioxane S o lu tio n s o f s u b s tr a te • • 8
A ssay u sin g C o llo id a l S uspensions o f S u b s tra te , 9
E x tra c tio n P rocedure ............................................ 10
D eterm in atio n o f C h o le s te ro l ..................................... 11
IV. EXPERIMENTAL RESU LTS........................................................... 14
C h o le s te ro l-E s te rify in g System s . . 14
D em onstration in L iv er E x t r a c t s ........................... 14
R equirem ents fo r A c t i v i t y ......................................... 16
E s t e r if i c a t i o n w ith I n t e s t i n a l E x tra c ts , . • 18
H y d ro ly sis o f C h o le ste ro l E s t e r s ............................ 23
D em onstration in L iv er E x t r a c t s ........................... 23
D em onstration in I n t e s t i n a l E x tra c ts , . . . 26
S e p a ra tio n o f Two F a c to rs involved in
H y d r o ly s is ................................................................... 30
CHAPTER PAGE
N ature o f th e H y d ro ly tic System in th e
B abbit • 38
N ature of th e Enzyae E naction . . . . . . . . 39
N ature o f th e G ofactor . . . . . . . . . . . . 43
Bole o f L e c ith in 43
V. DISCUSSION.................................................................................... 49
VI, S U M M A R Y A N D CONCLUSIONS .......................................05
BIBLIOGRAPHY........................................ 56
LIST OF TABLES
TABLE PAGE
I , E s t e r if i c a t i o n of C h o le s te ro l by L iv er
E x t r a c t .................................................................... . • . 15
I I . The E ffe c t o f th e B u ffer Ion on E s t e r if i c a t i o n
o f C h o le s te ro l by Crude L iv er E x tra c ts . . . . 17
I I I . The E ffe c t o f Adenosine T rip h o sp h a te and
G lu eo se-l-p h o sp h ate on E s t e r if ic a tio n o f
C h o le ste ro l by Crude L iv e r E x tra c ts • • • • • . 19
IV. Sources o f th e F a tty A cid R esidue of
C h o le ste ro l E s te rs ........................................................... 20
V. E s te r if y in g and H y d ro ly tic R eactio n s of Crude
E x tra c ts o f Rat Sm all I n te s tin e .............................22
VI. H y d ro ly sis o f C h o le s te ro l P a lm ita te by Crude
L iv e r E x tra c ts ............................................ 25
V II. Com parative A c tiv ity o f P u r if ie d L iv er E s te ra s e
A gainst E th y l A cetate and C h o le ste ro l
P a l m i t a t e .......................................................................................27
V III. E ffe c t o f Heat on H y d ro ly sis by I n te s t i n a l
E x tra c ts • • • • • • • • • • • . • • • • • • . 29
IX. F ra c tio n a tio n o f I n t e s t i n a l E ste ra sd by
D ia ly sis .............................................................. 51
X. Heat S t a b i l i t y o f H y d ro ly tic Enzyme and
C o facto r • • • • . ...................................................... • 32
LIST OF TABLES
( cont* d)
TABLE PAGE
XI. F ra c tio n a tio n of I n t e s t i n a l E x tra c ts w ith
Ammonium S u lfa te ............................................ 33
X II. F ra c tio n a tio n o f Sm all I n te s tin e o f th e
R a b b i t ........................................................................ .... . 40
X III. T rip ro p io n a se A c tiv ity o f th e H y d ro ly tic
S y s te m ........................................................................... 42
XIV. Chemical Data on C o facto r P re p a ra tio n 813 • . 44
XV. E f f e c ts of Acid and A lk alin e H y d ro ly sis on
u o f a e t o r ................................... 45
XVI. E ffe c t o f S u b s titu tio n s f o r L e c ith in in th e
H y d ro ly tic System ................................ ..... 47
LIST OF FIGURES
FIGURE PAGE
1* L im itin g C o facto r C o n cen tratio n
fo r A c tiv ity . . . . . . . . . . . . . 36
2. Optim al pH f o r H y d ro ly tic A c tiv ity . . . 37
CHAPTER I
BJTR03XJCTI0N
Tiie occurrence o f c h o le s te ro l e s te r s in th e body has
long been known. Many organs c o n ta in a p p re c ia b le amounts o f
f re e c h o le s te r o l, but th e e s t e r i f i e d form i s found in la rg e
q u a n titie s only in th e l i v e r , i n t e s t i n a l w a ll, and blood
serum of th e norm al anim al. Yet th e r a t i o betw een f re e and
e s t e r i f i e d c h o le s te ro l in norm al serum rem ains rem arkably
c o n s ta n t, and i t i s e v id e n t th a t a s e n s itiv e re g u la to ry
mechanism e x is ts to m ain tain t h i s r a t i o .
C e rta in ty p e s o f f a t t y l i v e r , n o ta b ly th o se produced
by th e fe e d in g of c h o le s te ro l o r by a d e fic ie n c y of u n s a t
u ra te d f a t t y a c id s , m eth io n in e, o r c h o lin e , a re c h a ra c te riz e d
by an in c re a se in l i v e r c h o le s te r o l, p redom inantly in th e
e s te r form . S ince th e in g e s tio n of c h o le s te ro l e s te r s in
th e d ie t i s u s u a lly a t a v ery low le v e l, i t i s ap p aren t th a t
t h i s e s t e r i f i c a t i o n can tak e p la c e in th e body. In a d d itio n ,
s in c e we n o te a d e crease in serum e s te r s d u rin g sev ere l i v e r
damage, i t i s no t im probable th a t t h i s e s t e r i f i c a t i o n of
c h o le s te ro l can tak e p lace in th e l i v e r i t s e l f .
In th e c o n d itio n known as a th e r o s c le r o s is , th e re i s
d e te r io r a tio n o f th e la r g e r blood v e s s e ls , p a r tic u la r ly th e
a o r ta , w ith d e p o s itio n o f l i p i d m a te ria l in th e w a ll of th e
v e s s e l. T his d e p o sit has been shown to have a high c h o le s
t e r o l c o n te n t, and much o f i t i s in th e e s te r form . I t i s
2
now thought th a t t h i s m a te r ia l is d ep o site d a t th e s i t e of
th e le s io n , no t formed th e re by th e c e l l s , b u t th e re aso n s
f o r c h o le s te ro l e s te r s b ein g among th e d e p o site d su b sta n ce s
i s unknown* I t may be m erely a r e f l e c t i o n o f th e e s t e r i f i e d
c h o le s te ro l p re se n t in th e serum.
In view o f th e se r e la tio n s h ip s , i t became o f i n t e r e s t
to in q u ire in to th e b io ch em istry involved in th e fo rm atio n
and d e s tru c tio n o f c h o le s te ro l e s te r s in th e body, and t h i s
work i s th e r e s u l t o f such a stu d y .
CHAPTER I I
REVIEW OF THE LITERATURE
In 1910, long b e fo re th e s tr u c tu r e o f c h o le s te ro l
i t s e l f was e s ta b lis h e d , Hondo (9) s tu d ie d th e e f f e c t o f
l i v e r e x tr a c ts on c h o le s te ro l e s te rs * As judged by modern
s ta n d a rd s , h is work was n o t c le a r cut* The in c u b a tio n
m ix tu re was not b u ffe re d , chloroform was used as a p re
s e r v a tiv e , he was w orking w ith th e n a tiv e c h o le s te ro l o f
th e l i v e r , which i s m o stly in th e u n e s te r if ie d form , as
a s u b s tr a te , and h is method f o r th e d e te rm in a tio n o f c h o le s
t e r o l u sin g a c e ty l numbers was u n c e rta in * However, h is
r e s u l t s in d ic a te d th a t c h o le s te ro l e s te r s could be s p l i t
by such ti s s u e e x tra c ts * H eating o f th e e x tr a c t d estro y ed
t h i s a c tiv ity *
T his work was re p e a te d in a more v a lid manner by
S c h u ltz in 1912 (1 6 ). U sing Windaus* d ig ito n id e method
f o r th e d e te rm in a tio n o f f r e e and t o t a l c h o le s te ro l, he
was ab le to show th a t l i v e r e x tr a c ts could hydrolyze
c h o le s te ro l e s te r s . The in c u b a tio n o f b lo o d gave no con
c lu s iv e r e s u l t s , a lth o u g h a b lo o d -liv e r m ix tu re brought
about alm ost com plete s p l i t t i n g o f th e e s te r s p re s e n t.
L a te r Shope (1 7 ), a tta c k in g th e problem by u sin g
th e c h o le s te ro l e s te r s o f bovine serum as a s u b s tr a te ,
t e s te d th e a c t i v i t y of s a lin e e x tr a c ts of a la rg e number
4
o f gu in ea p ig t i s s u e s . He found th a t l i v e r , k id n ey , m uscle,
and b ra in were r ic h e s t in e s t e r - s p l i t t i n g a c t i v i t y , b u t a l l
tis s u e s showed i t to some d eg ree.
Some o f th e most c a r e fu l work done on t h i s problem
came from K lein ( 7 ) , who p rep ared s a lin e e x tr a c ts o f anim al
t i s s u e s f o r a ssa y a g a in s t th e c h o le s te ro l e s te r s in serum or
a g a in s t c o llo id a l su sp en sio n s o f th e e s te r s . H is s e r ie s
was v ery c lo s e ly c o n tro lle d in m a tte rs o f pH and s u b s tr a te
a re
c o n c e n tra tio n . From h is work, i t appears th a t th e re ^ a t le a s t
two ty p e s o f e s te r a s e s whioh can a tta c k c h o le s te r o l e s te r s ,
one having a pH optimum a t 7, th e o th e r a t 5.3* Serum and
pancreas were found to have th e system a c tiv e a t pH 7,
w hile e x tr a c ts o f l i v e r , sp le e n , kidney, and i n t e s t i n a l mu
cosa were found to be a c tiv e a t th e low er pH a g a in s t th e
c h o le s te ro l e s te r s p re se n t in serum . When th e s e e x tr a c ts
were te s te d a g a in s t a c o llo id a l suspension o f c h o le s te ro l
e s t e r , only i n t e s t i n a l mucosa and sp lee n showed a c t i v i t y
a t pH 5 .3 .
The h is to r y o f th e o p p o site r e a c tio n , namely th e
e s t e r i f i c a t i o n o f c h o le s te r o l, i s s im ila r ly s p a rs e . L i t t l e
of Schoenheimer* s e a r ly and c l a s s i c a l work on c h o le s te ro l
m etabolism b e a rs on t h i s problem , bu t he d id observe th a t
i f an o i l s o lu tio n o f c h o le s te ro l was in je c te d in to th e
m uscle o f an an im al, e x c is io n of th e mass some mohths l a t e r
re v e a le d th a t th e c h o le s te ro l had been e s t e r i f i e d a t th e
s i t e o f in je c tio n (14)*
M ueller (1 2 ), in re p o rtin g work done on th e a b s o rp tio n
o f c h o le s te ro l from th e i n t e s t i n e u sin g a th o ra c ic duct f i s
t u l a , s ta te d th a t in th e th o ra c ic lymph a c o n sta n t r a t i o was
observed f o r f r e e to e s t e r i f i e d c h o le s te r o l, re g a rd le s s of
th e s ta t e in which i t was fe d to th e an im al. A s im ila r ex
perim ent c a r rie d out by IT S h lie h e r and sftllm ann (4) p ro v id ed
more of th e same ty p e o f evidence f o r th e e s t e r i f i c a t i o n o f
c h o le s te ro l durin g a b s o rp tio n , b u t th ey were not a b le to
s u b s ta n tia te M u eller’ s o b se rv a tio n s on a c o n sta n t f r e e to
combined r a t i o , A la rg e p a r t o f c h o le s te ro l fe d in th e f r e e
form appeared in th e chyle in th a t s t a t e , alth o u g h th e e s te r
c o n te n t o f th e chyle in d ic a te d e s t e r i f i c a t i o n had ta k e n p la c e
to some e x te n t.
L a te r, M ueller (13) c a r r ie d out an o th e r p ie c e o f work
in which he in cu b ated blood and l i v e r f o r s e v e ra l d a y s,u sin g
chloroform as a p r e s e r v a tiv e , and fo llo w ed th e d i s tr ib u tio n
o f th e n a tiv e f re e and e s te r c h o le s te ro l durin g th a t tim e .
He observed no s p l i t t i n g o f th e e s t e r , bu t ig n o re d th e d a ta in
f iv e out o f h is n in e experim ents which c le a r ly showed a r i s e
in c h o le s te ro l e s te r co n te n t o f l i v e r a f t e r in c u b a tio n ,
S p erry and h is group have done a good d eal o f work
on th e e s t e r i f i c a t i o n o f c h o le s te ro l in in cu b ated blood
serum , bu t work has n o t p ro g re ssed beyond th e d e s c rip tiv e
p h ase. S p erry and S to y an o ff (2 2 ), howOver, a re o f th e
o p in io n th a t th e re a re two enzym atic re a c tin n s in v o lv e d -
one, an e s t e r i f i c a t i o n , and th e second, a h y d ro ly s is of th e
e s t e r s , th e l a t t e r re q u irin g th e presen ce o f b ile s a l t s .
S p erry and Brand (20) s tu d ie d su sp en sio n s o f r a t l i v e r s , and
id e n t i f i e d th e e s t e r i f i c a t i o n r e a c tio n in th a t t i s s u e . The
pH optimum was found to be below 6 .4 .
No work has ev er been u n d ertak en in v o lv in g th e p u r i
f ic a t io n o f such system s except by K lein ( 8 ) , who achieved
a s ix - to s e v e n -fo ld in c re a se in a c t i v i t y of tis s u e e s te ra s e s
by e x tr a c tin g d rie d tis s u e powders w ith phosphate b u f fe r s .
F u rth e r work along th e se lin e s i s p re se n te d in th e p re se n t
stu d y .
CHAPTER I I I
MATERIALS AM) M ETH O D S
Pre p a ra tio n of C h o le s te ro l P a lm ita te . A m ix tu re of 9 gm.
c h o le s te ro l (Merck) and 11 gm. p a lm itic a c id (Eastm an te c h .)
was h e a te d f o r two ho u rs a t 200° C in an atm osphere o f carbon
d io x id e . The r e s u ltin g y ello w ish mass was co o led , d isso lv e d
in one l i t e r o f ho t 95$ a lc o h o l and allow ed to c r y s t a l l i z e .
Two b atch es of c r y s ta ls were o b ta in e d , w hite and s h in y , each
m e ltin g about 75°C. The 13 gm. of m a te ria l were r e c r y s t a l l i z e d
from h o t a lc o h o l, y ie ld in g 11.5 gm. of c h o le s te ro l p a lm ita te ,
w ith a m e ltin g p o in t o f 77°0 u n c o rre c te d . (P re v io u s ly re p o rte d
7 8 -7 9 .5°C .) Assay fo r c h o le s te r o l showed i t to be 86$ p u re ,
and to c o n ta in no d e te c ta b le f r e e c h o le s te ro l.
P re p a ra tio n o f C h o le s te ro l C o llo id . Five grams o f c h o le s te ro l
(Merck) was d isso lv e d in 200 m l. aceto n e, and warm w ater was
added u n t i l th e t u r b i d i t y did n o t d isap p e ar on h e a tin g . A
sm all a d d itio n a l p o rtio n of aceto n e was added, and th e c le a r
s o lu tio n was poured slow ly in to a l i t e r o f r a p id ly b o ilin g
w ater, w ith c o n sta n t s t i r r i n g . The su sp en sio n was f i l t e r e d
through paper and v a c u u m -d is tille d a t 40°C to a volume of
140 m l. A n aly sis showed i t to c o n ta in 1.87 gm. c h o le s te ro l
p er 100 m l. The f i n a l m ilky w hite suspension rem ained s ta b le
a t room tem p eratu re fo r over a y e a r.
8
P re p a ra tio n o f C h o le s te ro l P a lm ita te C o llo id , An aceto n e
s o lu tio n o f 0 ,5 gm, c h o le s te r o l p a lm ita te was d ilu te d w ith
warm w ater u n t i l th e t u r b i d i t y would no t c le a r up w ith
h e a tin g . At t h i s p o in t a l i t t l e more acetone was added.
T his m ix tu re was poured dropw ise in to 300 ml* b o ilin g w ater
and f i l t e r e d . A n aly sis showed 1.2 5 m g./m l. c h o le s te r o l,
o r 2,0 2 mg. as th e e s te r . No f r e e c h o le s te ro l could be
dem onstrated. The su sp en sio n showed a tendency to s e t t l e
out v e ry slo w ly a t room te m p e ra tu re , b u t has rem ained
s ta b le f o r s e v e ra l months in th e r e f r i g e r a t o r . No d e te c ta b le
h y d ro ly s is o ccu rred .
T issu e e x tr a c ts . A dult w hite r a t s from our colony were an es
th e tiz e d w ith nem butal and th e t i s s u e was q u ick ly removed
and c h ille d in ic e w a te r. A fte r a thorough washing w ith
c o ld w a te r, th e weighed tis s u e was m inced w ith s c is s o r s and
ground in a m o rta r w ith sand and an e q u al w eight o f 0*9% NaCl*
The su sp en sio n was c e n trifu g e d , and th e su p e rn a ta n t liq u id
s tr a in e d thro u g h gauze b e fo re i t was assayed or o th erw ise
tr e a te d . I t was found th a t m ost such crude e x tr a c ts c o n ta in
a sm all b u t s ig n if ic a n t amount o f f r e e c h o le s te ro l, so i t
was alw ays n e c e ssa ry to ru n an u n in cu b ated c o n tro l tu b e w ith
th e a ssa y .
A ssay u sin g Dioxane S o lu tio n s o f S u b s tr a te . A s o lu tio n o f
c h o le s te ro l and p a lm itic a c id in equim olar q u a n titie s was
made, u sin g 1 ,4 - dioxane as th e s o lv e n t, and a d ju s tin g th e
9
c o n c e n tra tio n so th a t one m i l l i l i t e r o f th e s o lu tio n con
ta in e d ap p ro x im ately 0*3 mg* c h o le s te ro l* In c ase s where
a c h o le s te ro l p a lm ita te s u b s tr a te was d e s ire d , th e same
ty p e o f s o lu tio n was made, and 5 mg*/ml* of soya l e c i t h i n
was a ls o d iss o lv e d in th e dioxane. The need f o r l e c i t h i n
w i l l be d isc u sse d under th e ex p erim en tal r e s u lts * To
a ssa y a g iv en p re p a ra tio n , th e fo llo w in g p ro p o rtio n s were
used:
0*5 m l. t is s u e e x tr a c t o r e q u iv a le n t, in 0 .9 $ NaCl
0*5 m l. M/20 b u ffe r - su c c in a te f o r h y d ro ly tic
re a c tio n o r phosphate f o r e s t e r i f i c a t i o n
1 .0 m l. s u b s tr a te s o lu tio n .
pH b e fo re in c u b a tio n was 6 .5 , a lth o u g h some l a t e r
experim ents were run a t d if f e r e n t v a lu e s .
F in a l dioxane c o n c e n tra tio n 50$.
The tu b e s were t i g h t l y sto p p e re d and in cu b a ted a t
37°C f o r 8-18 h o u rs, depending on th e a c t i v i t y o f th e en
zyme p re p a ra tio n . At th e end o f th a t tim e, th e y were r e
moved from th e in c u b a to r and th e r e a c tio n was stopped by
th e a d d itio n o f an a lc o h o l-e th e r s o lu tio n as d e sc rib e d
below .
A ssay u sin g c o llo id a l S uspensions o f S u b s tr a te . Much th e
same procedure was used f o r c o llo id a l s u b s tr a te s as f o r
dioxane s o lu tio n s . The c o n c e n tra tio n of c h o le s te ro l in
th e c o llo id s was about th re e tim es as h ig h as was f e a s ib le
w ith dioxane s o lu tio n s , and in a d d itio n th e h a za rd o f having
an o rg an ic so lv e n t p re s e n t in p u r if ie d system s was avoided.
10
Each a ssa y tube was p rep ared so as to c o n ta in :
0 .5 m l, tis s u e e x tr a c t o r e q u iv a le n t in 0*9$ NaCl
0 .5 m l. M/20 b u f f e r , as above
1 .0 m l. c o llo id a l s u b s tr a te in w ater - c h o le s te ro l
or c h o le s te ro l p a lm ita te
5 .0 mg, l e c i t h i n where c h o le s te ro l p a lm ita te was
used
3 .0 mg. p a lm itic a c id (o r th e amount equiraolar
w ith th e c h o le s te ro l) where c h o le s te ro l
was u sed .
The in c u b a tio n proceeded as b e fo re , and th e r e a c tio n was
sto p p ed by th e a d d itio n o f th e e x tr a c tio n s o lv e n t, a lc o h o l-
e th e r .
E x tra c tio n P ro ced u re. To th e 2 .0 m l. o f re a c tio n m ix tu re
in each a ssa y tu b e , 10 .0 m l. o f a 3 :2 m ix tu re o f a lc o h o l-
e th e r was added f o r c ib ly from a c a lib r a te d s y rin g e . T h is
s o lv e n t was found to be com pletely m isc ib le w ith th e a ssa y
re a g e n ts , and to e x tr a c t th e c h o le s te ro l and c h o le s te ro l
e s te r s q u a n tita tiv e ly w hile co m p letely p r e c ip ita tin g th e
p ro te in s which m ight i n t e r f e r e w ith th e c h o le s te ro l d e te r
m in a tio n l a t e r on. The e x tr a c tio n m ix tu re was allow ed to
sta n d f o r te n m in u tes, a f t e r which i t was c e n trifu g e d a t
2000 HUff f o r about a m in u te. The c le a r su p e rn a ta n t liq u id
was poured o ff in to a c le a n t e s t tu b e , and th e d e s ire d
a liq u o ts m easured in to 15-m l, c o n ic a l c e n trifu g e tu b es
f o r th e d e te rm in a tio n of f r e e and t o t a l c h o le s te ro l. In
cases where n o n -in cu b ated c o n tro ls were ru n , th e e x tr a c tio n
was made im m ediately a f t e r th e tu b e s had been s e t up , and
th e a liq u o ts were allow ed to sta n d a t room tem p eratu re u n t i l
XI
th o se o f th e in c u b a ted s e r ie s were re a d y , when th e whole s e t
was p ro cessed to g e th e r .
D eterm in atio n o f C h o le s te ro l, The method used f o r th e d e te r
m in atio n of f r e e and t o t a l c h o le s te ro l i s a m o d ified form of
th e Chaney method ( 1 ), in v o lv in g a c o lo rim e tric measurement
o f th e Lieberm ann-B urchard r e a c tio n . The a liq u o ts ta k e n of
th e o r ig in a l e x tr a c t were ev ap o rated to dryness w ith a g e n tle
stream of a i r in a b o red aluminum block which was therm o
s t a t i c a l l y c o n tr o lle d a t 60°C, When d ry , 3 m l, o f a m ixture
o f 1 :1 a le o h o l-a c e to n e was added, and th e tu b es which were
d e s tin e d f o r t o t a l c h o le s te ro ls were hyd ro ly zed w ith two drops
o f 30% K O H a t bO°C f o r 50 m in u te s. At th e end o f t h i s hydro
l y s i s p e rio d , th e c o n te n ts were made n e u tra l to p h e n o lp h th a le in
by th e dropw ise a d d itio n o f 15$ aqueous a c e tic a c id . The f re e
c h o le s te ro l was th e n p r e c ip ita te d from a l l tu b e s by th e ad
d itio n o f 1 m l, o f a 0 ,5 $ s o lu tio n o f d ig ito n in (Merck) in
50% a lc o h o l, fo llo w ed by a th re e -h o u r in c u b a tio n a t 35°C,
The p r e c ip ita te d d ig ito n id e s were c o lle c te d by cen
tr if u g in g th e tu b e s f o r te n m inutes a t 2500 KPM, A fte r th e
su p e rn a ta n t liq u id had been poured o f f and d isc a rd e d , th e
d ig ito n id e s were washed by th e f o r c ib le a d d itio n of 3 m l, o f
anhydrous e th e r from a s y rin g e . A nother c e n trifu g in g , t h i s
tim e f o r b m in u tes, was u sed to s e p a ra te th e e th e r , which
was a ls o d isc a rd e d . The d ig ito n id e s were th o ro u g h ly d rie d
in th e 60°C b lo ck and th e n d isso lv e d in 0 ,5 m l. o f g la c ia l
12
a c e tic acid* C o n sid erab le sh ak in g coupled w ith continuous
warming a t bO°C was n e c e ssa ry to e f f e c t t h i s s o lu tio n , and
o c c a s io n a lly i t became n e c essary to add a g la s s bead to th e
tu b e to break up a lump o f d ig ito n id e which would n o t r e a d ily
d isso lv e*
At t h i s sta g e th e tu b e s could be l e f t over n ig h t, a l
though u s u a lly th e c o lo r developm ent was c a r rie d out imme
d ia te ly * To each tube was added 3*0 ml* o f anhydrous c h lo ro
form , and th e t i g h t l y sto p p e re d tu b e s were warmed to 35°C in
th e b lo ck w h ile th e c o lo r re a g e n t was p re p a re d . A f la s k was
cooled in an i c e - s a l t b a th . The re a g e n t was made in m u ltip le s
o f 10 m l. depending on th e number o f d e te rm in a tio n s to be ru n ,
and had to be f r e s h ly p re p are d each tim e c o lo r developm ent
was c a r r ie d o u t. A cetic anhydride was co o led in th e f la s k ,
and when i t was th o ro u g h ly c h ille d , 1 m l. o f c o n c e n tra te d
HgS04 was added f o r each 9 m l. o f a c e tic an h y d rid e. The ad
d itio n o f th e c o lo r re a g e n t to th e warmed s o lu tio n s was made
a t te n second in te r v a ls , a c c u ra te ly tim ed . A fte r th e ad
d itio n o f 1 .0 m l. o f re a g e n t, ag ain u s in g th e c a lib r a te d sy
rin g e f o r f o r c ib le d e liv e ry , th e tu b e s were allow ed to
rem ain in th e 35°C b lo ck f o r 10.0 m in u tes. At th e end o f
th a t tim e, th e y were tr a n s f e r r e d to th e ic e b a th , ag ain a t
te n second in te r v a ls . Ten m inutes were allow ed to p ass
w h ile th e y cooled in th e bath b e fo re th e y were re ad in th e
K lett-Sum m erson c o lo rim e te r a g a in s t a chloroform b la n k ,
u sin g a o60 f i l t e r .
15
D u p licate d e te rm in a tio n s done in t h i s manner were round
to check w ith in 0.0 1 mg. c h o le s te ro l. C h o le s te ro l re c o v e rie s
oi* 0.0 2 o r low er were n o t co n sid ered s ig n if ic a n t because of
th e c lo se approxim ation to th e b la n k .
CHAPTER IY
EXPERIMENTAL RESULTS
I . CHOLESTERol- esterlfying systems
D em onstration in l i v e r e x t r a c t s . E x tra c ts o f l i v e r proved
v ery d i f f i c u l t to use in d em o n stratin g th e e s t e r i f i c a t i o n
of c h o le s te r o l, f o r th e re i s a ls o co n tain ed in most e x tr a c ts
some enzyme system which w i l l d e stro y c h o le s te ro l, th a t i s ,
re n d e r i t n o n -p re c ip ita b le w ith d ig ito n in under th e con^
d itio n s o f th e assay . In a d d itio n , th e re i s a lso p re s e n t an
a c tiv e h y d ro ly tic system which o b scu res any c le a r p ic tu re
o f e s t e r i f i c a t i o n . These two f a c to r s make q u a n tita tiv e
r e s u l t s im p o ssib le w ith u n p u rifie d e x tr a c ts , but th e y do
n o t s e rio u s ly in te r f e r e w ith a q u a lita tiv e d em o n stratio n o f
e s te r if y in g a c t i v i t y .
One such q u a lita tiv e d em o n stratio n may be seen in .
Table I . Three normal a d u lt m ale r a t s of th e sto c k colony
were a n e s th e tiz e d w ith nem butal and th e l i v e r s removed and
e x tra c te d in th e u su a l way, u sin g two volumes o f m/ 5 phosphate
b u f f e r , pH 5 .7 0 . t w o 2-m l. p o rtio n s o f th e e x tr a c t were
im m ediately ly o p h iliz e d , two o th e r 10-m l. p o rtio n s were
d ia ly z e d over n ig h t, one a g a in s t M/5 phosphate b u f fe r , pH 5 .7 0 ,
th e o th e r a g a in s t d i s t i l l e d w a te r. These d ia ly z e d p o rtio n s
and th e ly o p h iliz e d o nes, r e c o n s titu te d w ith w a te r, were
15
TABLE I
ESTERIFICATION OF CHOLESTEROL B Y LIVER EXTRACT
C h o le s te ro l (mg) mg.
P re p a ra tio n B u ffer T o ta l Free e s t e r i f i e d
N on-incubated
c o n tro l
Crude e x tr a c t
L y o p h ilize d
D ialyzed a g a in s t
M/5 phosphate
D ialyzed a g a in s t
w ater
M/20 phosphate 0*41 0*41
M/20 phosphate 0.38 0.31
M/20 phosphate 0.36 0.31
none 0 .4 1 0.36
M/5 phosphate 0.41 0.36
0.07
0.05
0.05
0.05
The s u b s tr a te u sed was c h o le s te ro l and p a lm itic a c id in dioxane
In c u b a tio n tim e 8 h o u rs. F in a l pH 6.5
S im ila r r e s u l t s were o b ta in e d in fo u r ex p erim en ts.
16
assay ed as denoted in tn e ta b le . A ll o f tn e se a ssay s fo r
enzyme a c t i v i t y were c a r r ie d o u t in d u p lic a te a g a in s t a
s u b s tr a te c o n s is tin g o f 0 ,4 1 m i./m l, c h o le s te ro l and 0*27 mg.
p e r m l. p a lm itic a c id in dioxane so lu tio n * in c u b a te d f o r 8
hours a t S7°C* I t may be seen th a t th e e s te r if y in g system
m ain tain ed i t s a c t i v i t y th ro u g h ly o p h iliz a tio n and d i a l y s i s .
There was i n i t i a l l y some lo s s o f c h o le s te ro l, presum ably
th ro u g h d e g ra d a tio n , but t h i s in te rf e r e n c e was n o t p re se n t
a f t e r d ia ly s is o f th e e x tr a c ts .
R equirem ents f o r a c t i v i t y . The phosphate ion i s n e c e ssa ry
fo r th e a c t i v i t y o f th e e s t e r i f i c a t i o n system s th u s f a r
found in e i t h e r th e l i v e r o r th e in t e s t i n e . T h is i s borne
ou t by Table I I . The tis s u e e x tr a c ts f o r th e s e experim ents
were p rep ared as b e fo re , except th a t one volume o f 0 .9 f o NaCl
was used as th e e x tr a c tin g ag en t in s te a d o f th e phosphate
b u lT e r. A ssaying th e se e x tr a c ts a g a in s t c h o le s te ro l and
p a lm itic a c id in dioxane, we fin d th a t as long as phosphate
b u ffe r is used to b rin g th e a ssa y tu b e s to th e f i n a l pH b.O,
a c t i v i t y is p r e s e n t. I f s u c c in a te b u ffe r i s em ployed, no
e s te r if y in g a c t i v i t y can be d em onstrated.
I t was th o u g h t th a t t h i s req u irem en t f o r phosphate
ion m ight in d ic a te th a t somewhere in th e p ro cess o f e s t e r i -
r ic a t io n th e re e x is te d a p h o sp h o ry la tio n of one o f th e sub
s ta n c e s in v o lv ed , xhe fo rm atio n of such compounds i s w e ll
known in b io lo g ic a l p ro c e sse s where energy i s re q u ire d to
b rin g th e r e a c tio n to co m p letio n . W ith t h i s in m ind, th e
17
TABLE II
THE EFFECT OF THE BUFFER IO N ON ESTER IFICATION
OF CHOLESTEROL BY CH U TE LIVER EXTRACTS
Exp. No. B u ffer
C h o le s te ro l (m g,)
C o n tro l in cu b ated
T o ta l Free T o ta l Free
mfe.
e s t e r i f i e d
408 M/5 phosphate 0 , d2 0 .6 2 0.62 0.34 0.28
M/20 su c c in a te 0 .6 2 0.62 0.62 0,58 0 . 04
450 M/20 phosphate 0.67 0.67 0.67 0.60 0.07
M/20 s u c c in a te 0.67 0.67 0.67 0.67 0
S u b stra te - c h o le s te ro l and p a lm itic a c id in dioxane.
In c u b a tio n tim e 21 h o u rs. F in a l pH 6 ,5
18
e f f e c t s o f adenosine trip h o s p h a te and g lu c o se -l-p h o sp h a te on
th e system were in v e stig a te d * (T able I I I ) I t would seem tb a t
e i t h e r o f th e se su b stan ce s i s in e f f e c tiv e in in c re a s in g th e
speed o f th e e s t e r i f i e a t i o n , alth o u g h th e p ro b ab le presen ce
o f ph o sp h atases in th e m ix tu re makes t h i s le s s c le a r cut
th a n i t appears*
The q u estio n o f th e f a t t y a e id source in th e c h o le s te ro l
e s te r was a lso in v e stig a te d * F ree f a t t y a c id s a re cap ab le of
speeding th e e s t e r i f i e a t i o n , and from th e d a ta p re se n te d in
Table 17, i t may be seen th a t i t e v id e n tly makes no d iffe re n c e
w hether th e f a t t y a c id i s s a tu r a te d or u n s a tu ra te d , o r w hether
a tr ig ly c e r id e was in tro d u c e d in s te a d , The f a c t th a t t r i s t e a r i n
could be used was in te r p r e te d as in d ic a tin g th a t th e crude
l i v e r e x tr a c t co n tain ed a lip a s e which could s p l i t o f f ‘ Che
n e c e ssa ry f a t t y a c id , and th a t t h i s l i p o l y t i c r e a c tio n p ro
ceeded f a s t enough so th a t i t was no t th e lim itin g r e a c tio n
in th e s e r i e s . O ccasio n al e x tr a c ts would show a c t i v i t y w ith
out an a d d itio n a l f a t t y a c id so u rce, presum ably because o f
th e p resen ce o f adequate so u rces of f a t t y a c id s in th e crude
e x tr a c t.
E s t e r if i e a t i o n w ith I n t e s t i n a l E x tr a c ts . C u rio u sly enough,
when e x tr a c ts of th e sm all i n t e s t i n e s o f r a t s which had been
m ain tain ed on a norm al sto c k d ie t were p rep ared and te s te d
f o r e s te r if y in g a c t i v i t y , th e r e s u l t was u n ifo rm ly n e g a tiv e .
However, when r a t s were used which had been fe d a d ie t con-
19
TABLE III
THE EFFECT OF ADENOSINE TRIPHOSPHATE A N D GHJCOSE-l-PHOSPHATE
O N ESTERI FI C A T I O N OF CHOLESTEROL
BY C RU D E LI IT E R EXTRACTS
Ch'oTeaterol (rag.) mg.
Added T o ta l Free e s t e r i f i e d
0 0.43 0.40 0.03
Adenosine
T rip h o sp h ate 0.43 0.4 1 0.02
G lucose-1-
Phosphate 0.43 0.40 0.03
S u b s tra te - c h o le s te ro l and p a lm itic a c id in dioxane.
B u ffer - M/20 ph o sp h ate, f i n a l pH o f m ix tu re 6 .5 .
In cu b a tio n Time - 21 h o u rs, 37°C
A denosine T rip h o sp h ate - 1.2 4 mg. disodium s a l t added,
o r a f i n a l c o n c e n tra tio n o f 1.12x10“® m olar
G lucose-1-phosphate - 0.67 mg. disodium s a l t added, o r
a f i n a l c o n c e n tra tio n o f 1.1x10“ ® m olar.
S im ila r r e s u l t s were o b ta in e d in a d u p lic a te experim ent.
20
TABLE IV
SOURCES OF THE FATTY ACID RESIDUE
OF CHOLESTEROL ESTERS
Added F a tty
Acid Source
C h o le s te ro l (m g.)
C o n tro l In cu b ated
T o ta l F ree T o ta l Free
mg.
e s t e r i f i e d
none 0,67 0.67 0.67 0.65 (0 .0 2 )
p a lm itic a c id 0 .6 5 0, 65 0.65
G O
lO
.
o
0.07
o le ic a c id 0.67 0.67 0.67 0.60 0.07
t r i s t e a r i n 0.67 0.67 0.67 0.60 0.07
S u b s tra te - -,6 7 m g,/m l, c h o le s te ro l in dioxane p lu s an
equim olar q u a n tity of th e f& tty a c id so u rce.
In cu b a tio n tim e - 21 h o u rs.
B u ffer - M/20 p h o sp h ate, f i n a l pH 6,5
Enzyme p re p a ra tio n - c e n trifu g e d e x tr a c t of r a t l i v e r in
an eq u al w eight of 0..9$ NaCl.
21
ta in in g e ith e r Vfo la n o lin , which c o n ta in s some c h o le s te ro l
e s t e r s , o r 1% f r e e c h o le s te r o l, i t was p o s s ib le a f t e r two
weeks to d em onstrate th e e x is te n c e o f a c h o le s te r o l- e s te r i-
Tying system in th e i n t e s t i n a l e x tr a c ts from such anim als*
One c u rre n t th e o ry on a b s o rp tio n m a in ta in s th a t only s te r o l s
which can be e s t e r i f i e d by th e in t e s t i n e a re ab so rb ed . (15)
T h is re c e iv e s a d d itio n a l su p p o rt h e re , fo r i t i s e v id en t th a t
th e e s te r if y in g system i s m arkedly in c re a se d in a c t i v i t y under
c o n d itio n s which re q u ire th e in c re a se d a b so rp tio n o f c h o le s
t e r o l . The f a c t th a t such in c re a s e d a c t i v i t y i s a ls o observed
a f t e r th e fe e d in g o f c h o le s te ro l e s te r s i s n o t c o n tra d ic to ry ,
s in c e i t i s known th a t th e re i s a n o n sp e c ific b u t v ery p o te n t
e s te r a s e p re se n t in th e p a n c re a tic s e c re tio n which r a p id ly and
th o ro u g h ly h y d ro ly zes c h o le s te ro l e s te r s as w e ll as o th e r
e s te r s (2 3 ). Presum ably, th e n , th e c h o le s te ro l e s te r s fe d
in th e form o f la n o lin are h y d ro ly zed b efo re th e y re a c h th e
s ta g e o f a b s o rp tio n .
The d a ta p e r ta in in g to th e d em o n stratio n of th e e s t e r i
fy in g system in i n t e s t i n a l w all a re p re se n te d in T able V. The
e x tr a c ts were p rep ared as b e fo re , u sin g 0 .9 fo NaCl as th e ex
t r a c t i o n s o lv e n t. The assay s were c a r rie d out a g a in s t c o l
l o id a l s u b s tr a te as w e ll as a g a in s t th e dioxane s o lu tio n , and
th e r e s u l t s a re com parable.
These crude e x tr a c ts were th en e le c tro d ia ly z e d a g a in s t
d i s t i l l e d w ater to remove any phosphate io n . E le c tr o d ia ly s is
was found to be more s a ti s f a c t o r y because i t sh o rten ed th e
d ia ly s is tim e and in c re a se d th e e f f ic ie n c y o f d ia ly s is . In
TABLE V
ESTERIFYING A N D H Y D RO LY TIC REACTIONS OF C R U D E SITRACTS OF RA T SM A L L INTESTINE
HYDROLYSIS ESTERIFICATION
S u b strate C ontrol Incubated mg. C ontrol Incubated mg.
Exp. No. P rep ara tio n Form T o tal Free T o tal Free freed T o tal Free T o tal Free e s te r if ie d
1204 crude dioxane 0.48 0.15 0.48 0.17 (0.02) 0.20 0.19 0.20 0.14 0.05
c o llo id 0.48 0.19 0.48 0.19 0 1.12 1.12 1,08 0.91 0.19
d ialy zed
v s. w ater c o llo id 0.56 0.07 0.56 0.12 0.05 1.12 1,03 1.12 1.02 (0.01)
106 crude c o llo id 0.52 0.19 0.52 0.19 0 1.12 1.12 1.12 1.02 0.10
d ialy zed
v s. w ater,
ly o p h iliz e d
c o llo id 0.40 0.10 0.40 0.14 0.04 1.12 1.12 1.12 1.12 0
These tis s u e e x tra c ts were from anim als fed 1% la n o lin in the d ie t fo r 16 and 21 days re s p e c tiv e ly
before th e experim ents.
H ydrolytic s u b s tra te - c h o le s te ro l p alm itate p lu s 5 mg. soya le c ith in , w ith M/20 su ccin ate b u ffe r.
E s te r if ie a tio n s u b s tra te - c h o le s te ro l p lus 3 mg. p a lm itic a c id , w ith M/20 phosphate b u ffe r.
F in a l pH 6.5 Incubated 8 hours, 3?°C
S im ilar r e s u lts in a t o t a l o f four experim ents.
23
th e course o f t h i s p ro ced u re, which was co n tin u ed a t 300 v o lts
f o r f iv e h o u rs, th e p r o te in was p re c ip ita te d * The m a te ria l
in s id e o f th e cello p h an e c a sin g a t th e end o f th e d ia ly s is was
brought to ap p ro x im ately 1f0 NaCl c o n c e n tra tio n by th e a d d itio n
o f th e s o lid s a l t , and th e s o lu tio n was s t i r r e d fo r f iv e
m in u tes. A fte r th e in s o lu b le d e b ris was removed by c e n t r i -
f ig in g , th e e x tr a c t was re a ssa y e d f o r a c t i v i t y . I t may be
seen th a t t h i s tre a tm e n t d e s tro y s th e e s te r if y in g a c t i v i t y
o f th e p re p a ra tio n , re g a rd le s s o f th e f a c t th a t th e assay s
were c a r rie d out u sin g phosphate b u f fe r . T h is f o rtu n a te
r e s u l t made i t p o s sib le to dem onstrate th e presence o f th e
h y d ro ljrtic system as w e ll in th e same e x tr a c ts , which was
not ap p aren t b e fo re d ia ly s is . Presum ably t h i s re p re s e n te d
th e m asking e f f e c t of th e o p p o site r e a c tio n .
The f a c t th a t such opposing re a c tio n s are going on
a t th e same tim e makes i t im p o rtan t th a t th e se r e s u l t s , to o ,
should be in te r p r e te d q u a l i t a t i v e l y in s te a d o f q u a n tita tiv e ly .
The lo s s o f c h o le s te ro l thro u g h d eg ra d ativ e r e a c tio n s which
nad been observed w ith l i v e r e x tr a c ts was n o t c o n s is te n tly
found w ith i n t e s t i n a l e x tr a c ts , alth o u g h o c c a s io n a lly th e re
was a p re p a ra tio n which showed a s l i g h t l y low er rec o v ery
a f t e r in c u b a tio n . F u rth e r work w ith t h i s d e g ra d a tiv e sy s
tem i s re p o rte d by Marx and L ip s e tt (1 0 ).
I I . HYDROLYSIS OF CHOLESTEROL ESTERS
D em onstration in L iv e r E x tr a c ts . The h y d ro ly s is o f c h o le s te ro l
24
e s te r s can be c a r rie d ou t by s a lin e e x tr a c ts o f l i v e r t i s s u e
p rep a red in th e u s u a l manner and assayed e i t h e r a g a in s t
dioxane s o lu tio n s o r a g a in s t c o llo id a l su sp en sio n s o f c h o le s
t e r o l p a lm ita te . In T able VI we fin d th e r e s u l t s o f two
such ex p erim en ts. Here may be found evidence th a t c h o le s te ro l
p a lm ita te in i t s e l f i s not s u f f ic ie n t f o r th e dem o n stratio n
o f t h i s r e a c tio n , b u t th a t some component o f soya l e c i t h i n
i s a ls o n e c e ssa ry i f th e a c t i v i t y o f th e e x tr a c t i s to be
a p p a re n t. The s t a t e o f p u r if ic a tio n o f th e enzyme system
in flu e n c e s t h i s re q u irem e n t, p o s s ib ly because o f th e p resen ce
o f p h o sp h o lip id s in u n p u rifie d l i v e r e x tr a c ts .
I t may a ls o be seen from T able VI th a t th e phosphate
io n i s not re q u ire d f o r th e h y d ro ly s is , sin c e t h i s r e a c tio n
proceeds e q u a lly w e ll when b u ffe re d w ith s u c c in a te .
The s p e c i f i c i t y o f t h i s h y d ro ly tic system was no t
s tu d ie d d i r e c t ly , because th e l i v e r p re p a ra tio n s were f a r
from p u re , and a stu d y o f a c t i v i t y a g a in s t o th e r s u b s tr a te s
would no t prove w hether o r no t th e same enzyme system s in
th e crude e x tr a c t were in v o lv ed each tim e. However, a
p u r if ie d p re p a ra tio n o f e s te ra s e from r a t l i v e r was made
and found to be in a c tiv e a g a in s t c h o le s te ro l p a lm ita te .
The p ro ced u re was as fo llo w s: (2) Two r a t l i v e r s were
m inced ? /ith sand in bO m l. o f 0 .9 $ s a lin e and allow ed to
s ta n d a t room te m p eratu re f o r 30 m in u tes. The p re p a ra tio n
was s tr a in e d thro u g h gauze and th e liq u id brought to pH 5 .7 .
25
TABLE VI
HYDROLYSIS OF CHOLESTEROL PALMITATE
BY C RU D E LIVER EXTRACTS
Free C h o le s te ro l (m g,)
Exp. No. L e c ith in B u ffer C o n tro l In cu b ated
414 0 M/20 phosphate 0 0
0 M/20 su c c in a te 0 (0 .0 2 )
/
M/20 phosphate 0 0.12
i
M/20 s u c c in a te 0 0.10
421 0 M/20 phosphate 0 0*05
/
M/20 phosphate 0 0.14
S u b s tra te - 0 .8 mg. c h o le s te ro l p a lm ita te in d io x an e, p lu s 5 mg.
soya l e c i t h i n where n o ted .
F in a l pH 6 .5
In c u b a tio n - 21 h o u rs, 37^C
26
A fte r c e n tr ifu g in g , th e p r e c ip ita te was washed fo u r tim es
w ith sm all p o rtio n s o f w ater a t pH 5 .7 , and th e f i n a l wash
r e ta in e d no c o lo r. The p r e c i p i ta te was suspended in 50 m l.
w ater and brought to pH 9 .5 b efo re s to ra g e in th e r e f r i g e r a to r
over n ig h t. The n ex t m orning i t was brought to pH 5 .0 , c e n tr i
fu g ed , and th e s u p e rn a ta n t assay ed .
The a ssa y method u sed was th a t employed, by fa lc o n e r (3 ).
A p h o sp h a te -b o ra te b u ffe r of pH 7 was p re p a re d , u sin g 0.05M
NaHgPO^. and 0.025M NagB^Oy, and 20 m l. o f t h i s b u ffe r was
in cu b ated w ith 0 .5 m l. e th y l a c e ta te and 0 .5 to 1*0 m l. e s te ra s e
s o lu tio n . The in c u b a tio n was c a r r ie d ou t a t 37°C, and sm all
sam ples were removed a t in te r v a ls f o r th e d e te rm in a tio n of
th e pH. In F a lc o n e r's term s, a f a l l o f 0.001 pH u n it p e r
m inute or 0.06 p e r hour i s d esig n a te d as one u n it o f e s te r a s e
a c t i v i t y . T his p a r t ic u la r s o lu tio n was found to have 5 .5 -b .O
u n its p er m i l l i l i t e r o f e s te ra s e a c t i v i t y (T able V II), w h ile
an a ssay f o r h y d ro ly tic o r s y n th e tic a c t i v i t y tow ard c h o le s
t e r o l e s te r s was n e g a tiv e . Thus i t would appear th a t th e
c h o le s te ro l-p a lm ita te -h y d ro ly z in g system d e sc rib e d in t h i s
d is s e r ta tio n i s not th e system u s u a lly d e sig n a te d as l i v e r
e s te r a s e . I t does not fo llo w , o f c o u rse , th a t th e c h o le s te ro l
e s te r a s e system i s a t a l l s p e c if ic .
D em onstration in I n t e s t i n a l E x tr a c ts . The d em o n stratio n of
h y d ro ly tic a c t i v i t y tow ard c h o le s te ro l p a lm ita te in e x tr a c ts
o f th e sm all in t e s t i n e o f th e r a t was no t c o n s is te n tly
27
TABLE VII
COM PARATIVE ACTIVITY OF HJRIFIED LIVER ESTERASE
AGAINST ETHYL ACETATE A N D CHOLESTEROL PALMITATE
I* A c tiv ity o f E s te ra s e a g a in s t E th y l A cetate
(See te x t f o r method)
Amount
E s te ra s e
S o lu tio n pH re a d in g s
(m l.) i n i t i a l 1.4 2 h r s . 2.03 h rs . u n its /m l.
0 .5 7.00 6.42 6.31 6.02
0 .5 6.98 6.42 6.31 6.02
1 .0 6.92 6. 22 6.03 5 .2
1 .0 6.92 6.17 5.96 5 .7
I I . A c tiv i ty of E s te ra s e jL ^ jC h o lestero l S y stem
days
S u b s tra te in c u b ated
C h o le ste ro l )
T o ta l Free
mg#
changed
c h o le s te r o l, 1 0.28 0.27 (0 .0 1 )
p a lm itic a c id ,
dioxane 3 .9 0.28 0.28 0
c h o le s te ro l 1 0.34 0 0
p a lm ita te ,
soya l e c i t h i n , 3.9 0.35 0 0
dioxane
28
s u c c e s s fu l f o r a long w h ile . A ctiv e p re p a ra tio n s were o b tain ed
o fte n enough to le a v e no doubt th a t such a r e a c tio n could occur*
I t was not u n t i l th e e s t e r i f i e a t i o n r e a c tio n had been dem onstrated
in c h o le s te r o l- f e d r a t s th a t i t was r e a liz e d th e p re v io u s nega
t i v e r e s u l t s m ight have re p re s e n te d a masking o f th e h y d ro ly tic
a c t i v i t y by tr a c e s o f e s t e r i f i e a t i o n in e x tr a c ts from normal
anim als* A cco rd in g ly , th e attem p t was made to in a c tiv a te e s -
t e r i f i c a t i o n and th u s allow th e a c t i v i t y o f th e h y d ro ly tic
system to appear* T y p ica l experim ents o f t h i s ty p e have a l
read y been p re se n te d in T able V. Here th e o r ig in a l crude ex
t r a c t s e x h ib ite d no a c t i v i t y a g a in s t e i t h e r th e c o llo id a l
su sp en sio n o r th e dioxane s o lu tio n o f c h o le s te ro l p a lm ita te ,
b u t a f t e r e l e c t r o d i a l y s i s , which was a good d e al more thorough
th a n th e u s u a l s t a t i c o v e r-n ig h t d i a l y s i s , th e e s te r if y in g
system was no lo n g e r a c tiv e , and th e h y d ro ly tic a c t i v i t y
became apparent*
O c c asio n a lly a crude p re p a ra tio n was o b ta in e d which
e x h ib ite d h y d ro ly tic a c tiv ity * Such a one i s shown in T able
V III. The i d e n t i f i c a t i o n o f th e h y d ro ly s is as a tr u e en
zym atic r e a c tio n i s made by comparing th e a c t i v i t y o f th e
crude e x tr a c t w ith th a t o f e x tr a c t h ea te d in a b o ilin g w ater
b a th fo r 15 m in u tes. I f th e h y d ro ly s is o f c h o le s te ro l
p a lm ita te under th e se c o n d itio n s were no t due to enzym atic
a c t i v i t y , th e re would be no reaso n to ex p ect th a t h e a t
d e n a tu ra tio n o f th e p ro te in s p re se n t in th e e x tr a c t would
29
TABLE VIII
EFFECT OF HEAT O N HYDROLYSIS BY INTESTINAL EXTRACTS
C h o le s te ro l (m g.)
C o n tro l Incu b ated mg*
P re p a ra tio n T o ta l F ree T o ta l F ree fre e d
crude e x tr a c t 0.3 2 0.0 2 0.32 0.11 0.09
♦
h e a te d 0.32 0.02 0.3 2 0.02 0
*Heated a t 100°C f o r 15 m in u tes b e fo re a d d itio n o f
s u b s tr a te .
S u b stra te - c h o le s te ro l p a lm ita te p lu s 5 mg. l e c i t h i n
in dioxane
B u ffer - M/20 s u c c in a te , f i n a l pH 6 .5
S im ila r r e s u l t s o b ta in e d in th re e ex p erim en ts.
30
have any e f f e c t on tn e p ro c e ss.
Separ a t i o n ojf t w o F a c to rs in v o lv e d in H y d ro ly sis. In th e
course o f d ia ly s is ex p erim en ts, i t was decided to t r y to
p u r if y th e system by s e p a ra tio n o f th e w a te r-in s o lu b le g lo
b u lin s from th e crude d ia ly z e d e x t r a c t . U nexpectedly, th e
s e p a ra tio n o f th e w a te r-s o lu b le and w a te r-in s o lu b le com
ponents a f t e r d ia ly s is y ie ld e d two in a c tiv e f r a c tio n s .
Recom bination o f th e two would r e s to r e th e a c t i v i t y o f th e
p re p a ra tio n , as may be seen from T able IX. I t i s e v id e n t th a t
th e re a re a t l e a s t two f a c to r s , both n o n d ia ly sa b le , which
a re n e c e ssa ry f o r a c t i v i t y o f th e system .
The n ex t p o in t in v e s tig a te d was th e h e a t s t a b i l i t y
o f th e com ponents. The w a te r-s o lu b le and w a te r-in s o lu b le
f r a c tio n s were h e a te d to g e th e r and s e p a ra te ly in a b o ilin g
w ater b ath f o r 15 m inutes b e fo re th e a d d itio n o f th e assay
s u b s tr a te . As may a ls o be seen in T able X, th e w a te r-
in s o lu b le f r a c tio n lo s e s i t s a c t i v i t y upon h e a tin g under
th e se c o n d itio n s , w h ile th e w a te r-s o lu b le f r a c tio n i s s ta b le
to h e a t.
The f a c t t h a t th e h e a t l a b i l e "enzyme* p o rtio n o f
t h i s system e v id e n tly behaves as a © uglobulin su g g ested
th e p o s s ib le s e p a ra tio n o f th e two components by ammonium
s u lf a te f r a c tio n a tio n . (T able XI) A s a lin e e x tr a c t o f r a t
in te s tin e was d ilu te d w ith one volume o f d i s t i l l e d w ater
and cooled in an i c e - s a l t b ath . Then th e c a lc u la te d
51
TABLE IX
FRACTIONATION OF INTESTINAL ESTERASE BY DIALYSIS
mMa mmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmma mFs s a s s &a a s
P re p a ra tio n C h o le ste ro l F reed
(mg.)
n o n -in cu b ated 0
c o n tro l
crude e x tr a c t 0.09
d ia ly z e d :
H gO -soluble f r a c tio n 0
H gO *insoluble f r a c tio n (0 .0 5 )
combined f r a c tio n s 0.07
See te x t f o r te c h n iq u e . D ia ly s is o f u s u a l 0.9% NaCl
e x tr a c t o f r a t sm all in t e s t i n e a g a in s t d i s t i l l e d
w ater f o r 24 h o u rs, f r a c tio n a te d by c e n tr ifu g in g .
S u b stra te - 0 .8 mg. c h o le s te ro l p a lm ita te p lu s 5 mg.
l e c i t h i n in dioxane
In c u b a tio n - 8 h o u rs 32°C
38
TABLE X
HEAT STABILITY OF HYDROLYTIC EN ZY M E AM) COFACTOR
Enzyme C o facto r C h o le s te ro l F reed
(mg«)
X X 0.05
£ '
X (0 .0 1 )
X
A
0.05
A A
-
X
- -
-
X (0 .0 1 )
/ \ in d ic a te s p re p a ra tio n was h e a te d a t
100°C f o r 15 m inutes b e fo re a d d itio n
o f s u b s tr a te .
These f r a c tio n s were p re p a re d by tn e d ia ly s is
te c h n iq u e as d e sc rib e d in th e t e x t .
S u b s tra te - c h o le s te ro l p a lm ita te c o llo id p lu s
b mg. l e c i t h i n
S im ila r r e s u l t s in 5 ex p erim en ts.
33
TABLE XL
FRACTIONATION OF INTESTINAL EXTRACTS
WITH A M M O N IU M SULFATE
Ammonium S u lfa te
{% Sat*n)
F ra c tio n s
O-bO b0-100 1 0 0 -so lu b le C h o le s te ro l fre e d (m g.)
X X X 0.0 4
X
- -
0
-
X 0
X
-
X 0.05
X X
- (-0 .0 1 )
-
X X 0
- X
- 0
P rocedure as in t e x t .
S u b s tra te - 0 .8 mg. c h o le s te ro l p a lm ita te p lu s b mg. soya
l e c i t h i n in dioxane
In c u b a tio n - 8 h o u rs, 32°C
S im ila r r e s u l t s in th re e ex p erim en ts.
amount o r s a tu r a te d aqueous ammonium s u lf a te s o lu tio n was ad
ded dropw ise, w ith c o n sta n t s t i r r i n g , and th e m ix tu re s t i r r e d
fo r s e v e ra l m inutes a f t e r com pletion o f tn e a d d itio n . The
m ix tu re was c e n trifu g e d in c n ille d cups, and tn e p r e c ip ita te
re d is s o lv e d in w ater and d ia ly z e d u n t i l am m onia-free. The
su p e rn a ta n t s o lu tio n was cooled a g ain and more ammonium s u ir a te
added u n t i l tn e c o n c e n tra tio n reach ed th e next le v e l. The
f i n a l s a tu r a tio n o f th e s o lu tio n was c a r rie d out by adding
s o lid ammonium s u lf a te ana allo w in g th e p re p a ra tio n to sta n d
over n ig h t in th e r e f r i g e r a to r b efo re c e n trifu g in g down
th e p r e c ip ita te w itn th e u n d isso lv e d ammonium s u lf a t e . The
f r a c tio n s from t h i s f i n a l p rocedure were a ls o d ia ly z e d u n t i l
am m onia-free and ly o p n iliz e d to e lim in a te la r g e volum es,
i t may be seen t h a t one p o rtio n o f th e system p r e c ip ita te s
w itn tn e g lo b u lin s in th e 0-00% s a tu r a tio n range w itn am
monium s u lf a t e , w h ile th e o th e r component i s s o lu b le in s a tu
r a te d ammonium s u lf a t e . Here a g a in , both f r a c tio n s a re
n e c e ssa ry f o r th e a c t i v i t y of tn e system .
The r e l a t i v e c o n c e n tra tio n s n e c e ssa ry f o r maximal
a c t i v i t y were of i n t e r e s t in d ecid in g w hether a c t i v i t y o f tn e
h e a t- s ta b le * co facto r* was due to i t s a c tu a l p a r tic ip a tio n
in tn e enzyme system o r w hether i t was se rv in g as some s o r t
o f s u b s tr a te . A cco rd in g ly , tn e a c t i v i t y of a given amount
o f a c tiv e enzyme p re p a ra tio n made by th e d i a ly s is method was
35
assayed a g a in s t p ro g re s siv e d ilu tio n s o f one o f tn e p u re s t
p re p a ra tio n s o f th e c o f a c to r, p rep ared by s a tu r a tin g tn e
e x tr a c t w itn ammonium s u if a te follow ed by d ia ly s is and ±yo-
p n iliz a tio n o f tn e s u p e rn a ta n t. The r e s u l t s a re shown in
F igure I . H ere, when tn e c o n c e n tra tio n o f c o fa c to r r a i l s
below 1 m g ./m l., co rresp o n d in g to tn e amount from 138 mg.
t i s s u e , th e a c t i v i t y o f th e m ix tu re b eg in s to f a l l o r r , a l
though some a c t i v i t y i s n o te d u n t i l th e c o n c e n tra tio n re a c n e s
0.025 m g./m l. Below t h i s le v e l th e r e s u l t s a re no lo n g e r
s i g n i f i c a n t , alth o u g h th e y in d ic a te a s t i l l f u r th e r d ecrease
in a c t i v i t y . The amount of enzyme used in t h i s s e r ie s c o r
responded to th a t d e riv e d from 400 mg. o f wet t is s u e and
weighed ap p ro x im ately 3 .5 mg. P a rt o f t h i s w eight i s due to
sodium c h lo rid e , sin c e th e p re p a ra tio n was ly o p h iliz e d from
s a lin e s o lu tio n .
The d e te rm in a tio n o f th e o p tim al pH fo r th e h y d ro ly tic
r e a c tio n was a ls o o f i n t e r e s t . T h is procedure was im p o ssib le
w ith crude p re p a ra tio n s , because i t was not f e a s ib le to b u ffe r
th e m ix tu re s tro n g ly enough to p re v e n t a marked d r i f t i n g
tow ard th e a c id s id e as th e in c u b a tio n p roceeded, no doubt be
cause o f th e accum ulation o f a c id s p l i t p ro d u cts o f p r o te in s
and c a rb o h y d ra te s. When p u re r p re p a ra tio n s were a v a ila b le ,
however, th e experim ent was t r i e d , and th e r e s u l t s may be seen
in F ig u re I I . U sing u /Z Q phosphate b u f f e r s , th e re was s t i l l
some d r i f t tow ard th e a c id s id e d u rin g th e 18-hour in c u b a tio n
“ 2*0 - 1.0 0
Logarithm o f eo fac to r co n cen tratio n
{mg. p er 2 m l. t o t a l volume)
FIGURE 1. LIMITING C O FA C T O R CO N CEN TRA TIO N FO R ACTIVITY
O P T I M A L p H F O R H Y D R O L Y T IC ACTIVITY
C h o le s te ro l f re e d (mg*)
0 / % O Q O I - *
• ' • • « •
o o o o o
N 05 00 O
>
C l
o 0 5
C l
o
to
o >
♦ -
o
0 0 to
05
05:
05
S3
O
0 5
S J
O
A2
38
p e rio d , but i t was v ery much l e s s m arked. The pH o f th e f i n a l
m ix tu re was ta k e n b e fo re in c u b a tio n , and d u p lic a te tu b e s were
in c u b ated . At th e end o f th a t tim e , th e pH was ag ain rec o rd ed
and th e c o n te n ts o f th e tube saved f o r a n a ly s is o f f r e e and
t o t a l c h o le s te r o l. The a c t i v i t y curves when p lo tte d a g a in s t
th e pH were seen to be v ery s im ila r w hether we used th e i n i t i a l
pH o r th e f i n a l pH, alth o u g h th e maxima o ccu rred a t d if f e r e n t
p o in ts . T h erefo re on t h i s f ig u r e th e two curves a re su p er
im posed, and we see th a t th e optimum pH f o r th e h y d ro ly s is o f
c h o le s te ro l p a lm ita te under th e s e c o n d itio n s l i e s betw een 0.7
and 7 .1 . In c u b a tio n o f th e s u b s tr a te alo n e thro u g h t h i s pH
range le d to no s p l i t t i n g o f th e e s te r .
H ature of th e H y d ro ly tic System in th e R a b b it. An a ttem p t
was made to id e n tif y t h i s system in an e x tr a c t from th e sm all
i n t e s t i n e o f th e r a b b i t. W hile no p o s itiv e i d e n t i f i c a t i o n i s
y e t p o s s ib le , i t ap p ears th a t th e r a b b it p o sse sse s an enzyme
system capable o f h y d ro ly sin g c h o le s te ro l p a lm ita te , and a ls o
th a t i t c o n ta in s th e w c o f a c to r w which i s n e ce ssa ry f o r th e
a c tio n o f th e r a t enzyme. The experim ent was c a r r ie d out as
fo llo w s:
A young r a b b it was a n e s th e tiz e d w ith nem butal and k i l l e d
by pneum othorax. The sm all i n te s tin e was removed and c h ille d
in c o ld w a te r. A fte r thorough w ashing, 75 gm. o f th e t is s u e
were ground w ith sand in a m o rta r w ith an eq u al w eight o f
0 .9 fa NaCl. The su sp en sio n was c e n trifu g e d , and 85 m l. o f
th e e x tr a c t was re c o v e re d . A p o rtio n o f t h i s was e le e tr o d ia ly z e d
a g a in s t d i s t i l l e d w ater f o r 4 h o u rs b e fo re f r a c tio n a tio n on th e
b a s is of water solubility The water-so lu b le p o r tio n was ob-
39
ta in e d by c e n trifu g in g tn e m ix tu re , and tn e w a te r-in s o lu b le
p o rtio n by e x tr a c tio n o f th e rem ain in g p r e c ip ita te w itn s a lin e .
These two f r a c tio n s were assay ed a g a in s t c o llo id a l c h o le s te ro l
p a lm ita te (T able X II). The rem ainder o f th e o r ig in a l e x tr a c t
was ly o p h iiiz e d .
S in ce th e r e s u l t s o f th e f i r s t assay were in c o n c lu siv e
and in d ic a te d th a t i f th e r a b b it system were th e same a s th a t
in th e r a t , each f r a c tio n h e re was contam inated w ith th e o th e r,
an o th e r experim ent was c a r r ie d o u t. A p re p a ra tio n of r a t en
zyme which was known to have a c t i v i t y was assay ed a g a in s t an
a c tiv e r a t c o fa c to r p re p a ra tio n and a ls o a g a in s t a sample o f
th e r a b b it enzyme f r a c tio n which had been h e a te d in a b o ilin g
w ater b a th f o r 15 m inutes to d e stro y any enzyme a c t i v i t y . I t
may be seen from th e r e s u l t s in T able X II th a t th e h e a te d
r a b b it p re p a ra tio n co n tain ed th e c o fa c to r n e c e ssa ry to a c tiv a te
th e r a t enzyme f r a c tio n . A more c o n clu siv e i d e n t i f i c a t i o n o f
th e system s in th e two s p e c ie s w ill have to aw ait th e com plete
s e p a ra tio n o f th e f a c to r s in th e r a b b i t.
N ature of th e Enzyme F r a c tio n . As has a lre a d y been p o in te d
o u t, th e enzyme p o rtio n of th e system in the sm all i n t e s t i n e
o f th e r a t which can h y d ro ly ze c h o le s te ro l p a lm ita te i s a
h e a t l a b i l e , w ater in s o lu b le , s a lin e s o lu b le p ro te in p re
c i p i t a t i n g betw een 30-50^ s a tu r a tio n w ith ammonium s u lf a t e .
In view of th e d em o n stratio n by Gyotoku (5) th a t an
e s te r a s e found in g a s tr ic mucosa and in p an creas c o n s is te d
40
TABLE XII
FRACTIONATION OF SM A LL INTESTINE OF THE RABBIT
I . A ssays o f th e e le c tr o d ia ly z e d f r a c tio n s from r a b b it in t e s t i n e
W ater- W ater- C h o le s te ro l (m g,) mg*
in s o lu b le s o lu b le T o ta l Free fre e d
X - 0.48 0.0 6 0.06
-
X 0.50 0.05 0.05
X X 0.50 0.08 0.08
NOTE incom plete s e p a ra tio n , d e s p ite f iv e
hour d ia ly s is period*
II* C r o s s - a c tiv ity between r a t enzyme and r a b b it c o fa c to r
Rat
w a te r-in s o l
R at
w a te r -s o l.
R abbit C h o le s te ro l (m g.)
w a te r -in s o l. T o ta l Free
mg.
fre e d
X X 0.42 0.07 0.07
X
-
X * 0.4 2 0.06 0.06
X - 0.40 0 0
h e a te d to 100°C f o r lb m inutes b e fo re u s e .
41
o f two m u tu ally e s s e n tia l com ponents, one h e a t s ta b le and th e
o th e r h e a t l a b i l e , an attem p t was made to e s ta b lis h w hether o r
n o t t h i s system was th e sa m e a s th a t h ere d e s c rib e d . S ince
Gyotoku1s system was a c tiv e a g a in s t t r i b u t y r i n , a stu d y was
made o f th e a c t i v i t y o f th e two f r a c tio n s o f th e c h o le s te r o l-
p a lm ita te -h y d ro ly sin g system , is o la te d by d ia ly s is a g a in s t
w a te r, on a trip r o p io n in s u b s tr a te . These r e s u l t s a re given
in T able X III, and in d ic a te th a t w hile th e a c t i v i t y a g a in s t
c h o le s te ro l p a lm ita te i s shown only by a com bination o f th e
two f r a c tio n s h ere is o la te d , th e trip rto p io n a se a c t i v i t y seemed
to be d is tr ib u te d in b o th f r a c tio n s and appeared r e g a rd le s s
o f w hether th e second component of th e system was p re s e n t.
The assay f o r trip r o p io n a s e a c t i v i t y was c a r r ie d out
sta la g m o m e tric a lly . The c o n tro l tu b e s and th e in c u b a te d
ones were c e n trifu g e d a t 2500 RPM f o r f iv e m in u te s, and th e
c le a r su p e rn a ta n t f l u i d was drawn up in to a f in e - tip p e d
p ip e tte which had a second c a lib r a tio n p o in t n e a r th e t i p .
The mouth o f th e p ip e tte was wiped d ry , and th e n th e liq u id
was allow ed to ru n out dropw ise. The number of drops con
ta in e d w ith in th e c a lib r a te d a re a o f th e p ip e tte was used
as a m easure o f th e r e l a t i v e s u rfa c e te n s io n o f th e liq u id ,
which in t h i s s e r ie s depends on th e amount of tr ip r o p io n in
p r e s e n t. S ince t h i s s e r ie s was ru n a t an a c id pH, i t i s
u n lik e ly th a t p ro p io n ic a c id lib e r a te d would e x e rt much
e f f e c t on th e s u rfa c e te n s io n .
43
TABLE XIII
TRI PROP IONASE ACTIVITY OF THE HYDROLYTIC SYSTEM
Humber o f Drops
Enzyme C o facto r T rip ro p io n in C o n tro l In cu b ated D iffe re n c e
- -
X 27 28 1
X X
• m
21 22 1
X X X 26 23 -3
X -
z 25 22 -3
-
X X 26 23 -3
A ssay: 0.25 m l. enzyme (o r s a lin e )
0*25 ml* c o fa c to r (o r M/5 phosphate)
0*5 m l. l e c i t h i n in w a te r, f i l t e r e d , 10 m g./m l.
1 .0 m l. tr ip r o p io n in in w a te r, s a tu r a te d s o lu tio n .
In c u b a tio n - 22 h o u rs, 37°G*
Enzyme p re p a ra tio n - as in T able XV.
C o facto r p re p a ra tio n - as in T able XV.
43
N ature o f th e C o fa c to r. The chem ical com position o f th e
c o fa c to r which i s re q u ire d f o r t h i s r e a c tio n p re se n te d i t s e l f
as an i n te r e s tin g problem . The f a c t th a t i t i s b o th non-
d ia ly jta b le and so lu b le in s a tu r a te d ammonium s u lf a te would
seem to f i x i t s m o lecu lar w eight between r a th e r narrow
l i m i t s , and th e h eat s t a b i l i t y of th e compound to g e th e r
w ith i t s s o l u b i l i t y in s a tu r a te d ammonium s u lf a te makes i t
u n lik e ly th a t th e re i s any g re a t p ercen tag e o f p r o te in
m a te ria l p r e s e n t, alth o u g h p e p tid e lin k a g e s a re in d ic a te d
by th e p o s itiv e b iu re t r e a c tio n .
The chem ical d a ta in T able XIV were o b ta in e d by
s ta n d a rd p ro ce d u res, u sin g c o fa c to r p re p a ra tio n 813, which
was p re p are d from an e x tr a c t of r a t in t e s t i n e by s a tu r a tio n
w ith ammonium s u lf a t e and subsequent d ia ly s is and l y o p h ili-
z a tio n o f th e s u p e rn a ta n t m a te r ia l.
The s t a b i l i t y o f th e c o fa c to r tow ard a c id and a lk a lin e
h y d ro u s i s was a ls o in v e s tig a te d . From T able XV, i t may be
seen th a t h y d ro ly s is in 5$ aqueous K O H a t 100°C f o r 15 m inutes
d e stro y s th e a c tiv a tin g a b i l i t y o f th e n e u tr a liz e d c o f a c to r.
H y d ro ly sis in 6N HC1 under th e same c o n d itio n s , fo llo w ed by
n e u tr a liz a tio n , does n o t ap p ear to in a c tiv a te th e su b sta n c e .
ROLE o f L e c ith in . The approxim ate com position o f soya
l e c i t h i n i s re p o rte d (6) a s :
44
TABLE XIV
CHEMICAL D ATA O N CO FA CTO R PREPARATION 813
N itro g e n (N e ssle r) 26*2$
B iu re t (Mehl) 23$ o f egg album in
re a d in g
T y ro sin e (F o lin -G io c a lte u x ) 2.1 3 $
C arbohydrate (O rc in o l) 1 .0 $
45
TABLE XV
EFFECTS OF ACID A N D ALKALINE HYDROLYSIS O N COFACTOR
C o facto r
Treatm ent
C h o le s te ro l (m g.}
T o ta l Free
m *
rre e d
u n tre a te d 0.42 0.04 0.04
3$ aqueous K D H
100°C, 15 m in.
0.4 0 0.01 (0 .0 1 )
6NHC1, 100°C,
15 m in.
0.4 2 0.04 0.0 4
S u b s tra te - c h o le s te ro l p a lm ita te c o llo id
p lu s 5 mg* l e c i t h i n
In c u b a tio n - 18 h o u rs, 37°C.
Enzyme p re p a ra tio n - 0*9% NaCl e x tr a c t o f r a t
i n t e s t i n e , e le c tr o d i a l l e d a t 300V f o r f iv e
h o u rs a g a in s t d i s t i l l e d w a te r. P r e c ip ita te
c o lle c te d by c e n trifu g in g and washed once
w ith co ld d i s t i l l e d w a te r. E x tra c te d w ith
0 .9 $ NaCl, e x tr a c t ly o p h iliz e d . Amount
e q u iv a le n t to 0 .5 m l. o r ig in a l e x tr a c t
d iss o lv e d in M/20 s u c c in a te b u f fe r b e fo re use*
C o facto r p re p a ra tio n - am m onium -sulfate-soluble
f r a c tio n o f 0 .9 $ NaCl e x tr a c t o f r a t in t e s t i n e
d ia ly z e d and ly o p h iliz e d . R ed isso lv ed in
w ater b e fo re u s e , 4 m g./m l.
45
l e c i t h i n 20$
c e p h a lin 20
o i l 30
p h y to s te r o l 2
i n o s i t o l 15
c arb o h y d rate 10
C arbohydrate may be ru le d o u t, because an a lc o h o lic
e x tr a c t o f soya l e c i t h i n has th e same a c t i v i t y as th e crude
l e c i t h i n , i n o s i t o l and t r i a c e t i n are no t a b le to s u b s titu te
f o r soya l e c i t h i n , as may be seen in T able XVI* I t i s im
p ro b ab le th a t p h y to s te ro ls have any v i t a l r o le in th e anim al
body, e x p e c ia lly in an enzyme system . I n c id e n ta lly , th e
s t e r o l s p re se n t in soya l e c i t h i n cannot give a f a l s e p o s itiv e
t e s t f o r c h o le s te ro l because th e y a re not p r e c ip ita te d by
d ig ito n in , alth o u g h th e y g iv e th e Lieberm ann-B urchard c o lo r.
T h e refo re i t i s e v id en t th a t l e c i t h i n i t s e l f , o r
c e p h a lin , i s th e a c tiv e component o f soya l e c i t h i n . In an
a ttem p t to e v a lu a te th e r o le in t h i s system , i t was decided
to see w hether l e c i t h i n could be re p la c e d by o th e r compounds
which were s im ila r ch em ically . T h erefo re assa y s were s e t
up u sin g g ly c e ro l m onoecetate and g ly c e ro l d ia c e ta te in s te a d
o f th e u su a l l e c i t h i n . The d ata in T able XVI in d ic a te th a t
th e se two compounds a re in a c tiv e in th e system .
I t sh o u ld n o t be tho u g h t th a t t h i s i s some ex tran eo u s
component which i s b ein g added to th e system , f o r i t i s ob
serv ed th a t th e crude u n d ia ly z ed e x tr a c ts do n o t alw ays r e
q u ire supplem entary l e c i t h i n . { As in T able V I .) I t i s on ly
47
TABLE XVI
EFFECT OF SUBSTITUTIONS FOR LECITHIN
IN THE HYDROLYTIC SYSTEM
na— — — — ■— n a i . i ,a i- t- -•7i itrat ■ i:r''i,'i':rw n a i ii.-anra
S ubstance Added C h o le s te ro l Treed (m g,)
5 mg, l e c i t h i n 0,08
2 mg, m onoacetin
4 mg. d ia c e tin
2 mg. in o s i t o l
4 mg. t r i a c e t i n
S u b s tra te - c h o le s te ro l p a lm ita te c o llo id
In c u b a tio n - 18 h o u rs, 30°C
Enzyme p re p a ra tio n - w a te r-in s o lu b le f r a c tio n o f
e le c tr o d ia ly z e d crude e x t r a c t , d iss o lv e d in
0 ,9 ^ NaCl and ly o p h iliz e d .
C o fa cto r p re p a ra tio n - A m m onium -sulfate-soluble
f r a c tio n o f crude e x t r a c t , d ia lv z e d and ly o
p h iliz e d .
48
a f t e r th e system Has been p u r if ie d by thorough d ia ly s is or
s a l t f r a c tio n a tio n th a t th e e s s e n tia l need f o r l e c i t h i n ap p e ars.
T h e refo re i t seems t h a t th e l e c i t h i n p re s e n t in th e i n t e s t i n a l
w a ll may be s u f f i c i e n t in some cases to allo w maximum a c t i v i t y
of th e h y d ro ly tic system , and on ly when t h i s n a tu r a l supply
i s removed does su p p lem en tatio n become n e c e ssa ry .
CHAPTER V
DISCUSSION
The occu rren ce o f enzyme system s in r a t l i v e r and
in t e s t i n e capable o f b rin g in g about th e s y n th e s is and
h y d ro ly s is o f c h o le s te ro l e s te r s in v i t r o has been demon
s tra te d * I t i s d i f f i c u l t to say w hether th e two system s
h ere d e sc rib e d a re id e n tic a l w ith th o se p re v io u sly men
tio n e d in th e l i t e r a t u r e . The work of K lein (8) in d ic a te s
th a t th e re a re a t l e a s t two s e ts of e s te r a s e s in t i s s u e .
One group has a pH optimum around pH 5 .3 , and b rin g s about
th e h y d ro ly s is of c h o le s te ro l e s te r s . A ccording to h is
work, t h i s group i s l a b i l e tow ard d i a l y s i s , b u t in view
of th e f in d in g s in t h i s stu d y , where th e m a jo rity of
p re p a ra tio n s a ia ly z e d in th e u s u a l manner became in a c tiv a te d ,
t h i s sta te m e n t cannot be accep ted w ith o u t q u a lif ic a tio n .
U sing e l e c t r o d i a i y s i s , where th e d ia ly s is tim e i s cu t to
about one te n th th a t o f th e u s u a l p e rio d , lo s s e s in a c t i v i t y
from t h i s pro ced u re are r a r e in our e x p e rie n c e . T h erefo re
t h i s e s te r a s e system as re p o rte d by K lein may o r may n o t
be th e one d e sc rib e d h e re .
The second group o f K le in ’ s e s te r a s e s , cap ab le of
b rin g in g about th e e s t e r i f i c a t i o n of c h o le s te r o l, has a pH
optimum around 7 .0 , and i s re p re s e n te d by th o se system s
50
p re se n t in serum and p an crea s. The system d e riv e d from tn e
p an creas ap p ears to be a r e v e r s ib le one, capable o f p roceeding
in e ith e r d ir e c tio n under c e r ta in c o n d itio n s . I t i s e v id e n tly
v ery s im ila r to th e n o n s p e c ific e s te r a s e which was s tu d ie d in
such d e t a i l by Sym (2 3 ). S ince aceto n e and ch lo ro fo rm were
used in th e a ssa y p rocedure to s o lu b iliz e th e s u b s tr a te
m a te r ia ls , and sin c e th e s e su b stan ce s have in v a r ia b ly in
a c tiv a te d tn e system s d e sc rib ed in t h i s d i s s e r ta ti o n , th e
i d e n t i t y o f th e p a n c re a tic system w ith them i s ru le d o u t.
S p erry and h is cow orkers re p o rte d e s s e n t i a l l y th e
same two g ro u p in g s of c h o le s te ro l e s te r if y in g system s in a
s e r ie s o f p ap e rs (18, 19, 20, 21, 2 2 ). The e s t e r i f i c a t i o n
o f c h o le s te r o l by serum e x h ib ite d a maximum a c t i v i t y a t pH 8 ,
w hereas th e a c t i v i t y o f th e l i v e r su sp en sio n was s a id to in
c re ase as th e pH dropped below th e i n i t i a l v a lu e o r approx
im a te ly 0 .4 , alth o u g h ex a c t d e te rm in a tio n s o f optimum a c t i v i t y
were no t re p o rte d . T his l a t t e r system i s p ro b ab ly tn e one
w ith w hich work was done in th e p re se n t stu d y .
dyotoku ( 5 ) , w orking on a q u in in e f r a c tio n a tio n of
lip a s e s from blood serum , g a s tr ic m ucosa, and p a n c re a s, found
th a t th e re were two f r a c tio n s which could be o b ta in e d .
These were a c tiv e to g e th e r , but in a c tiv e s e p a ra te ly . One,
w hich ne seemed to re g a rd as an a c t i v a t o r , could be p re
c i p i t a t e d w ith q u in in e . The o th e r, re g a rd e d as th e enzyme,
was so lu b le in th e q u in in e s o lu tio n s employed. Both f r a c tio n s
ol
were round to "be s ta b le r o r 15 m inutes a t 51°C. The pH op
timum r o r canine g a s tr i c mucosa e s te r a s e was round to be 5.8*
I t m ust be borne in m ind, however, th a t th e s e f r a c tio n s ex
h ib ite d a c t i v i t y tow ard t r i b u t y r i n as th e a ssa y s u b s tr a te , a
compound which i s no t as r a p id ly s p l i t by ty p ic a l e s te r a s e s .
T his system does n o t appear to be th e same as th e h y d ro ly tic
system from r a t i n t e s t i n e which was s tu d ie d in tn e p re se n t
work, f o r s e v e ra l re a so n s. F i r s t , th e f r a c tio n s of th e r a t
h y d ro ly tic system se p a ra te d in to w a te r-s o lu b le and w a te r-
in s o lu b le components e x h ib it a c t i v i t y a g a in s t c h o le s te ro l
p a lm ita te on ly when th e y a re recom bined, w hile tr ig ly c e r a s e
a c t i v i t y i s shown by e i t h e r f r a c tio n . F u rth erm o re, a l l of
th e lip a s e a c t i v i t y was observed by Gyotoku to rem ain in
th e ammonium s u lf a t e p r e c i p i ta te r e s u ltin g from 50$ s a tu r a tio n
o f th e s o lu tio n , a f r a c tio n which i s known to c o n ta in only
th e enzyme p o rtio n o f th e h y d ro ly tic system .
The r o le of l e c i t h i n in tn e system i s open to wide
s p e c u la tio n . There a re two p o s s ib le in te r p r e t a t i o n s . F i r s t ,
th e p o s s i b i l i t y e x i s t s th a t th e re may be two r e a c tio n s going
on, as f o r exam ple,
(1) c h o le s te ro l e s te r ------ — —» c h o le s te r o l + f a t t y a c id
{ 2 } l e c i t h i n (o r d e riv a tiv e ) + f a t t y a c id ----- > ? ? ?
However, even i f t h i s i s th e c a se , r e a c tio n I should go tow ard
th e r ig h t to some e x te n t in th e absence o f l e c i t h i n . T h is has
never been observed in p u r if ie d system s w ith o u t l e c i t h i n , but
th e amount o f f r e e c h o le s te ro l l ib e r a te d m ight be to o sm all
52
to be d e te c te d by th e methods used*
The second h y p o th e sis i s th a t l e c i t h i n a c tu a lly ta k e s
p a r t in tn e p rim ary r e a c tio n in s te a d o r a c tin g th ro u g h mass
a c tio n e f f e c ts by rem oving one o f th e prim ary end products*
Thus we m ight have a r e a c tio n such a s :
(3) c h o le s te ro l e s te r + l e c i t h i n (o r d e riv a tiv e ) ------- *
c h o le s te ro l ? ? ?
The d a ta do n o t p erm it o f making a choice betw een th e s e two
p o s s i b i l i t i e s a t t h i s tim e.
C lin ic a l d a ta must a ls o be re c o n c ile d w ith any i n t e r
p r e ta tio n o f th e r o le o f l e c i t h i n in t h i s system . I t has
been found t h a t in cases o f f a t t y l i v e r which have been caused
e i t h e r by fe e d in g o f c h o le s te ro l or by a d e fic ie n c y o f one o f
th e components needed f o r l e c i t h i n fo rm a tio n , namely un
s a tu r a te d f a t t y a c id s , c h o lin e , o r m e th io n in e, c h o le s te ro l
e s te r s accum ulate in th e l i v e r . T h is cannot re p re s e n t p r i
m a rily a d e fic ie n c y in th e tr a n s p o r ta tio n o f c h o le s te r o l,
f o r we would expect to f in d th e f re e form p ilin g up as w e ll
as th e e s t e r . The in d ic a tio n i s th a t e s t e r i f i c a t i o n i s p ro
ceeding f a s t e r th e n h y d ro ly s is . W hether t h i s means th a t th e
d e a rth o f l e c i t h i n i s in h ib itin g th e h y d ro ly tic r e a c tio n or
w hether th e e s t e r i f i c a t i o n i s pushed by sh ee r mass a c tio n
e f f e c ts o f th e accum ulating c h o le s te ro l and f a t t y a c id s i s
im p o ssib le to say. At any r a t e , i t ap p ears to be th e
53
p h o sp h o ry lch o lin e p o rtio n o f tn e l e c i t h i n m olecule which
e x e r ts th e a c t i v i t y , sin c e mono- and d i-g ly c e rid e s a re in
capable o f re p la c in g l e c i t h i n .
The d em o n stratio n o f th e a b i l i t y o f th e sm all i n te s tin e
to e s t e r i f y c h o le s te ro l and th e f a c t t h a t t h i s a c t i v i t y i s
in c re a se d on c h o le s te ro l fee d in g seems to b e a r out th e th e o ry
o f Schoenheim er th a t s te r o l s must be e s t e r i f i e d in th e p ro c e ss
of a b s o rp tio n (1 5 ), However, th e evidence on which t h i s th e o ry
r e s t s i s o ld and should be re e v a lu a te d . The work of M u eller
(12) and o f J to h lic h e r and Sullm ann (4) w ith th o ra c ic duct
f i s t u l a anim als in d ic a te s th a t an a p p re c ia b le amount o f th e
c h o le s te r o l ab so rb ed appears in th e lymph in th e f r e e s t a t e ,
alth o u g h a p o rtio n o f i t i s undo u b ted ly e s t e r i f i e d durin g
p assag e th ro u g h th e i n t e s t i n a l t r a c t and w a ll.
W hile tn e i n t e s t i n a l mucosa may p la y a r o le in th e
m aintenance of th e norm al e s te r le v e l in th e body, i t i s
o b v io u sly no t th e o n ly organ concerned. The l i v e r i s im
p o rta n t f o r t h i s p ro c e ss , b o th on th e b a s is o f th e c l i n i c a l
o b s e rv a tio n s o f d ecreased e s te r le v e ls in l i v e r damage and
on th e b a s is o f th e d a ta p re se n te d h e re . W hether th e
e s te r if y in g system s are p re s e n t in c o n c e n tra tio n in o th e r
tis s u e s of th e body rem ains to be seen , b u t evidence a lre a d y
c ite d from th e l i t e r a t u r e in d ic a te s th a t i t i s p ro b ab le .
A ll o f th e s e t i s s u e s , th e n , must c o n trib u te to th e m aintenance
54
o f th e rem arkably ste a d y r a t i o o f f r e e to e s t e r i f i e d c h o le s
t e r o l in th e b lo o d stream .
CHAPTER VI
S U M M A R Y A N D CONCLUSIONS
A ssay m ethods have been developed f o r th e e s tim a tio n o r
enzyme system s capable o f form ing o r h y d ro ly z in g c h o le s
t e r o l e s te r s .
E x tr a c ts from l i v e r and sm all i n t e s t i n e o f th e r a t have
been found a c tiv e in th e in v i t r o e s t e r i f i c a t i o n o f
c h o le s te r o l. T h is p ro ce ss r e q u ire s th e p resen ce o f th e
phosphate io n and can be c a r rie d ou t w ith f r e e f a t t y
a c id .
Under c e r ta in c o n d itio n s , s im ila r e x tr a c ts can hyd ro ly ze
c h o le s te r o l e s te r s . T h is system does n o t r e q u ire th e
p resen ce o f p h o sp h ate, bu t some component o f soya l e c i
t h in was found to be n e c e ssa ry f o r a c t i v i t y .
The h y d ro ly tic system i s composed o f two f a c to r s . The
f i r s t behaves as a g lo b u lin and i s h e a t l a b i l e .
The second f a c to r i s a c tiv e in ex trem ely sm all am ounts,
i t i s h e a t s ta b le , n o n d ia ly z a b le , and s o lu b le in s a tu
r a te d ammonium s u lf a t e . Some chem ical d a ta a re p re
s e n te d .
l*he r o le o f th e se system s in th e m aintenance o f th e le v e l
o f c h o le s te r o l e s te r s in th e body i s d isc u sse d .
BIBLIOGRAPHY
1. Chaney, A. L ., In p ress#
2. F a lc o n e r, Ja c q u e lin e S . , and T a y lo r, D .3 ., "The I n i t i a l
S tag es in tn e P u r if ic a tio n of P ig L iv e r E s te r a s e ,"
B iochem ical J o u rn a l, 40:831, 1946.
3. F alc o n e r, Ja c q u e lin e S ., and T a y lo r, D. B . , "A S p e c ific
P ro p e rty S o lu b ility T est f o r P ro te in P u r ity , and I t s
A p p lic a tio n to th e P re p a ra tio n o f Pure L iv e r E s te r a s e ,"
B iochem ical J o u rn a l, 40:835, 1945.
4 . F ro h lic h e r, E ., and Sullm ann, H ,, "Die V eresteru n g von
C h o le s te rin b e i d er R eso rp tio n aus dem Darm," B lo-
chem ische Z e i t s c h r i f t 274:21, 1934
5. Gyotoku, K ., T erashim a, S ., "S tu d ien u b er d ie L ip ase.
VI. Die T re n n b a rk e it d er L ipase in zwei unwirksame
B e s ta n d te ile ," Biochem ische Z e i t s c h r i f t 217:306, 1930.
6. K esten , H. D ., and S ilb o w itz , R ., "E xperim ental A thero
s c le r o s is and Soya L e c ith in ," P ro ceed in g s o f th e
S o c ie ty f o r E x p erim en tal B iology and M edicine 49 :7 1 ,
1942
7. K le in , W ., "Uber d ie enzym atische H ydrolyse d er C h o le s te rin -
e s te r des m enschlichen Serum s," Z e i t s c h r i f t filr phy-
s io lo g is c h e Ohemie 254:1, 1938
8 . K le in , W., "Uber d ie enzym atische H ydrolyse d er C h o le s te rin -
e s t e r . I I . , " Z e it s c h r if t f u r p h y sio lo g isc h e Chemie
259:268, 1939
9. Kondo, K ., "Zur K enntnis d er in E s s ig a th e r l$ s lic h e n S to ffe
t i e r i s c h e r Organe und ih re s V erh a lte n s b e i der A u to ly se.
I I . E n h a lt d ie Leber e in C h o le s te rin e s te r sp a lte n d e s
Enzym?," Biochem ische Z e it s c h r if t 26:243, 1910.
10. Marx, W .H., and L ip s e tt, M. B*, U npublished o b s e rv a tio n s .
11. M ic h a e lis, L . , Rona, P ., "Uber E s te r - und F e tts p a ltu n g im
B lu te und im Serum ," Biochem ische Z e it s c h r if t
31:345, 1911.
12. M u e ller, J . H . , "The Mechanism o f C h o le s te ro l A b so rp tio n ,"
J o u rn a l o f B io lo g ic a l C hem istry 27:453, 191b.
13.
14.
lb .
16.
17.
18.
19.
20.
21.
22.
m
M u e lle r, J . t i. , "The In flu e n c e o f A u to ly sis upon C holes
t e r o l E s t e r s ," J o u rn a l o f B io lo g ie a l C hem istry 25:
b o l, 1916.
Schoenheim er, R ., "Cher d ie Bedeutung d er P fla n z e n s te rin e
f 3 r den tie r is e h e n G rganism us, n Z e it s c h r if t fu r
p h y aio lo g iso h e Chemie 18 0 :1 , 1929.
Schoenheim er, R ., "New C o n trib u tio n s in S te r o l M etabolism ,"
S cien ce 74:579, 1931.
S c h u ltz , J . h . , "U ntersuchungen b e tre ffe n d das Yorkommen
e in e s G h o le ste rin sp a lte n d e n Ferm entes in B lu t und
L e b e r," Biochem ische Z e i t s c h r i f t 42:255, 1912.
Shops, R. E ., " C h o le ste ro l E s te ra s e in Animal T is s u e s ,"
J o u rn a l o f B io lo g ic a l C hem istry 80:127, 1928.
S p e rry , W . M ., " C h o le ste ro l E s te ra s e in B lo o d ," J o u rn a l
o f B io lo g ic a l C hem istry l l l : 4 6 v , 193b.
S p e rry , w. M . , "The E ffe c t of T issu e E x tra c ts on E s t e r i -
f ic a t io n o f C h o le s te ro l in Blood Serum ," J o u rn a l or
B io lo g ic a l C hem istry 113:599. 1936.
S p e rry , M ., and Brand, F. c . , "A Study of C h o le s te ro l
E s te ra s e in L iv e r and B ra in ," J o u rn a l o f B io lo g ic a l
C hem istry 137:377, 1941.
S p erry , W . M ., and S to y an o ff, V. A ., "The In flu e n c e o f
B ile S a lts on th e Enzym atic S y n th e sis and H y d ro ly sis
o r c h o le s te r o l E s te rs in a lo o d Serum ," J o u rn a l o f
B io lo g ic a l o h em istry 121:101, 1937.
S p e rry , W. m. , and S to y a n o ff, v. A ., "The Enzym atic S y n th e sis
and H y d ro ly sis of c h o le s te ro l E s te r s in Blood Serum,"
J o u rn a l o f B io lo g ic a l C hem istry 12o:77, 1938.
23. Syrn, E. A ., "A ction of E s te ra se in th e P resen ce o f o rg a n ic
S o lv e n ts ," B iochem ical J o u rn a l 30:609, 1936.
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Creator Nieft, Marie Louise (author)

Core Title Studies on cholesterol esterases

Contributor Digitized by ProQuest (provenance)

School Graduate School

Degree Doctor of Philosophy

Degree Program Biochemistry

Degree Conferral Date 1948-06

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

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c26-385979

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