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Studies on the biosynthesis of glycoprotein in reticulocyte stroma

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Studies on the biosynthesis of glycoprotein in reticulocyte stroma

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Content STUDIES ON THE BIOSYNTHESIS OF
GLYCOPROTEIN IN RETICULOCYTE STROMA
b y
M a rio n T h e r e s a L a ic o
A D i s s e r t a t i o n P r e s e n t e d to t h e
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
I n P a r t i a l F u l f i l l m e n t o f t h e
R e q u i r e m e n t s f o r t h e D e g re e
DOCTOR OF PHILOSOPHY
( B i o c h e m l s t r y )
J u n e 1967
UNIVERSITY O F S O U T H E R N CA LIFO RN IA
T H E G R A D U A TE S C H O O L
U N IV E R S IT Y PA R K
L O S A N G E L E S . C A L IF O R N IA 9 0 0 0 7
This dissertation, written by
...................... MARION„THERESA LAICO..............................
under the direction of h.er.-.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 T O R OF P H I L O S O P H Y
g ................
D ate 8 , 1967............................
DISSERTATION COM MITTEE
.........
Chairman
ACKNOWLEDGMENTS
I w o u ld l i k e t o t h a n k D r. E y l a r , n o t o n l y f o r h i s
c o n t i n u o u s h e l p and e n c o u r a g e m e n t w i t h t h i s p a r t i c u l a r w o rk ,
b u t e s p e c i a l l y f o r t h e i n t e r e s t I n r e s e a r c h he h a s g i v e n to
me by h i s c r e a t i v e a n d s t i m u l a t i n g a p p r o a c h t o e v e r y p r o b le m
t h a t we h a v e e n c o u n t e r e d . F u r t h e r , I am g r a t e f u l f o r h i s
s u p p o r t i n a l l t h e p r o b le m s r e l a t e d t o g r a d u a t e s t u d y t h a t
e x t e n d b e y o n d t h e r e a l m o f t h e l a b o r a t o r y .
I w o u ld a l s o l i k e to t h a n k D o c t o r s W a l t e r Marx and
E a r l M cN all who h a v e a i d e d me g r e a t l y by B e r v in g on my d o c
t o r a l c o m m itte e .
An o f f e r o f t h a n k s i s a l s o g i v e n to Mr. J e s s e J a c k
so n f o r t h e p r e p a r a t i o n s o f g l y c o p r o t e i n r e c e p t o r s an d f o r
t h e h e l p he h a s so o f t e n g i v e n to me, and to D r. G. M a t i o l i
and D r. H. N ie w is c h f o r h e l p w i t h t h e r a b b i t s and m i c r o
s c o p i c e x a m i n a t i o n s .
F i n a l l y , I am g r a t e f u l to my f a m i l y and t o Lucy Ja o
f o r t h e i r c o n t i n u i n g m o r a l s u p p o r t . A s p e c i a l w ord o f
t h a n k s g o e s t o my p a r e n t s , w i t h o u t w hose e n c o u r a g e m e n t and
h e l p t h e s e y e a r s o f g r a d u a t e s t u d y w o u ld n o t h a v e b e e n p o s
s i b l e .
i i
TABLE OF CONTENTS
P age
ACKNOWLEDGMENTS......................................................................................................... i i
L IST OF T A B L E S ......................................................................................................... i v
LIST OF ILLU STRA TIO N S......................................................................................... v i
I . HISTORICAL INTRODUCTION ..................................................................... 1
D e f i n i t i o n o f a G l y c o p r o t e i n
B i o s y n t h e s i s o f N o n - c o n j u g a t e d P r o t e i n s
S t r u c t u r a l R e q u i r e m e n t s f o r G l y c o p r o t e i n
B i o s y n t h e s i s
S t u d i e s on G l y c o p r o t e i n B i o s y n t h e s i s
I I . STATEMENT OF PROBLEM AND EXPERIMENTAL PLAN . . . 16
I I I . MATERIALS AND METHODS........................................................................ 18
M a t e r i a l s
M e th o d s
IV. R E S U L T S .............................................................................................................. 35
W hole C e l l S t u d i e s
C e l l F r e e E x p e r i m e n t s
R e l e a s e o f P r o d u c t s fro m S tro m a
b y P r o n a s e
F r a c t i o n a t i o n o f S tro m a a n d S o l u b i l i
z a t i o n o f T r a n s f e r a s e
V. DISCUSSIONS AND CONCLUSIONS ........................................................ 80
V I. SUMMARY.............................................................................................................. 90
A P P E N D I X ......................................................................................................................... 93
LITERATURE C I T E D ................................................................................................... 98
i l l
LIST OF TABLES
T a b le P age
1. The C e l l -Free B i o s y n t h e s i s o f R a b b i t
R e t i c u l o c y t e Membrane G l y c o p r o t e i n ............................ 44
14
2 . The C e l l F r e e I n c o r p o r a t i o n o f S e r i n e - C
i n t o P r o t e i n o f R a b b i t R e t i c u l o c y t e s ........................ 45
3. U D P -G a la c to s e I n c o r p o r a t i o n i n t o G ly c o
p r o t e i n i n a C e l l F r e e S y s te m o f
R a b b i t R e t i c u l o c y t e s .................................................................. 48
4 . T r a n s f e r o f G a l a c t o s e fro m U D P -G a la c to s e
t o E x o g e n o u s R e c e p t o r s ................................................................ 56
5 . S o l u b i l i z a t i o n o f S t r o m a l C o m p o n en ts:
E f f e c t s on T r a n s f e r a s e A c t i v i t i e s ........................... 58
6 . U D P -G a la c to s e T r a n s f e r a s e A c t i v i t y o f
T r it o n - X - 1 0 0 T r e a t e d S t r o m a l F r a c t i o n s . . . . 60
7 . R e l e a s e o f U D P -G a la c to s e T r a n s f e r a s e
A c t i v i t y fro m S tro m a by T r i t o n - X - 1 0 0 ...................... 6 l
8 . P u r i f i c a t i o n o f U D P -G a la c to s e T r a n s f e r a s e
A c t i v i t y ..................................................................................... 62
9 . S e p a r a t i o n o f E n d o g en o u s R e c e p t o r o f G a l a c
t o s e fro m t h e U D P -G a la c to s e T r a n s f e r a s e . . . 66
10. T r a n s f e r o f G a l a c t o s e fro m U D P -G a la c to s e
t o S t r o m a l R e c e p t o r I s o l a t e d
a f t e r P h e n o l E x t r a c t i o n ....................................................... 67
11. S t a b i l i t y o f t h e U D P -G a la c to s e T r a n s f e r a s e
i n V a r i o u s S t a t e s .......................................................... 69
14
12. I n c o r p o r a t i o n w i t h Time o f U D P -G a la c to s e -C
i n t o G l y c o p r o t e i n R e c e p t o r ................................................. 74
i v
LIST OP ILLUSTRATIONS
F i g u r e P age
1. I n c o r p o r a t i o n o f L a b e l l e d S u g a r s a n d S e r i n e
i n t o S t r o m a l P r o t e i n ............................................................. 36
lii
2 . I n c o r p o r a t i o n o f S e r i n e - C a n d G l u c o s a m i n e -
C^4 i n t o S t r o m a l L i p i d s .................................................. 37
14
3 . I n c o r p o r a t i o n o f G lu c o s a m in e - C a n d S e r i n e -
C i n t o U nw ashed R ib o s o m a l P e l l e t s . . . . 38
4 . S u c r o s e G r a d i e n t A n a l y s i s o f L a b e l l e d
R i b o s o m e s ......................................................................................... 40
14
5 . I n c o r p o r a t i o n o f G lu c o s a m in e - C a n d
S e r i n e - C l ^ i n t o R ib o s o m a l S u p e r
n a t a n t P r o t e i n s ........................................................................ 4 l
6 . E l u t i o n o f L a b e l l e d G l y c o p e p t i d e s fro m
S e p h a d e x - G - 2 5 a f t e r L a b e l l i n g
i n I n t a c t C e l l s ........................................................................ 43
14
7 . I n c o r p o r a t i o n o f G a l a c t o s e - C fro m UDP-
G a l a c t o s e - C 1^ i n t o S tro m a i n a
C e l l F r e e S y B t e m ............................................................... 49
8 . S u g a r C o n c e n t r a t i o n E f f e c t on G a l a c t o s e
T r a n s f e r t o t h e S t r o m a l R e c e p t o r ............................ 51
9 . M e ta l I o n C o n c e n t r a t i o n E f f e c t on t h e
T r a n s f e r o f G a l a c t o s e t o S t r o m a l P r o t e i n s . . 52
1 0 . M e ta l I o n C o n c e n t r a t i o n E f f e c t on t h e
T r a n s f e r o f G a l a c t o s e t o S t r o m a l L i p i d s . . 53
11. E l u t i o n o f L a b e l l e d G l y c o p e p t i d e s fro m
S e p h a d e x - G - 2 5 a f t e r L a b e l l i n g i n a
C e l l F r e e S y s t e m ............................................................... 54
12 . E l u t i o n o f U D P -G a la c to s e T r a n s f e r a s e
fro m a S e p h a d e x -G -1 0 0 C o l u m n .............................. 63
1 3 . M e ta l I o n C o n c e n t r a t i o n E f f e c t on
P u r i f i e d U D P -G a la c to s e T r a n s f e r a s e ...................... 70
14 . E f f e c t o f Enzyme C o n c e n t r a t i o n on G a l a c
t o s e T r a n s f e r t o a ^ - G l y c o p r o t e l n ............................ 72
v
F ig u r e P age
15. T e m p e r a tu r e E f f e c t s on G a l a c t o s e
T r a n s f e r t o a ^ - G l y c o p r o t e i n ...................................... 73
1 6 . C o n c e n t r a t i o n E f f e c t o f c x ^ - G ly c o p r o te ln
T h e o r e t i c a l R e c e p t o r S i t e s on t h e
T r a n s f e r o f G a l a c t o s e - C 1^ fro m UDP-
G a l a c t o s e - C ^ a t V a r i o u s T i m e s ................................. 75
17. C a l c u l a t i o n o f an A p p ro x im a te Km V a lu e
f o r a i - G l y c o p r o t e i n T h e o r e t i c a l
R e c e p t o r S i t e s ............................................................................ 77
1 a
18. C o n c e n t r a t i o n E f f e c t o f U D P -G a la c to s e -C
on t h e T r a n s f e r o f G a l a c t o s e - C ^ ^ to t h e
a ^ - G l y c o p r o t e i n R e c e p t o r a t V a r i o u s
T i m e s .................................................................................................. 78
19. C a l c u l a t i o n o f an A p p ro x im a te Km V a lu e
f o r U D P - G a l a c t o s e - C ^ ....................................................... 79
20 . Summary o f S t r o m a l R e a c t i o n s .............................................. 88
v i
LIST OF ABBREVIATIONS
ATP — A d e n o s in e t r i p h o s p h a t e
AMP — A d e n o s in e m o n o p h o s p h a te
CTP — C y t i d i n e t r i p h o s p h a t e
CMP — C y t i d i n e m o n o p h o s p h a te
OTP — G u a n o s in e t r i p h o s p h a t e
UTP — U r i d i n e t r i p h o s p h a t e
GSH — G l u t a t h i o n e
TCA — T r i c h l o r a c e t i c a c i d
T r i s — T r l s ( h y d r o x y m e t h y l) a m in o m e th a n e
TKM b u f f e r — T r i s HC1 b u f f e r , 0 . 1M, pH 7 . 2 - 7 . ^ ,
0.05M KCl, 0.002M MgCl2
DNA — D e s o x y r i b o n u c l e i c a c i d
RNA — R i b o n u c l e i c a c i d
sRNA — S o l u b l e r i b o n u c l e i c a c i d
mRNA — M e s s e n g e r r i b o n u c l e i c a c i d
v i i
I . HISTORICAL INTRODUCTION
C l e a r c u t d i s t i n c t i o n s b e tw e e n v a r i o u s t y p e s o f
c a r b o h y d r a t e - p r o t e i n c o m p le x e s h a v e b e e n e s t a b l i s h e d by
s t r u c t u r a l a n a l y s e s ; among t h e s e a r e t h e g l y c o p r o t e i n s .
G l y c o p r o t e i n s c a n be d e f i n e d a s ( 1 , 2 , 3 ) c o n j u g a t e d p r o t e i n s ,
c o n t a i n i n g a s p r o s t h e t i c g r o u p s , o n e o r m ore h e t e r o s a c c h a
r i d e s w i t h a r e l a t i v e l y low nu m b er o f s u g a r r e s i d u e s .
T h e se u n i t s a r e b o u n d c o v a l e n t l y t o t h e p o l y p e p t i d e c h a i n ,
l a c k a s e r i a l l y r e p e a t i n g s e q u e n c e , a n d do n o t c o n t a i n s u l
f a t e g r o u p s o r u r o n i c a c i d s .
The i n t r a c e l l u l a r s i t e an d b a s i c m e c h a n ism s r e s p o n
s i b l e f o r t h e b i o s y n t h e s i s o f t h e n o n - c o n j u g a t e d p r o t e i n
m o l e c u l e h a v e b e e n e s t a b l i s h e d b y s e v e r a l i n v e s t i g a t o r s .
U s in g t h i s k n o w le d g e a s a fra m e o f r e f e r e n c e , i t i s p o s
s i b l e to f o r m u l a t e s p e c i f i c q u e s t i o n s r e l e v a n t t o g l y c o p r o
t e i n s ( 4 ) . At w h a t c e l l u l a r s i t e o r s i t e s i s t h e c a r b o h y -
d r a t e - p r o t e i n b o n d fo rm e d , a n d t h e c a r b o h y d r a t e a s s e m b le d ?
A t w h a t l e v e l o f p r o t e i n s y n t h e s i s i s t h e c a r b o h y d r a t e -
p r o t e i n b o n d fo rm e d ? I s t h e c a r b o h y d r a t e a d d e d o ne u n i t a t
a t i m e , o r a s h e t e r o s a c c h a r i d e u n i t s , o r b o t h ? I n t h e l a s t
few y e a r s , a t t e n t i o n h a s b e e n f o c u s e d on a n s w e r i n g t h e s e
a n d o t h e r b a s i c q u e s t i o n s c o n c e r n i n g t h e m e c h a n ism o f s y n
t h e s i s o f g l y c o p r o t e i n s .
2
The b i o s y n t h e s i s o f n o n - c o n J u g a t e d p r o t e i n s , c l a r
i f i e d i n t h e l a s t t e n y e a r s , h a s b e e n r e v i e w e d b y many
a u t h o r s ( f o r r e c e n t r e v i e w s s e e 5, 6 , 7 , 8 ) . B r i e f l y , i n
d i v i d u a l am ino a c i d s a r e a c t i v a t e d i n t h e p r e s e n c e o f Mg++
by r e a c t i o n w i t h ATP. T h is r e a c t i o n , m e d i a t e d b y s p e c i f i c
B o lu b le e n zy m es, c r e a t e s an am ino acid-AM P c o m p le x . The
com plex t h e n r e a c t s w i t h a s p e c i f i c B o lu b le RNA (sRNA),
l i n k i n g t h e am ino a c i d t o t h e r i b o B e o f th e t e r m i n a l b a s e
o f t h e RNA. T h is a m i n o - a c y l RNA i s t h e n l i n e d up a t s p e
c i f i c p o s i t i o n s on t h e r ib o s o m e b o u n d m e s s e n g e r RNA, t r a n s
c r i b e d p r e v i o u s l y fro m t h e g e n e t i c m a t e r i a l , DNA. B e g i n
n i n g a t t h e am ino t e r m i n a l e n d , th e p r o t e i n 1 b b u i l t u p on
t h e r i b o s o m e . E ach s u c c e e d i n g am ino g r o u p o f t h e a m in o -
a c y l - s o l u b l e RNA r e a c t s w i t h t h e COOH o f t h e p r e c e d i n g p e p -
t i d y l sRNA, fo rm in g a new bo nd an d r e l e a s i n g t h e p e p t i d e
b o und sRNA. A t l e a s t two e n z y m e s, GTP and GSH a r e r e
q u i r e d . F i n a l l y , t h e c o m p le te d p r o t e i n i s r e l e a s e d fro m
t h e r i b o s o m e . T h u s, p r o t e i n s y n t h e s i s r e q u i r e s b o t h s o l u b l e
and p a r t i c u l a t e c e l l f r a c t i o n s , i s g e n e t i c a l l y c o n t r o l l e d
b y a t r a n s c r i b e d t e m p l a t e m ec h an ism , and r e q u i r e s e n e r g y
f o r a c t i v a t i o n o f t h e am ino a c i d s . I n t h e p o l y m e r i z a t i o n
p r o c e s s , o n l y one t y p e o f b o n d , t h e s e c o n d a r y am ide b o n d ,
i s fo rm e d , and b r a n c h e d c h a i n s and o t h e r l i n k a g e s a r e n o t
fo u n d i n p r o t e i n p r i m a r y s t r u c t u r e .
T h e re i s no e x i s t i n g d a t a show ing t h e s p e c i f i c b i o
s y n t h e s i s o f t h e p r o t e i n m o ie ty o f a d e f i n e d g l y c o p r o t e i n .
T h e re i s en o u g h i n d i r e c t e v i d e n c e h o w e v e r, b o t h fro m s t u d i e s
on p la s m a g l y c o p r o t e i n s y n t h e s i s and i t s i n h i b i t i o n by a n t i
b i o t i c s ( 9 , 1 0 , 1 1 , 1 2 ) an d fro m s t u d i e s on t h e s y n t h e s i s o f
t h e c a r b o h y d r a t e p o r t i o n o f g l y c o p r o t e i n s , o u t l i n e d b e lo w ,
t o i n d i c a t e t h a t t h e p r o t e i n m o ie t y i s s y n t h e s i z e d i n th e
same m an n e r a s n o n - c o n j u g a t e d p r o t e i n m o l e c u l e s . The q u e s
t i o n o f when an d how t h e c a r b o h y d r a t e i s a d d e d p r e s e n t s
new p r o b l e m s . S t r u c t u r a l a n a l y s e s show , t h a t i n c o n t r a s t
t o n o n - c o n j u g a t e d p r o t e i n s , g l y c o p r o t e i n s y n t h e s i s r e q u i r e s
t h e f o r m a t i o n o f m ore t h a n o ne t y p e o f c h e m ic a l l i n k a g e .
Bonds a r e fo rm ed b e tw e e n am ino a c i d s and c a r b o h y d r a t e s , a s
w e l l a s b e tw e e n d i f f e r e n t c a r b o h y d r a t e s ( 1 3 )*
The p r o t e l n - c a r b o h y d r a t e l i n k i n v o l v e s a t l e a s t
two a n d v e r y p o s s i b l y t h r e e d i f f e r e n t t y p e s o f b o n d s . The
f i r s t , shown to e x i s t i n egg a lb u m i n , a n d p r o b a b l y a l s o i n
7 - g l o b u l i n , o v o m u co id , f e t u i n , o ro s o m u c o id , an d i n p o r c i n e
b l o o d g ro u p A s u b s t a n c e , i n v o l v e s an N - a c y l g l y c o s y l a m i n e
l i n k a g e to t h e am ide n i t r o g e n o f a s p a r a g i n e . The s e c o n d ,
shown t o e x i s t i n a p - a m y l a s e an d i n o v i n e and b o v i n e s u b -
m a x i l l a r y m u c in , i n v o l v e s t h e h y d r o x y l g r o u p o f s e r i n e o r
t h r e o n i n e i n an O - g l y c o s i d i c l i n k a g e to a s u g a r r e s i d u e . A
t h i r d p o s s i b i l i t y , s u g g e s t e d f o r s u b m a x i l l a r y g l y c o p r o t e i n s
o f s h e e p an d o x , i n v o l v e s th e r e d u c i n g en d o f t h e B ug ar,
j o i n e d i n g l y c o s i d i c e B t e r l i n k a g e t o p o r 7 COOH g r o u p s o f
a s p a r t i c o r g l u t a m i c a c i d r e s p e c t i v e l y .
4
L in k a g e s b e tw e e n t h e c a r b o h y d r a t e m o i e t i e s a r e n o n -
u n i f o r m g l y c o s i d i c b o n d s , a n d t h e o l i g o s a c c h a r i d e r e s i d u e s
may b e e i t h e r l i n e a r o r b r a n c h e d . F u r t h e r , t h e c a r b o h y
d r a t e may h a v e v a r y i n g s e q u e n c e s a n d c h a i n l e n g t h s , and may
be a t t a c h e d t o t h e p r o t e i n a t one o r s e v e r a l p o i n t s .
S t u d i e s on t h e s i t e an d m ethod o f g l y c o p r o t e i n b i o
s y n t h e s i s h a v e b e e n a p p r o a c h e d b o t h b i o c h e m i c a l l y and w i t h
t h e e l e c t r o n m i c r o s c o p e . E a r l y s t u d i e s r e v e a l e d t h e l i v e r
t o b e t h e s i t e o f s y n t h e s i s o f t h e p la s m a g l y c o p r o t e i n s .
G r a d u a l l y t h e s e s t u d i e s h a v e e v o l v e d i n t o a B tudy o f t h e
s u b c e l l u l a r s i t e s and m o l e c u l a r m e c h an ism s r e s p o n s i b l e f o r
t h e s y n t h e s i B o f g l y c o p r o t e i n s i n g e n e r a l . The b i o c h e m i c a l
s t u d i e s h a v e made u s e o f i s o t o p i c s u g a r s and s u g a r n u c l e o
t i d e s , a n d w ere c a r r i e d o u t by o b s e r v i n g t h e i n c o r p o r a t i o n
o f h e x o s e s a n d h e x o s a m in e s i n t o t h e g l y c o p r o t e i n o f v a r i o u s
s u b c e l l u l a r s i t e s .
W ith i s o l a t e d p e r f u s e d r a t l i v e r , S a r c i o n e ( l 4 , 1 5 ) ,
l4
s t u d i e d t h e i n c o r p o r a t i o n o f g l u c o s e - C i n t o t h e g a l a c t o s e ,
m annose an d g l u c o s a m in e r e s i d u e s o f t h e g l y c o p r o t e i n f r a c
t i o n s . W h ile g a l a c t o s e an d m annose i n c o r p o r a t i o n was r a p i d
and e q u a l , t h e g l u c o s a m in e i n c o r p o r a t i o n was much s l o w e r .
The r e s u l t s w ere i n a g re e m e n t w i t h t h o s e o f B o s tru m e t a l .
( l 6 ) and Kohn e t a l . ( 1 7 ) . Richm ond (18 ) fo u n d t h a t l a b e l
l e d g lu c o s a m in e was i n c o r p o r a t e d f a s t e r i n t o p la s m a g l y c o
p r o t e i n s t h a n t h e am ino a c i d s , an d i n a g re e m e n t w i t h Kohn,
t h a t g lu c o s a m in e was a b e t t e r p r e c u r s o r t h a n N - a c e t y l g l u c o B -
5
am ine. Macbeth e t a l . (1 9 ) c o n c lu d e d , t h a t B in ce serum
14
p r o t e i n - b o u n d N - a c e t y l n e u r a m i n i c a c i d - C a n d g l u c o s a m i n e -
14 14
C , a s p r o d u c t s o f i n j e c t e d g l u c o s a m i n e - l - C p e a k e d a t
t h e same p o i n t s , t h e r e was no tim e l a g i n t h e tim e b e tw e e n
t h e i n c o r p o r a t i o n o f e a c h i n t o t h e m a c r o m o l e c u l e s .
Richm ond (2 0 ) co m p a red t h e t u r n o v e r o f p la s m a g l y
c o p r o t e i n s w i t h a lb u m in i n i n t a c t r a t s and i n t h e i s o l a t e d
p e r f u s e d l i v e r s . He was t h e f i r s t t o show t h a t w h i l e am ino
a c i d i n c o r p o r a t i o n was s u b s t a n t i a l l y i n h i b i t e d b y p u ro m y c in ,
c a r b o h y d r a t e i n c o r p o r a t i o n , u n d e r t h e same c o n d i t i o n s , was
n o t . S im k in a n d J a m ie s o n (2 1 ) d e m o n s t r a t e d t h a t L u b r o l e x
t r a c t s o f m ic r o s o m a l f r a c t i o n s o f l i v e r r e a c t e d w i t h a n t i
se ru m t o g u i n e a p i g a c i d g l y c o p r o t e i n s . T h y r o id p a r t i c u l a t e
14
f r a c t i o n s i n c o r p o r a t e d g l u c o s e - C i n t o p a r t i c l e - b o u n d
t h y r o g l o b u l i n p r e c u r s o r ( 2 2 ) . The bound l a b e l c o u l d b e r e
l e a s e d w i t h d e s o x y c h o l a t e .
14
The i n c o r p o r a t i o n o f g a l a c t o s e - C i n t o s u b c e l l u l a r
f r a c t i o n s was i n v e s t i g a t e d by S a r c i o n e ( 2 3 ) , s i n c e g a l a c
t o s e i s i n g l y c o p r o t e i n s b u t n o t m u c o p o l y s a c c h a r i d e s . He
u s e d d e s o x y c h o l a t e t o s o l u b i l i z e t h e m ic r o s o m a l m em branes
a n d t h u s d i s t i n g u i s h b e tw e e n r i b o s o m a l and m ic r o s o m a l i n
c o r p o r a t i o n . The r e s u l t s i n d i c a t e d t h a t t h e s u b c e l l u l a r
s i t e o f i n c o r p o r a t i o n o f t h e g a l a c t o s e was t h e m em branous
p o r t i o n o f t h e r o u g h e n d o p la s m ic r e t i c u l u m , r a t h e r t h a n
t h e r i b o s o m e s . T h i s d a t a t h e n , d o e s n o t s u p p o r t a m ec h an -
6
is m I n v o l v i n g t h e i n c o r p o r a t i o n o f a s u g a r i n t o p r o t e i n a t
t h e r i b o s o m a l l e v e l *
I t h a d b e e n shown t h a t g l u c o s a m in e was i n v o l v e d i n
t h e c a r b o h y d r a t e - p r o t e i n l i n k a g e o f many g l y c o p r o t e i n s
( 2 4 * 2 5 ,2 6 ) and t h a t g a l a c t o s e o f t e n o c c u p i e d a p e n u l t i m a t e
p o s i t i o n ( 2 7 ) . S a r c i o n e e t a l . (2 8 * 2 9 ) r e c o g n i z i n g t h a t
t h e h e x o s a m in e i n c o r p o r a t i o n m ec h an ism may d i f f e r fro m t h e
h e x o s e m echanism * s t u d i e d t h e i n c o r p o r a t i o n o f g l u c o s a m i n e -
14
C i n t o r a t l i v e r s u b c e l l u l a r p a r t i c l e s . The r e s u l t s p a r
a l l e l e d t h o s e o b t a i n e d f o r g a l a c t o s e * s i n c e t h e g l u c o s a m in e
waB i n c o r p o r a t e d i n t o t h e m em branous p a r t o f t h e r o u g h e n d o
p l a s m i c r e t i c u l u m . S h e t l a r * C apps a n d H ern ( 3 0 ) f o l l o w e d
14
t h e i n v iv o i n c o r p o r a t i o n o f g l u c o s a m i n e - l - C I n t o seru m
p r o t e i n s o f r a t s and r a b b i t s . The 22$ I n c o r p o r a t e d a s N-
a c e t y l n e u r a m i n i c a c i d e x h i b i t e d a go o d p r e c u r s o r r e l a t i o n
sh ip * w h i l e t h e 7 8$ I n c o r p o r a t e d a s g l u c o s a m in e d i d n o t .
M olnar* R o b in s o n a n d W I n z l e r ( 3 1 )* s t u d y i n g t h e
14
i n v iv o i n c o r p o r a t i o n o f g lu c o s a m in e - C i n t o r a t l i v e r s *
f o l l o w e d i t s i n c o r p o r a t i o n i n t o a c i d s o l u b l e an d a c i d i n
s o l u b l e p r o d u c t s . They fo u n d no I n c o r p o r a t i o n i n t o a c i d
s o l u b l e o l i g o s a c c h a r i d e s . They o b t a i n e d 7 1 - 9 5 $ I n h i b i t i o n
14
w i t h pu ro m y cin * i n d i c a t i n g t h a t g l u c o s a m i n e - l - C I n c o r p o r
a t i o n was d e p e n d e n t on de novo p r o t e i n s y n t h e s i s . U s in g
E h r l i c h a s c i t e s tu m o r c e l l s h o w e v e r, M olnar* L u te s and
W in z le r ( 3 2 ) showed t h a t p u ro m y c in i n h i b i t e d t h e i n c o r p o r a
t i o n o f g l u c o s a m in e - C " ^ i n t o p r o t e i n o n l y 12-20$* and t h a t
r
b y p r e i n c u b a t i o n t h i s i n h i b i t i o n c o u ld b e i n c r e a s e d to 50$ .
W h ile f a i l u r e t o f i n d l a b e l l i n g o f o l i g o s a c c h a r i d e s d o e s
n o t r u l e o u t t h e i r i n c o r p o r a t i o n , a s s u c h , i n t o p r o t e i n , i t
a t l e a s t s u g g e s t s t h a t t h e s u g a r s may b e a d d e d one a t a
t i m e . R ichm ond (3 3 )* s t u d y i n g t h e r o l e o f s u g a r n u c l e o
t i d e s i n t h e i n c o r p o r a t i o n o f su g a rB i n t o g l y c o p r o t e i n s , was
a l s o u n a b l e t o i s o l a t e o r d e t e c t any n u c l e o t i d e o l i g o s a c
c h a r i d e s .
I n o t h e r s t u d i e B , i t was c o n c l u d e d t h a t i n c o r p o r a
t i o n o f g l u c o s a m i n e , t a k i n g p l a c e b e f o r e r e l e a s e o f t h e
p r o t e i n from t h e l i v e r p a r t i c u l a t e f r a c t i o n , a c t u a l l y d o e s
t a k e p l a c e i n p a r t on t h e r i b o s o m e s (3 ^ * 3 5 )* H ow ever, t h e
i n c o r p o r a t i o n o b t a i n e d w i t h t h e r i b o s o m e s was e x t r e m e l y
lo w , and i t i s d i f f i c u l t t o r u l e o u t c o n t a m i n a t i o n . I n
c o r p o r a t i o n d i d n o t f i t t h e t h e o r e t i c a l c u r v e c a l c u l a t e d
f o r a p r e c u r s o r - p r o d u c t r e l a t i o n s h i p . The t o t a l p r o t e i n -
bo und r a d i o a c t i v e s i a l i c a c i d , a r i s i n g i n v iv o fro m t h e
14
g l u c o s a m i n e - 0 i n c r e a s e d p r o g r e s s i v e l y fro m r ib o s o m e s to
ro u g h e n d o p la s m ic r e t i c u l u m , t o sm ooth e n d o p la s m ic r e t i c u
lum , t o p la s m a m em brane. The r e s u l t s w ere i n t e r p r e t e d t o
mean t h a t t h e g lu c o s a m in e was a d d e d p r o g r e s s i v e l y , b e g i n
n in g a t t h e r i b o s o m e s , and c o n t i n u i n g i n t h e e n d o p la s m ic
r e t i c u l u m , and t h a t t h e m em branes a r e m ore a c t i v e i n s i a l i c
a c i d i n c o r p o r a t i n g a c t i v i t y t h a n t h e r i b o s o m e s .
H e l g e l a n d 's ( 3 6 ) r e s u l t s fro m i n v i v o i n c o r p o r a t i o n
o f g l u c o s a m i n e - C 1^ i n t o r a t l i v e r f r a c t i o n s , showed 3$ i n
8
r i b o s o m e s , 28$ i n t h e d e s o x y c h o l a t e e x t r a c t , a n d 6 9 $ i n t h e
u l t r a s o n i c e x t r a c t , an d l e d him to s p e c u l a t e t h a t t h e mem
b r a n e s o f t h e m ic ro so m e s may b e t h e s i t e o f g l y c o p r o t e i n
s y n t h e s i s .
S i n o h a r a a n d S k y -P e c k (37) a s s a y e d s u b c e l l u l a r
m ouse l i v e r f r a c t i o n s f o r t h e i r a b i l i t y t o i n c o r p o r a t e
14
g l u c o s a m i n e - l - C . The d e s o x y c h o l a t e s o l u b l e p o r t i o n o f
t h e m ic r o s o m a l p r e c i p i t a t e was t h e m o st a c t i v e , th o u g h t h e
m i t o c h o n d r i a l f r a c t i o n a l s o h a d c o n s i d e r a b l e a c t i v i t y .
S o l u b l e RNA f r a c t i o n s w e re i s o l a t e d fro m t h e s o l u b l e p o r
t i o n o f t h e c y t o p l a s m , and fro m t h e r i b o s o m e s . N e i t h e r
p r e p a r a t i o n o f sRNA h a d i n c o r p o r a t e d l a b e l , t h u s r u l i n g o u t
a sugar-sR N A p r e c u r s o r i n t h e s y n t h e s i s o f g l y c o p r o t e i n .
I n w h o le c e l l i n c o r p o r a t i o n s t u d i e s w i t h E h r l i c h
a s c i t e s tu m o r c e l l s ( 3 8 ) , i t was f o u n d t h a t t h e m ic r o s o m a l
membrane f r a c t i o n a p p e a r e d to be t h e s i t e o f g l u c o s a m in e
i n c o r p o r a t i o n . R ib o s o m e s , s e p a r a t e d on a g r a d i e n t , w ere
n o t l a b e l l e d . P r o t e i n s y n t h e s i s c o u l d be c o m p l e t e l y i n
h i b i t e d w h i l e i n c o r p o r a t i o n o f s u g a r i n t o g l y c o p r o t e i n was
i n h i b i t e d a t m ost by a b o u t 30$* i n d i c a t i n g a p o o l o f p r e
fo rm e d p r o t e i n . I n h i b i t i o n o f p r o t e i n s y n t h e s i s f o r l o n g e r
t i m e s , h o w e v e r, g a v e c o m p le te i n h i b i t i o n o f s u g a r i n c o r p o r
a t i o n , i n d i c a t i n g t h a t , a t l o n g e r t i m e s , w i t h p u ro m y c in
b l o c k i n g de novo p r o t e i n s y n t h e s i s , t h e p o o l b ecom es e x
h a u s t e d .
9
H a l l i n a n ( 3 9 ) j i n i n v iv o e x p e r i m e n t s , f o l l o w e d t h e
l 4
i n c o r p o r a t i o n o f g l u c o s a m l n e - l - C i n t o t h e g l y c o p r o t e i n o f
r a t s u b c e l l u l a r f r a c t i o n s , and c o n c l u d e d t h a t t h e sm ooth
e n d o p la s m ic r e t i c u l u m w as t h e m o st i m p o r t a n t , th o u g h n o t
e x c l u s i v e , s i t e f o r i n c o r p o r a t i o n . He n o t e d t h a t t h e ro u g h
e n d o p la s m ic r e t i c u l u m c o u l d b e t h e i m p o r t a n t s i t e , b u t
t h e r e w o u ld h a v e to e x i s t a t t h a t s i t e a p o o l o f p r e c u r s o r s ,
14
t o w h ic h g l u c o s a m in e - C h a d d e l a y e d a c c e s s .
More r e c e n t l y , L aw fo rd and S c h a c t e r (4 0) h a v e i s o
l a t e d l a b e l l e d r i b o s o m e s fro m r a t l i v e r a f t e r i n j e c t i o n o f
l 4
g l u c o s a m i n e - l - C . F u r t h e r , i f c e l l h o m o g e n a te s , fro m
w h ic h t h e s e l a b e l l e d r i b o s o m e s c o u ld b e i s o l a t e d , w ere i n
c u b a t e d w i t h p u r o m y c in , 1 2 - 50# o f t h e l a b e l c o u l d b e r e
l e a s e d . From t h e s p e c i f i c a c t i v i t y o f t h e g l u c o s a m in e h y
d r o l y z e d o f f t h e i s o l a t e d r i b o s o m e s , a n d fro m t h e p u ro m y c in
14
r e s u l t s , i t was c o n c l u d e d t h a t g l u c o s a m i n e - l - C i n c o r p o r a
t i o n b e g i n s w h i l e t h e p r o t e i n m o ie ty i s s t i l l on t h e r i b o
so m es. H ow ever, d e t e r m i n a t i o n s w ere p e r f o r m e d a t t h e l e v e l
o f d e t e c t i o n , m ak ing t h e s p e c i f i c a c t i v i t y d a t a u n r e l i a b l e .
The p u ro m y c in r e s u l t s c o u ld a l t e r n a t i v e l y b e i n t e r p r e t e d a s
i n v i t r o p r o t e i n B y n t h e s i s d u r i n g h o m o g e n a te i n c u b a t i o n , o r
b i n d i n g o f f r e e g l u c o s a m in e to r i b o s o m e - b o u n d g l y c o p r o t e i n
p r e c u r s o r s .
B o u c h i l l o u x and C h e f t e l ( 4 l ) h a v e r e c e n t l y r e p o r t e d
r e s u l t s i n a g r e e m e n t w i t h t h o s e o f Cook e t a l . U s in g i n c u
b a t e d t h r y o i d s l i c e s , i t waB fo u n d t h a t w h i l e r i b o s o m e s d i d
10
h a v e l a b e l l e d s u g a r a s s o c i a t e d w i t h th em , t h e l a b e l was r e
moved w i t h s u c c e s s i v e a c i d w a s h e s . They s u g g e s t e d t h a t t h e
com pound re m o v e d w as p r o b a b l y a s u g a r n u c l e o t i d e . F u r t h e r ,
i n two h o u r i n c u b a t i o n s t u d i e s , t h e y w e re a b l e t o i n h i b i t
p r o t e i n s y n t h e s i s by 9 8$ w i t h p u r o m y c i n , w h i l e c a r b o h y d r a t e
i n c o r p o r a t i o n i n t o p r o t e i n w as i n h i b i t e d b y o n l y 18$ .
I n r a t l i v e r m ic r o s o m a l f r a c t i o n s ( 4 2 ) , t r a n s f e r a s e
a c t i v i t y was f o u n d w h ic h c a t a l y z e d t h e t r a n s f e r o f g a l a c
t o s e fro m U D P - g a l a c t o s e t o e i t h e r e n d o g e n o u s r e c e p t o r s o r
t o d e s i a l y z e d , p - g a l a c t o s i d a s e t r e a t e d , a ^ - a c i d g l y c o p r o
t e i n , o r t o N - a c e t y l g l u c o s a m i n e . Up t o 8 5 $ o f t h e g a l a c -
to B e rem o v e d fro m t h e a - ^ - g l y c o p r o t e i n c o u l d b e r e p l a c e d i n
t h e i r s y s t e m . T h e s e r e s u l t s w o u ld t e n d to s u g g e s t t h a t t h e
s u g a r r e s i d u e s may be a d d e d t o t h e g l y c o p r o t e i n s o n e a t a
t i m e , r a t h e r t h a n a s o l i g o s a c c h a r i d e u n i t s .
U s in g a c e l l f r e e s y s t e m fro m E h r l i c h a s c i t e s tu m o r
c e l l s , E y l a r a n d Cook (4 3 ) s u g g e s t e d t h a t o l i g o s a c c h a r i d e s
a r e s y n t h e s i z e d b y p a r t i c u l a t e enzy m es i n a " p o s t - m i c r o
so m a l p a r t i c u l a t e " f r a c t i o n , t h e n a t t a c h e d to p r o t e i n w i t h
i n t h e n e tw o r k o f t h e e n d o p l a s m i c r e t i c u l u m . R ibosom e
s t i m u l a t i o n w as i n t e r p r e t e d t o mean t h a t p r o t e i n r e c e p t o r
w as b e i n g p r o v i d e d to t h e s y s te m by t h i s f r a c t i o n . On t h e
o t h e r h a n d , S a r c i o n e a n d Carmody ( 4 4 ) , u s i n g a r a t l i v e r
c e l l f r e e s y s t e m , h a v e o b t a i n e d r e s u l t s c o m p a t i b l e w i t h
t h o s e o f M cG uire e t a l . , f i n d i n g m ic r o s o m a l e n zy m es t h a t
c a t a l y z e t h e i n c o r p o r a t i o n o f g a l a c t o s e fro m U D P - g a l a c t o s e
11
I n t o p r o t e i n . No o t h e r s u g a r n u c l e o t i d e was r e q u i r e d ,
a g a i n s u g g e s t i n g t h a t s u g a r m o i e t i e s a r e a d d e d o n e a t a
t i m e . I t w as f u r t h e r fo u n d t h a t t h i s t r a n s f e r a s e a c t i v i t y
c o u l d be s o l u b i l i z e d away fro m t h e membrane w i t h d e s o x y c h o
l a t e .
O t h e r s t u d i e s a l s o i n d i c a t e t h a t t h e s u g a r s may be
a d d e d o n e a t a t i m e . Thus G r e b n e r e t a l . (4 5 ) h a v e shown
t h a t a t r a n s f e r a s e e x i s t s i n h e n o v i d u c t t h a t c a t a l y z e s t h e
t r a n s f e r o f x y l o s e fro m U D P -x y lo se i n t o g l y c o p r o t e i n . Prom
g o a t c o l o s t r u m , J o h n s t o n e t a l . (46 ) h a v e i s o l a t e d a t r a n s
f e r a s e t h a t c a t a l y z e s t h e t r a n s f e r o f N - a c e t y l g l u c o s a m i n e
fro m i t s UDP d e r i v a t i v e t o l a r g e m o l e c u l a r w e i g h t g l y c o p r o
t e i n r e c e p t o r s . H ow ever, o n l y 1 8 - 2 1 $ o f t h e t h e o r e t i c a l
r e c e p t o r s i t e s c o u l d b e s a t u r a t e d . A s u r v e y o f r a t t i s s u e
h o m o g e n a te s show ed t h a t t h i s a c t i v i t y e x i s t s i n a v a r i e t y
o f t i s s u e s .
I n s t u d i e s on t h e t r a n s f e r o f s i a l i c a c i d to g l y c o
p r o t e i n s , B a r t h e t a l . (4 7 ) fo u n d t h a t a s o l u b l e g o a t c o l o s
tru m t r a n s f e r a s e c o u l d r e p l a c e 28$ o f t h e s i a l i c a c i d r e
moved fro m o r o s o m u c o id , w h i l e a r a t mammary p a r t i c u l a t e
f r a c t i o n w i t h e n d o g e n o u s a c t i v i t y was n o t a c t i v e a t a l l
w i t h o r o s o m u c o id . T h i s p a r t i c u l a t e enzyme h a d b e e n p r e
v i o u s l y r e p o r t e d (48 ) t o be a b l e to t r a n s f e r N - a c e t y l n e u -
r a m l n i c a c i d o r N - g l y c o l y l n e u r a m i n i c a c i d fro m t h e i r CMP
d e r i v a t i v e s to p - D - g a l a c t o p y r a n o B y l en d g r o u p s , b u t w as n o t
a c t i v e w i t h s e v e r a l d e s i a l y z e d g l y c o p r o t e i n s . I t was c o n -
12
e l u d e d t h a t N - a c e t y l n e u r a m i n i c a c i d was I n c o r p o r a t e d a s t h e
f i n a l s t e p i n g l y c o p r o t e i n s y n t h e s i s . , a n d t h a t t h e r e q u i r e d
g a l a c t o s i d i c e n d g r o u p w as n o t t h e o n l y d e t e r m i n a n t o f
t r a n s f e r a s e s p e c i f i c i t y , O’B r i e n e t a l . ( 4 9), h a v e demon
s t r a t e d t h e e x i s t e n c e , i n l i v e r m ic r o s o m a l p r e p a r a t i o n s , o f
an enzyme t h a t t r a n s f e r s N - a c e t y l n e u r a m i n i c a c i d fro m CMP-N-
a c e t y l n e u r a m i n i c a c i d t o i n c o m p l e t e se ru m g l y c o p r o t e i n s .
S t u d i e s on t h y r o g l o b u l i n b i o s y n t h e s i s by S p i r o an d
S p i r o (5 0 ) h a v e shown t h a t t h e p e p t i d e an d c a r b o h y d r a t e p o r
t i o n s o f t h i s g l y c o p r o t e i n a r e made on p a r t i c u l a t e f r a c
t i o n s , w i t h t h e c a r b o h y d r a t e b e i n g a d d e d a f t e r t h e p e p t i d e
p o r t i o n i s s y n t h e s i z e d . F u r t h e r m o r e , t h e r a t e s o f i n c o r
p o r a t i o n i n d i c a t e a g a i n t h a t t h e s u g a r s a r e m o st p r o b a b l y
a d d e d s t e p w i s e .
W h ile s t u d i e s c o n c e r n i n g t h e b i o s y n t h e s i s o f g l y c o
p r o t e i n s i n d i c a t e t h a t t h e i r s u g a r s a r e a d d e d one a t a
t i m e , s t r u c t u r a l a n a l y s e s i n d i c a t e t h e y may be a d d e d a s
o l i g o s a c c h a r i d e s . S e v e r a l g l y c o p r o t e i n B fro m v a r i o u s
s o u r c e s h a v e s i m i l a r o l i g o s a c c h a r i d e u n i t s , e . g . , o ro so m u
c o i d ( 2 7 ) , g a s t r i c m ucin ( 5 1 ) ^ b l o o d g r o u p s u b s t a n c e s ( 5 2 ,
5 3 ) , an d m i l k ( 5 4 ) . The p o s s i b i l i t y t h a t t h e s e u n i t s may
b e a d d e d a s o l i g o s a c c h a r i d e s i s s t r e n g t h e n e d by t h e f a c t
t h a t n u c l e o t i d e o l i g o s a c c h a r i d e s h a v e a c t u a l l y b e e n i s o
l a t e d fro m m i l k a n d c o l o s t r u m . J o u r d i a n e t a l . (55) h av e
i s o l a t e d n u c l e o t i d e d e r i v a t i v e s w i t h c a r b o h y d r a t e m o i e t i e s
s i m i l a r t o t h o s e fo u n d i n f e t u i n a n d o r o s o m u c o id , w h i l e
13
K a b a ta ( 5 6 ) h a s i s o l a t e d two w i t h s t r u c t u r e s s i m i l a r to
th o B e fo u n d i n t h y r o g l o b u l i n a n d o r o s o m u c o id .
W h ile b i o c h e m i c a l d a t a i m p l i c a t e s t h e e n d o p la s m ic
r e t i c u l u m a s a s u b c e l l u l a r s i t e o f g l y c o p r o t e i n s y n t h e s i s ,
r a d i o a u t o g r a p h i c w o rk w i t h t h e e l e c t r o n m ic r o s c o p e h a s l e d
P e t e r s o n and L eB lond to c o n s i d e r t h e G o lg i r e g i o n a s a n o t h e r
p o s s i b i l i t y . U s in g r a t i n t e s t i n a l c e l l s , t h e y f o l l o w e d t h e
f a t e o f g l u c o s e - l - H ^ (5 7 ) o r g l u c o s e - 6 - H ^ ( 5 8 ) , p a r t o f
w h ic h was r e c o v e r e d i n s e c r e t e d r a d i o a c t i v e g l y c o p r o t e i n s ,
and s u g g e s t e d t h e f o l l o w i n g s e q u e n c e f o r g l y c o p r o t e i n b i o
s y n t h e s i s :
F o rm in g p r o t e i n on C o m p le te d
r o u g h e n d o p la s m ic ---------------------- ► p r o t e i n s
r e t i c u l u m ^
m i g r a t e s G o lg i ______________ Becom es
to R e g io n g l y c o p r o t e i n
E v id e n c e t h a t a n t i - f e r r i t i n a n t i b o d i e s a r e b o t h s y n t h e s i z e d
and s t o r e d i n t h e m ic ro s o m e s w as s u p p l i e d by P e t r i s e t a l .
( 5 9 ) . T h ese d a t a o b t a i n e d w i t h t h e e l e c t r o n m ic r o s c o p e
h o w e v e r, w ere n o t s u f f i c i e n t t o e x c l u d e s y n t h e s i s i n o t h e r
r e g i o n s . S t o r a g e i n t h e G o lg i r e g i o n a f t e r s y n t h e s i s i n
t h e e n d o p la s m ic r e t i c u l u m w o u ld be c o n s i s t e n t w i t h P e t e r s o n
and L e B lo n d 1s d a t a . P e t r i s ' d a t a c o u l d b e i n t e r p r e t e d to
i n d i c a t e l i m i t e d a c c e s s o f t h e f e r r i t i n m o l e c u l e s t o t h e i r
a n t i b o d i e s . I t m ig h t b e a d d e d t h a t t h e b i o c h e m i c a l d a t a
doeB n o t e x c l u d e t h e G o lg i r e g i o n aB a s i t e . No a t t e m p t
waB made i n a n y o f t h e s t u d i e s to l o c a l i z e t h e G o lg i r e g i o n
i n t h e s u b c e l l u l a r f r a c t i o n s a s s a y e d .
I n b a c t e r i a l s y s t e m s , c e l l w a l l g l y c o p e p t i d e s y n
t h e s i s h a s b e e n shown t o o c c u r a c c o r d i n g t o t h e f o l l o w i n g
s e q u e n c e s , by c e l l m em brane enzym es ( 6 0 , 6 1 , 6 2 , 6 3 , 6 4 ) :
i n M ic r o c o c c u s l y B o d l e k t i c u s ,
(U D P -N -a c e ty lm u ra m ic a c i d - p e n t a p e p t i d e ) + membrane
1
( m e m b r a n e - p h o s p h o l i p i d - p e n t a p e p t i d e - N - a c e t y l m u r a m i c a c i d )
| + U D P - N - a c e ty lg lu c o s a m in e
( m e m b r a n e - p h o s p h o l i p i d - p e n t a p e p t i d e - N - a c e t y l m u r a m i c a c i d -
N - a c e t y l g l u c o s a m i n e ) ;
i n S t a p h y l o c o c c u s a u r e u s t h e p r o d u c t i s
( m e m b r a n e - p h o s p h o l i p i d - p e n t a g l y c i n e - p e n t a p e p t i d e - N - a c e t y l
m uram ic a c i d - N - a c e t y l g l u c o s a m i n e ) .
The b a c t e r i a l s y s te m s s u g g e s t t h a t p e r h a p s a n a l o g o u s mem
b r a n e b o u n d i n t e r m e d i a t e s o c c u r i n g l y c o p r o t e i n b i o s y n t h e
s i s i n m am m alian B y ste m s. E s p e c i a l l y i n t e r e s t i n g i n t h i s
r e g a r d i s t h e f a c t t h a t e v en s o l u b l e g l y c o p r o t e i n s h a v e
b e e n Bhown t o be s y n t h e s i z e d i n a p a r t i c u l a t e b o u n d s t a t e
b e f o r e a p p e a r i n g i n s o l u b l e fo rm .
I n summary t h e n , i t a p p e a r s t h a t i n m am m alian s y s
tem s t h e c a r b o h y d r a t e p o r t i o n o f g l y c o p r o t e i n s i s m ost
p r o b a b l y a d d e d a f t e r t h e p r o t e i n m o i e t y i s s y n t h e s i z e d b y
r i b o s o m e s , e i t h e r a s t h e p r o t e i n p r o g r e s s e s a l o n g t h e n e t
w ork o f t h e e n d o p la s m ic r e t i c u l u m , o r i n t h e G o lg i a p p a r a
t u s , o r b o t h . G l y c o p r o t e i n s a r e c h a r a c t e r i s t i c a l l y e x t r a
c e l l u l a r (6 5 ) , an d i t i s c o n c e i v a b l e t h a t w h i l e t h o s e p r o
t e i n s d e s t i n e d f o r e x p o r t a r e s h u n t e d i n t o one r e g i o n f o r
c a r b o h y d r a t e a d d i t i o n , t h o s e d e s t i n e d f o r t h e p la s m a mem
b r a n e may b e a s s e m b le d a t a n o t h e r B i t e , w h ere m em brane iB
b e i n g a s s e m b l e d .
The d a t a i n d i c a t e s t h a t s u g a r s a r e a d d e d s e q u e n t i a l
l y , b y s p e c i f i c s u g a r n u c l e o t i d e t r a n s f e r a s e s , and w i t h o u t
t h e i n v o l v e m e n t o f sRNA. T r a n s f e r o f o l i g o s a c c h a r i d e u n i t s ,
h o w e v e r, c a n n o t be r u l e d o u t . S t i l l d e b a t a b l e a l s o , i s
t h e q u e s t i o n o f w h e t h e r o r n o t c a r b o h y d r a t e a d d i t i o n i s b e
g u n , th o u g h n o t c o m p l e t e d , w h i l e t h e p r o t e i n i s s t i l l a s s o
c i a t e d w i t h t h e r i b o s o m e s . T h is i s a c r i t i c a l q u e s t i o n ,
s i n c e no i n f o r m a t i o n h a s b e e n o b t a i n e d on t h e f o r m a t i o n o f
c a r b o h y d r a t e - p r o t e i n b o n d s . F u r t h e r m o r e , i t i s an e x
t r e m e l y d i f f i c u l t one to r e s o l v e s i n c e t h e i n v i t r o s i t e o f
t h e b o n d 's f o r m a t i o n i s n o t e a s i l y d e f i n e d . On one h a n d ,
r i b o s o m e s a r e known t o be i n t i m a t e l y a s s o c i a t e d w i t h mem-
b r a n c e s d u r i n g p r o t e i n s y n t h e s i s , p o s s i b l y w i t h t h e i r p h o s
p h o l i p i d ( 6 6 , 6 7 , 6 8 ) , a n d c a n b e r e l e a s e d w i t h d e s o x y c h o l a t e
(6 9 ) . On t h e o t h e r h a n d , one o f t h e membrane b o u n d B ugar
t r a n s f e r a s e s h a s a l s o b e e n r e l e a s e d w i t h d e s o x y c h o l a t e ( 4 4 ) .
I I . STATEMENT OP PROBLEM AND EXPERIMENTAL PLAN
S i n c e g l y c o p r o t e i n B i n m am m alian system B em b ra ce
s u c h a w id e v a r i e t y o f f u n c t i o n s , i n c l u d i n g s u c h i m p o r t a n t
r o l e B a s h o rm o n e s , a n t i b o d i e s , p la s m a p r o t e i n s , c e l l s u r
f a c e a n t i g e n s an d v i r u s r e c e p t o r s i t e s , t h e i r b i o s y n t h e s i s
p r e s e n t s f o r u s a b i o c h e m i c a l p r o b le m w hose s o l u t i o n s h o u l d
h a v e much r e l e v a n c e t o o u r u n d e r s t a n d i n g o f t h e c e l l and
d i s e a s e B t a t e s . F u r t h e r m o r e , a b a s i c p r o b le m t h a t m e r itB
s p e c i a l em p haB is on a h i g h e r p l a n e i s t h e p r o b le m o f t h e
b i o s y n t h e s i s o f c e l l u l a r m em branes t h e m s e l v e s , t h a t i s ,
how t h e g l y c o p r o t e i n i s i n t e g r a t e d w i t h t h e l i p o p r o t e i n ,
t r a n s p o r t p r o t e i n a n d enzym es i n t o an o r g a n i z e d f u n c t i o n a l
e n t i t y w h ic h we c a l l a m em brane. I n d e e d , I t I s p o s s i b l e
t h a t t h e two p r o c e s s e s , membrane a s s e m b ly and c a r b o h y d r a t e
a d d i t i o n to i t s p r o t e i n , may b e i n t i m a t e l y r e l a t e d .
I n o r d e r t o u t i l i z e a s i m p l e r s y s te m t h a n t h o s e
t h a t h a v e b e e n B t u d i e d , r a b b i t r e t i c u l o c y t e s w ere c h o s e n .
T h ese c e l l s c o n t a i n no ro u g h e n d o p la s m ic r e t i c u l u m , t h e y
do n o t s e c r e t e p r o t e i n s , and t h e i r m em branes a r e e a s i l y
I s o l a t e d .
I t i s t h e p u r p o s e o f t h e s e s t u d i e s t o I n v e s t i g a t e
t h e s i t e a n d n a t u r e o f t h e enzymeB I n v o l v e d i n t h e s y n t h e
16
s i s o f t h e g l y c o p r o t e i n o f t h e r a b b i t r e t i c u l o c y t e m em brane.
T h is w i l l b e a p p r o a c h e d i n i t i a l l y by t h e i n c o r p o r a t i o n o f
r a d i o a c t i v e p r e c u r s o r s i n t o i n t a c t c e l l s f o l l o w e d b y d e
v e lo p m e n t and u t i l i z a t i o n o f c e l l f r e e s y s t e m s . F i n a l l y ,
a t t e m p t s w i l l b e made t o i s o l a t e an d s t u d y p u r e enzyme s y s
te m s , b o t h w i t h n a t u r a l and a r t i f i c i a l r e c e p t o r s w here p o s
s i b l e . I t i s a l s o h o p e d t h a t i n t h i s way s o m e th in g c an be
l e a r n e d a b o u t t h e s t r u c t u r a l o r g a n i z a t i o n o f t h e c e l l mem
b r a n e .
I I I . MATERIALS AND METHODS
M a t e r i a l s
Anim alB
F i v e t o s i x m onth o l d m ale and f e m a le New Z e a la n d
w h i t e r a b b i t s w e i g h in g a p p r o x i m a t e l y s e v e n p o u n d s w ere u s e d .
C h e m ic a ls
A l l c h e m i c a l s u s e d w ere M a l l i n k r o d t R e a g e n t G ra d e ,
u n l e s s o t h e r w i s e n o t e d .
T riz m a Base-RG ( T r i s ( h y d r o x y m e th y l) a m in o m e th a n e ) ,
was a p r o d u c t o f Sigm a C o r p o r a t i o n . l - A c e t y l - 2 - p h e n y l h y d r a -
z i n e web o b t a i n e d fro m M a th e s o n , Colem an a n d B e l l . Sodium
d e s o x y c h o l a t e was M.A. s p e c i a l enzyme g r a d e fro m Mann C o r
p o r a t i o n . H e p a r i n ( a q u e o u s l l p o h e p a r i n , 200 m g/m l = 2 0 ,0 0 0
U .S .P . u n i t s ) , was a p r o d u c t o f R i k e r L a b o r a t o r i e s . S ucro se
was a s p e c i a l enzyme g r a d e from Mann. O c t y l p h e n o x y p o l y -
e th o x y ( 9 - 1 0 ) e t h a n o l w as s u p p l i e d a s T r i t o n - X - 1 0 0 by Rohm
a n d H aas C o r p o r a t i o n .
N u c l e o t i d e t r i p h o s p h a t e s , g l u t a t h i o n e , an d 6 - a z a -
u r i d i n e w ere a l l A g r a d e fro m C a lb io c h e m . M e r c a p t o e t h a n o l
was a l s o a p r o d u c t o f C a lb io c h e m .
18
19
R a d i o c h e m i c a l s
D - g l y c o s a m l n e - U - C 1^ , 180 mC/mM, w as o b t a i n e d fro m
2 1 l .
t h e Y o lk C o r p o r a t i o n . D -m anno se-C , 2 . 3 mC/mM, w as fro m
l2 i
New E n g la n d N u c l e a r C o r p o r a t i o n a n d D - g a l a c t o s e - C , 34 mC/
14 /
mM, fro m C a lb io c h e m C o r p o r a t i o n . L - s e r i n e - U L - C , 0 . 1 mC/
O.O 876 mg, L - l e u c i n e - U L - C 1^ , 0 . 1 m C /0 .0 5 9 mg* a n d UDP-
g a l a c t o s e - C " ^ , 40 mC/mM, w e re p r o d u c t s o f New E n g la n d Nu-
14
c l e a r C o r p o r a t i o n . U D P - N - a c e t y lg l u c o s a m i n e - C an d UDP-N-
14
a c e t y l g a l a c t o s a m i n e - C w e re p r e p a r e d a s o u t l i n e d i n t h e
l 4
A p p e n d ix . U D P - g a l a c t o s e - C w as r o u t i n e l y c h e c k e d b y e l e c
t r o p h o r e s i s f o r b re a k d o w n d ue t o s t o r a g e a n d f r e e z e - t h a w i n g .
R i b o n u c l e a s e , p h o s p h a t a s e f r e e , was R i b o n u c l e a s e A,
l y o p h i l i z e d RAF 6 0 4 7 fro m W o r t h i n g t o n . P ro n a B e w as B g r a d e
fro m C a lb i o c h e m . a -A m y la s e w as B a c t e r i a l , Type I I fro m
B a c i l l u s s u b t i l i s , C r y s t a l l i z e d , L y o p h i l i z e d , fro m Sigm a
C h e m ic a l Company.
O t h e r M a t e r i a l s
S e p h a d e x f i l t r a t i o n g e l s a n d i o n e x c h a n g e g e l s w ere
p r o d u c t s o f P h a r m a c ia F i n e C h e m ic a lB , I n c . C e l l u l o s e d i a l
y s i s t u b i n g w as a p r o d u c t o f U n io n C a r b i d e C o r p o r a t i o n
( d i a . 0 . 2 5 i n c h ; a v g . p o r e B i z e , 2 4 $ ) . P o l y e t h y l e n e g l y c o l
was s u p p l i e d a s C arbow ax 2 0 ,0 0 0 ( f l a k e d ) , fro m t h e U n io n
C a r b i d e C o r p o r a t i o n .
20
M ethods
C h e m ic a l D e t e r m i n a t i o n s
P r o t e i n s w ere r o u t i n e l y a s s a y e d b y t h e m eth o d o f
Lowry e t a l . (7 0 ) o r b y t h e m i c r o b i u r e t m eth o d o f Goa ( 7 1 ) .
S i m i l a r r e s u l t s w ere o b t a i n e d w i t h b o t h m e th o d s e x c e p t i n
sam pleB w h ic h c o n t a i n e d T r i t o n - X - 1 0 0 . T h is d e t e r g e n t
c a u s e d n o n - l i n e a r s t a n d a r d c u r v e s o b t a i n e d b y t h e m i c r o
b i u r e t m e th o d .
S i a l i c a c i d s w ere r e l e a s e d by h y d r o l y s i s w i t h 0 . 1 0 N
HgSO^ a t 80° f o r o n e h o u r . They w e re a s s a y e d b y t h e m eth o d
o f W arren ( 7 2 ) , u s i n g p u r e N - a c e t y l n e u r a m i n i c a c i d a s a
s t a n d a r d . The s t a n d a r d s w e re h y d r o l y z e d i n t h e same m ann er
a s t h e s a m p le s .
Amino a c i d s w ere d e t e r m i n e d by t h e m eth o d o f R osen
( 7 3 )* u s i n g p u r e 1 - l e u c i n e a s a s t a n d a r d .
C o u n tin g o f R a d i o a c t i v e S a m p le s
D r i e d p r e c i p i t a t e s w ere d i s s o l v e d i n m in im a l a m o u n ts
o f 1 N NaOH. S u i t a b l e a l i q u o t s w e re p i p e t t e d o n to Whatman
No. 3 mm f i l t e r p a p e r ( l cm x 1 . 2 cm) o r o n t o g l a s s f i l t e r
p a p e r d i s c s ( 2 . 4 cm d i a m .) w h ic h w ere t h e n d r i e d b y e x p o s u r e
t o an i n f r a r e d h e a t l i g h t . The p a p e r c o n t a i n i n g t h e d r i e d
sa m p le was t h e n p l a c e d i n t o t h e g l a s s v l a l B , on one s i d e i n
t h e c a s e o f t h e f i l t e r p a p e r , a n d a t t h e b o t t o m i n t h e c a s e
o f t h e g la s B f i b e r p a p e r . E i t h e r 15 ml o r 5 ml o f t h e
s c i n t i l l a t i o n f l u i d was a d d e d , r e s p e c t i v e l y .
21
The s c i n t i l l a t i o n f l u i d c o n s i s t e d o f 5*0 gms o f
2 , 5 - d i p h e n y l o x a z o l e (PPO) a n d 0 . 3 gms o f 2 - p - p h e n y l e n e b l s
( 4 - m e t h y l , 5 - p h e n y l o x a z o l e ) ( d im eth y l-P O P O P ) d i s s o l v e d I n a
l i t e r o f t o l u e n e .
C o u n tin g w as done e i t h e r i n a N u c l e a r C h ic a g o t r i p l e
c h a n n e l s c i n t i l l a t i o n c o u n t e r , Model 7 2 4 , w i t h an e f f i c i e n c y
o f b e tw e e n 5 0 - 6 0 $ , o r i n a Beckman room t e m p e r a t u r e l i q u i d
s c i n t i l l a t i o n c o u n t e r , Model LS200, w i t h an e f f i c i e n c y o f
a b o u t 9 0 $ . R e s u l t s w ere e x p r e s s e d a s c o u n t s p e r m in u te i n
m o st c a s e s , o r i n m i l l i m i c r o m o l e s .
C e l l P r e p a r a t i o n
R e t i c u l o c y t e r e s p o n s e was i n d u c e d i n r a b b i t s by a
m o d i f i c a t i o n o f th e m ethod o f London e t a l . (7 4 ) by d a i l y
s u b c u t a n e o u s i n j e c t i o n o f a s a t u r a t e d s o l u t i o n o f 1 - a c e t y l -
2 - p h e n y l h y d r a z i n e i n 4 2 $ e t h a n o l , f o l l o w e d b y a r e s t i n g
p e r i o d . R e t i c u l o c y t e b u i l d u p i n t h e c i r c u l a t i o n w as f o l
low ed by d r a w in g b l o o d s a m p le s fro m t h e e a r v e i n i n t o
h e p a r i n i z e d 0.145M s a l i n e , b r o u g h t to pH 7 . 2 - 7 . 4 w i t h
NaHCO^ o r T r i s , w a s h in g t h e c e l l s w i t h s a l i n e , s t a i n i n g
w i t h b r i l l i a n t c r e s y l b l u e , a n d e x a m in in g w i t h t h e l i g h t
m i c r o s c o p e . The r a b b i t s w e re s a c r i f i c e d when t h e r e t i c u l o
c y t e c o u n t w as fro m 5 0 - 70$ .
B lo o d , o b t a i n e d fro m t h e r a b b i t s b y h e a r t p u n c t u r e ,
was draw n i n t o a h e p a r i n i z e d s a l i n e s o l u t i o n ( a p p r o x i m a t e l y
200 mg l i p o h e p a r i n f o r e a c h 180 ml b l o o d ) . A l l s a m p le s
w e re m a i n t a i n e d b e tw e e n 0 - 4 ° t h r o u g h o u t t h e w a s h in g p r o c e
d u r e . A r e f r i g e r a t e d S o r v a l l (h e a d # 4 . 7 6 ) w as u s e d f o r
c e n t r i f u g a t i o n . The c e l l s w e re c e n t r i f u g e d a t a b o u t 1 ,2 0 0 x
g f o r 15 m i n u t e s , r e s u s p e n d e d i n a b o u t s i x t i m e s t h e i r
volum e o f 0.145M B a l i n e (pH 7 . 2 - 7 . 4 w i t h 1M T riB o r 0.15M
NaHCO^) a n d r e c e n t r i f u g e d . T h is w a s h in g p r o c e d u r e w as c a r
r i e d o u t t h r e e more t i m e s , w i t h c a r e b e i n g t a k e n t o rem ove
t h e b u f f y c o a t e a c h t i m e . U s in g t h i s p r o c e d u r e , v i r t u a l l y
no l e u c o c y t e s c o u l d b e d e t e c t e d w i t h t h e l i g h t m i c r o s c o p e .
S t u d i e s w i t h I n t a c t C e l l s
I n c u b a t i o n . F o r e x p e r i m e n t s w i t h i n t a c t c e l l s ,
w ashed c e l l s w ere s u s p e n d e d i n an e q u a l volum e o f i n c u b a
t i o n medium , w h ic h c o n s i s t e d o f 0.13M N aC l, 0.005M KC1,
0.0074M MgCl2 *6 H2 0 , and 32 ml o f NaHCO^j t o w h ic h b o v i n e
se ru m a lb u m in (2 m g/m l) an d g l u c o s e ( l m g/m l) w e re a d d e d .
The pH was a d j u s t e d to 7 . 2 - 7 . 4 w i t h a few d r o p s o f HG1.
I n c u b a t i o n was c a r r i e d o u t f o r v a r y i n g tim e l e n g t h s , a t
37° i n a D u b n o ff m e t a b o l i c B h ak in g b a t h .
F r a c t i o n a t i o n . A t t h e end o f t h e i n c u b a t i o n t im e ,
a l i q u o t s o f c e l l s w ere p i p e t t e d i n t o i c e c o l d b u f f e r e d s a
l i n e (pH 7 . 2 - 7 .4 w i t h T r i s o r NaHCO^). A l l s u b s e q u e n t p r e
p a r a t i o n s w e re c a r r i e d o u t a t a p p r o x i m a t e l y 4 ° . The c e l l s
w e re c e n t r i f u g e d a t 2 ,0 0 0 x g i n t h e S o r v a l l ; t h i s w a s h in g
w as r e p e a t e d t h r e e t i m e s . The c e l l s w e re th e n l y s e d i n a p
p r o x i m a t e l y f o u r t i m e s t h e i r volum e o f 0.002M M gC ^J t h e y
w e re m ix ed I n t h e h y p o t o n i c s o l u t i o n a n d l e f t f o r 10 m in
u te s ,, w i t h o c c a s i o n a l g e n t l e s t i r r i n g . A f t e r s p i n n i n g a t
5 ,0 0 0 x g f o r 10 m i n u t e s t o o b t a i n s t r o m a , t h e 5^000 x g
s u p e r n a t a n t f l u i d was spu n f o r 15 m i n u t e s a t 1 5 ,0 0 0 x g .
T h is p e l l e t was d i s c a r d e d . The 1 5 ,0 0 0 x g s u p e r n a t a n t
f l u i d w as sp u n f o r f o u r h o u r s a t 1 0 0 ,0 0 0 x g i n t h e S p in c o
M odel L # 4 0 r o t o r , y i e l d i n g t h e r i b o s o m a l p e l l e t .
M e asu rem en t o f i n c o r p o r a t i o n i n t o s t r o m a . The
s t r o m a l p e l l e t was w a sh e d by r e p e a t e d r e s u s p e n s i o n and r e
c e n t r i f u g a t i o n i n o n e o f t h e f o l l o w i n g w a y s:
1. The p e l l e t was r e s u s p e n d e d i n i c e c o l d 0.002M
MgClg'SH^O and c e n t r i f u g e d a t 5 ,0 0 0 x g f o r 10 m i n u t e s .
T h is w a s h in g w as p e r f o r m e d t h r e e t i m e s . The p e l l e t was
t h e n e x t r a c t e d t h r e e t i m e s i n a c i d r a c e t o n e ( l ml c o n c e n
t r a t e d HC1:100 ml a c e t o n e ) , by t h o r o u g h l y s u s p e n d i n g w i t h
a g l a s s r o d , and th e n c e n t r i f u g i n g .
2. The s t r o m a l p r e c i p i t a t e was w ash e d t h r e e t i m e s
i n 50$ (NH^J^SO^ (j_n w h ic h h e m o g lo b in i s s o l u b l e ) o r i n
0.145M N aC l, f o l l o w e d by t h r e e w a s h e s w i t h 5$ t r i c h l o r a c e
t i c a c i d (TCA). I t was t h e n e x t r a c t e d w i t h a c i d r a c e t o n e ,
a s i n 1 .
3. The s t r o m a l p r e c i p i t a t e was waBhed t h r e e t im e s
w i t h 5$ TCA, f o l l o w e d by e x t r a c t i o n w i t h a c i d : a c e t o n e , a s
i n 1 .
A f t e r one o f th e above waBheB, th e str o m a l p e l l e t
24
was t h e n e x t r a c t e d t h r e e t i m e s w i t h C H C l^ tm e th a n o l ( 2 : 1 *
v / v ) ; th en * to a l l o w b e t t e r p a c k i n g o f t h e s t r o m a l p e l l e t *
t h e C H C l^ :m e th a n o l r a t i o waB a d j u s t e d t o 1 :1 ( v / v ) . T h is
e x t r a c t i o n was f o l l o w e d by t h r e e e t h a n o l : e t h e r ( 1 :1 v / v )
e x t r a c t s . The l i p i d e x t r a c t e d p e l l e t was th e n d r i e d * d i s
s o l v e d i n NaOH* and c o u n t e d .
Of t h e ab o v e w a s h e s , m eth o d 2 was c h o s e n b e c a u s e o f
r e s u l t B o b t a i n e d i n t h e c e l l f r e e system * an d t o m in im iz e
h e m o g lo b in c o n t a m i n a t i o n o f t h e s t r o m a l p e l l e t . I t was o b
s e r v e d t h a t low i o n i c s t r e n g t h w a s h e s p r o g r e s s i v e l y r e
l e a s e d a c o n s i d e r a b l e p o r t i o n o f n o n - l i p i d - e x t r a c t a b l e c a r
b o h y d r a t e r e c e p to r * w h ic h was i n s o l u b l e i n TCA* a l o n g w i t h
m o st o f t h e c o n t a m i n a t i n g h e m o g lo b in . On t h e o t h e r h a n d ,
t h i s r e c e p t o r was n o t r e l e a s e d w i t h NaCl o r NH^SO^ e x t r a c
t io n * w h e r e a s much o f t h e h e m o g lo b in c o u ld b e w a sh e d aw ay.
T r i c h l o r a c e t i c a c i d w ashes* o f c o u rs e * when p e r f o r m e d d i
r e c t l y * g av e v e r y h i g h h e m o g lo b in c o n t a m i n a t i o n o f t h e
s t r o m a l p e l l e t .
M e asu rem en t o f I n c o r p o r a t i o n i n t o r i b o s o m e s . The
r i b o s o m a l p r e c i p i t a t e s w ere p r e p a r e d f o r d i r e c t c o u n t i n g by
w a s h in g o n c e w i t h 0.002M MgClg, t w i c e w i t h 5$ TCA* th e n
d r i e d * d i s s o l v e d i n 1 N NaOH an d c o u n t e d .
The r i b o s o m a l p r e c i p i t a t e w as p r e p a r e d f o r d e n s i t y
g r a d i e n t a n a l y s i s b y s u s p e n s i o n i n 0 . 2 ml o f a s o l u t i o n o f
MgClg (0.001M ):K C 1 (0.05M ) o r 0 . 2 ml o f a s o l u t i o n o f MgClg
25
(0.001M ):K C 1 (0.05M ) made 1 .3 $ I n so d iu m d e s o x y c h o l a t e . An
a l i q u o t waB draw n a t t h i s p o i n t f o r t o t a l c o u n t s , p r e c i p i
t a t e d w i t h 10$ TCA, w ashed t h r e e tim eB w i t h 5$ TCA, d i s s o lv e d
i n NaOH a n d c o u n t e d . The r e s t o f t h e r i b o s o m a l s u s p e n s i o n
was l a y e r e d on t o p o f a 5$ - 20$ l i n e a r s u c r o s e g r a d i e n t ( i n
0.05M T r i s , 0.001M MgClg pH 7 . 2 ) . T h is g r a d i e n t was c e n
t r i f u g e d f o r 75 m i n u t e s a t 3 9 ,0 0 0 rpm i n t h e SW 39 r o t o r o f
t h e S p in c o M odel L u l t r a c e n t r i f u g e . The g r a d i e n t was r e
moved fro m t h e t u b e by p u n c t u r e o f t h e t u b e b o t t o m , f o l l o w e d
b y c o l l e c t i o n o f f r a c t i o n s o f 0 . 2 3 ml e a c h . The f r a c t i o n s
w e re d i l u t e d , an d t h e 0 * D*26o waB m e a s ijr e d * E ach f r a c t i o n
w as p r e c i p i t a t e d w i t h e q u a l v o lu m e s o f 10$ TCA, w ash ed two
t i m e s i n 5$ TCA, d i s s o l v e d i n NaOH, and t h e n c o u n t e d .
M e a su re m e n t o f i n c o r p o r a t i o n i n t o r i b o s o m a l s u p e r
n a t a n t f l u i d . A l i q u o t s o f t h e r i b o s o m a l s u p e r n a t a n t f l u i d
w e re p r e c i p i t a t e d w i t h 10$ TCA, waBhed t w i c e i n 5$ TCA, e x
t r a c t e d o f l i p i d i n t h e same m an n er a s t h e s t r o m a , d i s s o l v e d
i n NaOH and c o u n t e d .
R e l e a s e o f p r o d u c t s from s t r o m a . T h re e two h o u r
14
i n c u b a t i o n s w e re c a r r i e d o u t , u s i n g g lu c o s a m in e - C , g a l a c -
t o s e - C 1^ , a n d m a n n o se -C 1^ . The s tr o m a was i s o l a t e d , w a sh e d ,
and e x t r a c t e d o f l i p i d a s o u t l i n e d a b o v e i n " S t u d i e s w i t h
I n t a c t C e l l s . " I t was t h e n s u s p e n d e d i n 0.145M N aC l-0.00 5M
C a C lg . The pH was a d j u s t e d t o 7*2 w i t h 0 . 5M NaHCO^. A few
dropB o f t o l u e n e a n d 500 p g p r o n a s e p e r ml o f s u s p e n s i o n
26
w e re a d d e d . The s u s p e n s i o n was i n c u b a t e d f o r 48 h o u r s i n a
D u b n o ff m e t a b o l i c s h a k i n g b a t h , k e p t a t 3 7 ° . The pH was
a d j u s t e d to J.2 when n e c e s s a r y . A d d i t i o n a l p r o n a s e (500 ^ g /
m l) was a d d e d a t 24 h o u r s .
A t t h e end o f t h e i n c u b a t i o n t im e , t h e s u s p e n s i o n
w as c e n t r i f u g e d a t 2 0 ,0 0 0 x g f o r 15 m i n u t e s . G l y c o p e p t i d e s
w e re i s o l a t e d e s s e n t i a l l y a c c o r d i n g t o t h e m ethod o f M arks
e t a l . ( 7 5 )* The s u p e r n a t a n t f l u i d was t h e n c o n c e n t r a t e d
b y l y o p h i l i z a t i o n a n d a p p l i e d to a S e p h a d e x -G -2 5 colum n
( e q u i l i b r a t e d w i t h 0 . 1 N a c e t i c a c i d ) . The colum n waB
e l u t e d w i t h 0 . 1 0 N a c e t i c a c i d , a n d a s s a y e d f o r r a d i o a c t i v
i t y , s i a l i c a c i d , a n d am ino a c i d s .
C e l l F r e e E x p e r i m e n t s
F r a c t i o n p r e p a r a t i o n . I n i n i t i a l e x p e r i m e n t s , c e l l s
w e re l y s e d i n 0.002M MgCl^j t h e l y s a t e was th e n made 0.25M
I n s u c r o s e , a n d 0.075M i n KC1. I n t h e l a t e r e x p e r i m e n t s
r e p o r t e d h e r e , s u c r o s e , fo u n d to be n o n - e s s e n t i a l , was
o m i t t e d . The s tr o m a was spun down, w ash ed o n c e i n 0.002M
M gC^* an d s u s p e n d e d I n 0.1M T r i s - H C l , pH 7 * 2 - 7 . 4 , 0.05M
KC1, a n d 0.002M MgCl^ (TKM b u f f e r ) . The s u p e r n a t a n t f l u i d
was a d j u s t e d to 0 . 1M T r i s - H C l , d i a l y z e d a g a i n s t TKM b u f f e r ,
a n d c o n c e n t r a t e d i n d i a l y s i s t u b i n g , u s i n g C arbow ax . R ib o
somes w e re o b t a i n e d b y c e n t r i f u g a t i o n f o r t h r e e h o u r s a t
1 0 0 ,0 0 0 x g I n t h e #40 h e a d o f t h e S p in c o Model L u l t r a
c e n t r i f u g e , They w ere w ashed o n c e and r e s u s p e n d e d i n t h e
27
same TKM b u f f e r a s t h e s t r o m a . F o r r i b o s o m a l s u p e r n a t a n t
f r a c t i o n a t i o n , a l i q u o t s w ere c o n c e n t r a t e d i n d i a l y B i s t u b
i n g ( u s i n g C arbow ax) t o 1 / 2 t h e o r i g i n a l v o lu m e . They w ere
t h e n c e n t r i f u g e d a t 4 8 ,0 0 0 rpm i n t h e #5 0 r o t o r o f th e
S p in c o Model L u l t r a c e n t r i f u g e f o r 1 2 .5 h o u r s . T h is c e n
t r i f u g a t i o n y i e l d e d a g r a d a t i o n o f c o l o r e d l a y e r s , r a n g i n g
fro m a q u e o u s a t t h e t o p , t h r o u g h y e l l o w o r a n g e , t o a d e e p
r e d , d e n s e w i t h h e m o g lo b in . At t h e b o t to m o f t h e tu b e t h e
d e e p r e d p r e c i p i t a t e was c o v e r e d w i t h a t h i n w h i t e f l u f f y
l a y e r , o f t e n h a r d l y v i s i b l e . The tu b e w as a r b i t r a r i l y
d i v i d e d i n t o f i v e f r a c t i o n s , a q u e o u s , r e d - o r a n g e , r e d ,
w h i t e f l u f f y a n d p r e c i p i t a t e .
I n c u b a t i o n s . I n c u b a t i o n s w e re c a r r i e d o u t i n
m a r b l e - s t o p p e r e d t e s t t u b e s , i n a D u b n o ff m e t a b o l i c s h a k i n g
w a t e r b a t h a t 3 7 ° . R e a c t i o n s w ere t e r m i n a t e d by a d d i t i o n
o f 10$ t r i c h l o r a c e t i c a c i d i n e a r l y e x p e r i m e n t s , a n d b y a d
d i t i o n o f 1$ p h o s p h o t u n g s t i c a c i d ( i n 0 . 5 N HCl) i n e x p e r i
m e n ts w h e re e x o g e n o u s r e c e p t o r was UBed.
P r e c i p i t a t e w a s h in g s and e x t r a c t i o n s . P r e c i p i t a t e s
fro m c e l l f r e e i n c u b a t i o n s w e re r o u t i n e l y w a sh e d i n t h e
t u b e b y r e p e a t e d s u s p e n s i o n an d r e c e n t r i f u g a t i o n j th e
w a sh e s c o n s i s t e d o f e i t h e r f o u r 5$ TCA w a s h e s , f o l l o w e d by
heme a n d l i p i d e x t r a c t i o n s a s i n w h o le c e l l e x p e r i m e n t s , o r
t h r e e p h o s p h o t u n g s t i c a c i d waBhes (1 $ p h o s p h o t u n g s t i c a c i d
i n 0 . 5 N HCl, d i l u t e d 1 :5 w i t h 0.145M NaCl) f o l l o w e d by two
28
5$ TCA w a s h e s , an d t h e n heme an d l i p i d e x t r a c t i o n . I n e x
p e r i m e n t s w i t h p u r i f i e d enzym e, t h e p r e c i p i t a t e was a c i d
w a sh e d , f o l l o w e d b y one e t h a n o l : e t h e r ( 1 :1 v / v ) w ash , to
a l l o w d r y i n g o f t h e p r e c i p i t a t e . The p e l l e t s w ere t h e n
d r i e d , d i s s o l v e d i n NaOH, an d s u i t a b l e a l i q u o t s c o u n t e d .
P r o d u c t i s o l a t i o n . I n o r d e r t o o b t a i n a p r o d u c t
14
c o n t a i n i n g b o u n d g l u c o s a m in e - C , a c e l l f r e e i n c u b a t i o n
c o n t a i n i n g 0 . 1 ml (10 nC) o f g l u c o s a m in e - C ^ ^ , 1 ,0 ml o f
s t r o m a l s u s p e n s i o n ( a b o u t 1 0 -2 0 mg p r o t e i n ) , 0 , 5 ml s u g a r s ,
N - a c e t y l n e u r a m i n l c a c i d ( 6 . 2 m g /m l), an d g a l a c t o s e ( 3 - 6
m g /m l) , and 1 .0 ml o f s u p e r n a t a n t f l u i d was c a r r i e d o u t
f o r 90 m i n u t e s , a t 3 7 ° , i n t h e D u b n o ff m e t a b o l i c s h a k in g
14
b a t h . F o r t h e p r o d u c t o f U D P - g a la c to s e - C i n c o r p o r a t i o n ,
a s i m i l a r i n c u b a t i o n was s e t u p , c o n t a i n i n g 0 . 3 ml (3 n C ) ,
14
U D P - g a la c to s e - C , 1 .0 ml s t r o m a l s u s p e n s i o n , 0 . 1 ml MnC] 2
(20 nM ), 0 . 5 ml s u g a r B , N - a c e t y l g a l a c t o s a m i n e and s i a l i c
a c i d (10 m g/m l e a c h ) , and 1 .0 ml o f s u p e r n a t a n t f l u i d .
A f t e r i n c u b a t i o n , e a c h r e a c t i o n m i x t u r e was e x
h a u s t i v e l y d l a l y z e d a g a i n s t 0.05M T r is - H C l b u f f e r . The u n -
d i a l y z e d m a t e r i a l was t h e n p r e c i p i t a t e d w i t h TCA (1 0 $ ) and
w a sh e d i n 5$ TCA a n d l i p i d e x t r a c t e d , a s d e s c r i b e d e a r l i e r .
E ac h p r e c i p i t a t e was t h e n w a sh e d o n c e w i t h , a n d s u s p e n d e d
i n , 0.145M s a l i n e - 0 . 0 0 5 M C a C l2 , b r o u g h t t o pH 7 . 3 w i t h
NaHCO^. I n i t i a l l y , 0 . 5 mg p r o n a s e p e r ml s u s p e n s i o n was
a d d e d , a s w e l l a s a d ro p o f t o l u e n e , and t h e s u s p e n s i o n was
29
I n c u b a t e d f o r a t o t a l o f 36 h o u r s , a d d in g a d d i t i o n a l p r o n a s e
e v e r y 12 h o u r s . A l i q u o t s o f t h e r e l e a s e d g l y c o p e p t i d e s
w e re c o u n te d f o r t o t a l I n c o r p o r a t i o n , an d t h e r e m a in i n g
am ount was p l a c e d on a S e p h a d e x G-25 c o lu m n . The colum n
w as e l u t e d w i t h 0 . 1 N a c e t i c a c i d a n d t h e f r a c t i o n s w ere
a s s a y e d f o r r a d i o a c t i v i t y , am ino a c i d B , and s i a l i c a c i d s .
A r t i f i c a l r e c e p t o r s . T h ese w ere p r e p a r e d i n o u r
l a b o r a t o r y u n d e r D r. E y l a r ' s d i r e c t i o n , by m eth o d s o u t l i n e d
i n t h e A p p e n d ix .
S o l u b i l i z a t i o n o f U D P - g a la c t o s e t r a n s f e r a s e . S o l u
b i l i z a t i o n o f membrane c o m p o n e n ts h a s b e e n a c c o m p l i s h e d i n
s e v e r a l ways i n o t h e r s y s t e m s . S o n i c a t i o n h a s b e e n u s e d
s u c c e s s f u l l y t o d i s r u p t m em branes o f v a r i o u s t y p e s and i n
many c a s e s t o s o l u b i l i z e c e r t a i n p o r t i o n s o f i t ( 7 6 - 7 7 ) .
B u t a n o l e x t r a c t i o n h a s b e e n s u c c e s s f u l l y u s e d b y Murakami
and E y l a r (7 8 ) an d b y G re en a n d c o w o r k e r s w i t h m i t o c h o n d r i a l
m em branes ( 7 9 , 8 0 ) , and was a l s o r e c e n t l y Bhown t o s o l u b i l i z e
much o f t h e r e d c e l l m em brane ( 8 l ) . D i g i t o n i n h a s b e e n
s u c c e s s f u l l y u s e d i n t h e m i t o c h o n d r i a l s y s te m t o d i s r u p t
m em branes ( 8 2 ) . Sodium d o d e c y l s u l f a t e h a s b e e n w i d e l y
u s e d t o d i s s o c i a t e rlb o so m eB fro m t h e m em branous p o r t i o n o f
t h e ro u g h e n d o p la s m ic r e t i c u l u m by d i s s o l u t i o n o f much o f
t h e membrane ( 7 0 ) , a s w e l l a s i n t h e s o l u b i l i z a t i o n o f
o t h e r membrane c o m p o n e n ts ( 8 3 , 8 4 , 8 5 , 8 6 ) . T r i t o n - X - 1 0 0 , a
n o n - i o n i c d e t e r g e n t , h a s a l s o b e e n u s e d s u c c e s s f u l l y i n
30
many i n s t a n c e s i n s o l u b i l i z a t i o n and d i s r u p t i o n o f m em branes
( 8 7 - 9 1 ) . M o d i f i c a t i o n s o f t h e s e m e th o d s w e re t h e r e f o r e u s e d
i n a t t e m p t s t o s o l u b i l i z e p o r t i o n s o f t h e r e t i c u l o c y t e
s tr o m a w i t h o u t d e s t r o y i n g t h e t r a n s f e r a s e enzyme a c t i v i t y .
The r e t i c u l o c y t e s w ere e i t h e r b r o k e n a t 1 ,0 0 0 p s i
w i t h an Aminco F r e n c h p r e s s u r e c e l l , u s i n g t h e Pow er Lab
p r e s s o f t h e A m e ric an I n s t r u m e n t Company, o r l y s e d i n
0.005M T r i s - H C l , pH 7 . 2 - 7 - ^ i n 7 x 10 ^M m e r c a p t o e t h a n o l .
S o n i c a t i o n . S o n l c a t i o n o f 10 ml o f S t r o m a l s u s p e n
s i o n w as c a r r i e d o u t w i t h a B r a n s o n S o n i f i e r Model S110 a t
s e t t i n g # 3 f o r a t o t a l o f 300 s e c o n d s , u s i n g 5 s e c o n d
b u r s t s , w i t h 30 s e c o n d i n t e r v a l s . The s tr o m a was i n s u s
p e n s i o n i n 5 ml o f 0.1M T r i s - H C l , pH 7 . 2 - 7 . 4 , i n 6 . 8 x 10~^M
m e r c a p t o e t h a n o l an d k e p t i c e c o l d t h r o u g h o u t s o n i c a t i o n .
The s o n i c a t e d s u s p e n s i o n was t h e n c e n t r i f u g e d f o r h a l f an
h o u r a t 1 5 0 ,0 0 0 g i n t h e S p in c o # 5 0 h e a d , and d i v i d e d i n t o
a 1 5 0 ,0 0 0 x g p e l l e t an d i t s s u p e r n a t a n t f l u i d .
B u t a n o l e x t r a c t i o n . A p p r o x i m a t e l y 2 ml o f s tr o m a
was s u s p e n d e d i n 6 ml o f 0.1M T r is - H C l (pH 7 . 2 - 7 . 4 , c o n
t a i n i n g m e r c a p t o e t h a n o l ) a n d 2 ml o f b u t a n o l , t h u s y i e l d i n g
a 20$ b u t a n o l s u s p e n s i o n . E x t r a c t i o n was e f f e c t e d b y k e e p
i n g t h i s s u s p e n s i o n a t 4° f o r 20 m i n u t e s w i t h c o n t i n u a l
s t i r r i n g . T h is s u s p e n s i o n was c e n t r i f u g e d a t 3 0 ,9 0 0 x g i n
t h e S o r v a l l f o r 15 m i n u t e s . T h is y i e l d e d a lo w e r c l e a r
a q u e o u s p h a s e , a s o l i d l y p a c k e d r e d d i s h - b r o w n i n t e r p h a B e ,
a n d a c l e a r u p p e r b u t a n o l p h a s e . The b u t a n o l and a q u e o u s
p h a s e s w e re e x h a u s t i v e l y d i a l y z e d a g a i n s t t h e Bame T r l s
b u f f e r , w i t h f r e q u e n t c h a n g e s o f t h e b u f f e r . The I n t e r
p h a s e was w ashed t w i c e I n t h e same b u f f e r an d t h e n s u s
p e n d e d i n I t .
D i g i t o n l n t r e a t m e n t . A p p r o x i m a t e l y 2 ml o f s t r o m a l
s u s p e n s i o n ( i n t h e T r i s - m e r c a p t o e t h a n o l b u f f e r ) w as made
0 . 8 $ i n d i g i t o n l n , and c o n t i n u a l l y s t i r r e d a t 4 ° f o r 20
m i n u t e s . The r e s u l t a n t s u s p e n s i o n was t h e n spun a t 1 5 0 ,0 0 0
x g i n t h e S o r v a l l f o r 15 m i n u t e s , y i e l d i n g a 1 5 0 ,0 0 0 x g
s u p e r n a t a n t and p e l l e t .
Sodium d o d e c y l s u l f a t e . E q u a l v o lu m e s o f s t r o m a l
s u s p e n s i o n a n d f r e s h l y made 2$ so d iu m d o d e c y l s u l f a t e ( b o t h
i n th e T r i s - m e r c a p t o e t h a n o l b u f f e r ) w ere m ix e d an d s t i r r e d
c o n t i n u a l l y a t 4° f o r 20 m i n u t e s . The s u s p e n s i o n was th e n
sp u n f o r 30 m i n u t e s a t 1 5 0 ,0 0 0 x g i n t h e #5 0 h e a d o f t h e
S p i n c o , an d d i v i d e d i n t o a 1 5 0 ,0 0 0 x g p e l l e t and s u p e r n a
t a n t f l u i d .
U re a e x t r a c t i o n . A l i q u o t s o f t h e s t r o m a l s u s p e n
s i o n w ere c e n t r i f u g e d down an d t h e n r e s u s p e n d e d i n 6M u r e a .
The r e s u l t i n g s u s p e n s i o n was t h e n s t i r r e d g e n t l y a t 4° f o r
20 m i n u t e s , and c e n t r i f u g e d a t 30^900 x g f o r 15 m i n u t e s .
The s u p e r n a t a n t f l u i d was t h e n d i a l y z e d a g a i n s t 0 . 1M T r i s -
HCl b u f f e r , pH 7 . 2 - 7 . 4 , c o n t a i n i n g 7 x 10 m e r c a p t o -
3 2
e t h a n o l . The p e l l e t w as w a sh e d i n t h e same b u f f e r by r e
p e a t e d r e s u s p e n s i o n a n d c e n t r i f u g a t i o n .
T r i t o n - X - 1 0 0 e x t r a c t i o n . E q u a l v o lu m e s o f s t r o m a l
s u s p e n s i o n ( i n t h e T r i s - m e r c a p t o e t h a n o l b u f f e r ) a n d a 1$
( v / v ) s o l u t i o n o f T r i t o n - X - 1 0 0 , i n t h e same b u f f e r , w ere
m ix e d a n d s t i r r e d c o n t i n u a l l y f o r 20 m i n u t e s a t 4 ° . The
r e s u l t i n g s u s p e n s i o n was c e n t r i f u g e d a t 1 5 0 ,0 0 0 x g f o r 30
m i n u t e s , y i e l d i n g a 1 5 0 ,0 0 0 x g s u p e r n a t a n t f l u i d and p e l
l e t . C e n t r i f u g a t i o n f o r 1 - 1 / 2 h o u r s a t t h e same s p e e d was
l a t e r shown t o g i v e t h e same r e s u l t s .
P u r i f i c a t i o n o f t h e enzyme U D P - g a la c t o s e t r a n s f e r
a s e . S tr o m a , u s e d f o r t h e p u r i f i c a t i o n o f t h e U D P -g a la c
t o s e t r a n s f e r a s e , was o b t a i n e d i n one o f two w a y s: by l y s i s
o f t h e c e l l s i n 0 .005M T r i s b u f f e r i n 7 x 10~^M m e r c a p t o
e t h a n o l , o r by b r e a k i n g o f t h e c e l l s i n t h e F r e n c h p r e s s a t
1 ,0 0 0 p s i . T h is was do ne i n o r d e r to c h e c k f o r l o s s o f
enzyme a c t i v i t y o r e n d o g e n o u s r e c e p t o r a t t h e low i o n i c
s t r e n g t h s u s e d i n l y s i s . A f t e r o b t a i n i n g s t r o m a , i t was
w a sh e d t h r e e t i m e s i n t h e T r i s - H C l - m e r c a p t o e t h a n o l b u f f e r ,
t h e n e x t r a c t e d w i t h 0 . 2 5 $ T r i t o n - X - 1 0 0 . The T r i t o n e x
t r a c t e d p e l l e t was t h e n w a sh e d o n c e i n t h e e x t r a c t i n g s o l u
t i o n , a n d t h e s u p e r n a t a n t f l u i d was c o n c e n t r a t e d t o a p p r o x
i m a t e l y o n e - t e n t h i t s t o t a l v o lu m e . T h is c o n c e n t r a t e d s u
p e r n a t a n t f l u i d was t h e n a p p l i e d t o a S e p h a d e x G-100 co lu m n ,
e q u i l i b r a t e d w i t h 0 . 1M T r i s - H C l i n f x 10 m e r c a p t o e t h a n o l
33
(pH 7 . 2 - 7 . ^ ) . The colum n was e l u t e d w i t h t h e same b u f f e r
s o l u t i o n , an d e a c h f r a c t i o n was a s s a y e d f o r t r a n s f e r a s e a c
t i v i t y b o t h to w a rd e n d o g e n o u s r e c e p t o r a n d to w a rd t h e a ^ -
g l y c o p r o t e i n r e c e p t o r . T ho se s a m p le s c o n t a i n i n g enzyme a c
t i v i t y w ere c e n t r i f u g e d a t 1 5 0 ,0 0 0 x g f o r 1 - 1 /2 h o u r s i n
t h e S p in c o # 5 0 h e a d , a s a t e s t f o r B o l u b i l i t y a f t e r r e m o v a l
o f t h e d e t e r g e n t .
P r e p a r a t i o n o f t h e s t r o m a l r e c e p t o r f o r g a l a c t o s e .
The p o r t i o n o f t h e r e c e p t o r ( f o r g a l a c t o s e ) t h a t was r e
l e a s e d fro m t h e s tr o m a a lo n g w i t h t h e U D P - g a la c t o s e t r a n s
f e r a s e enzym e, was c o n s i d e r e d s o l u b l e b y t h e d e f i n i t i o n
t h a t i t w o u ld n o t s e d i m e n t , e v e n a t 1 5 0 ,0 0 0 x g f o r 1 .5
h o u r s . H ow ever, a f t e r r e m o v a l o f t h e T r i t o n - X - 1 0 0 i n th e
p u r i f i c a t i o n o f t h e enzyme on a S e p h a d e x -G -1 0 0 co lu m n , th e
r e c e p t o r c o u ld be c e n t r i f u g e d o u t o f s o l u t i o n , b u t a l s o
c o u l d b e a s s a y e d f o r i t s r e c e p t o r a b i l i t y a s a f i n e s u s
p e n s i o n .
I n a n o t h e r p r e p a r a t i o n , s tr o m a fro m 15 ml o f c e l l s
w a sh e d i n low i o n i c B t r e n g t h , was e x t r a c t e d i n c o l d , a c i d
i f i e d C H C l^ :m e th a n o l ( 2 : 1 , v / v ) w h ich was th e n made 1 : 1 ,
v / v t o a l l o w p a c k i n g o f t h e s tr o m a on c e n t r i f u g a t i o n . T h is
e x t r a c t i o n was p e r f o r m e d t w i c e . The l i p i d f r e e s tr o m a was
th e n waBhed w i t h 0 . 1M T r i s - H C l , pH 7 . 2 , a n d e x t r a c t e d t w i c e
i n 0 . 5 $ T r it o n - X - 1 0 0 i n t h e T r i s - H C l b u f f e r . A f t e r w a s h in g
away t h e T r i t o n - X - 1 0 0 fro m t h e e x t r a c t e d p e l l e t w i t h t h e
3^
0.1M T r is - H C l b u f f e r , t h e r e s i d u e was s u s p e n d e d i n t h i s
b u f f e r t o a t o t a l volum e o f 8 m l. To t h i s s u s p e n s i o n 8 ml
o f w a t e r - s a t u r a t e d p h e n o l was a d d e d , a n d h e a t e d a t 8 0 ° f o r
a b o u t 10 m i n u t e s . A f t e r t h e p h a s e s s e p a r a t e d , t h e w a t e r
p h a s e w as c l e a r l y re m o v e d , a n d d i a l y z e d e x h a u s t i v e l y a g a i n s t
t h e T r i s - H C l b u f f e r t o rem ove t h e p h e n o l . T h is d i a l y s a t e
was t h e n l y o p h i l i z e d , an d t a k e n up i n 2 ml o f 0.1M T r is - H C l
(pH 7*2 i n 7 x 1 0 - ^M m e r c a p t o e t h a n o l ) , t o a s s a y f o r r e c e p t o r
a b i l i t y .
Enzyme s t u d i e s . Enzyme a s s a y s w e re c a r r i e d o u t w i t h
t h e S e p h a d e x -G -1 0 0 p u r i f i e d enzym e, a f t e r r e c e n t r i f u g a t i o n
o f t h e a c t i v e p e a k s .
IV . RESULTS
W hole C e l l S t u d i e s
The i n c o r p o r a t i o n o f r a d i o a c t i v e am ino a c i d s an d
s u g a r s i n t o w h ole c e l l f r a c t i o n s w as s t u d i e d a s an i n i t i a l
s t e p i n g a i n i n g an i n s i g h t i n t o some o f t h e m ec h a n ism s r e
s p o n s i b l e f o r i n c o r p o r a t i n g g l y c o p r o t e i n i n t o t h e s tr o m a o f
14 i 4
r e t i c u l o c y t e s . G lu c o s a m in e -C , g a l a c t o s e - C and m annoBe-
14
C w ere i n c o r p o r a t e d i n t o t h e n o n - l i p i d p o r t i o n o f s tr o m a
l i n e a r l y u p to f o u r h o u r s . On t h e o t h e r h a n d , i n c o r p o r a -
14
t i o n o f s e r i n e - C i n t o t h i s same f r a c t i o n d e c r e a s e s m a rk
e d l y a f t e r h a l f an h o u r ( F i g . l ) . The k i n e t i c s o f i n c o r
p o r a t i o n o f g l u c o s a m in e i n t o l i p i d was l i n e a r t h r o u g h t h e
two h o u r s s t u d i e d , w h i l e s e r i n e i n c o r p o r a t i o n i n t o l i p i d
d e c r e a s e s a f t e r h a l f an h o u r ( F i g . 2 ) . Though s t e a d y l i n e
a r i n c o r p o r a t i o n o f s e r i n e w as o b s e r v e d i n t h e a c i d r a c e t o n e
e x t r a c t , no g l u c o s a m in e i n c o r p o r a t i o n was d e t e c t e d i n t h i s
f r a c t i o n .
14 i4
I n c o r p o r a t i o n o f g l u c o s a m i n e - l - C and s e r i n e - C
i n t o t h e u n w a sh e d r i b o s o m a l p e l l e t was f o l l o w e d w i t h tim e
( F i g . 3 ) . T h ese r i b o s o m e s w e re p r e p a r e d f o r c o u n t i n g u s i n g
5$ TCA b u t w e re n o t l i p i d e x t r a c t e d . L a b e l l e d am ino a c i d
i n c o r p o r a t i o n shows a r a p i d l a b e l l i n g o f th e r i b o s o m e s ,
35
O / 60 —
* GLUCOSAMINE
- GALACTOSE
- MANNOSE
- SERINE
K- 120
o too
1 2 0
TIME (MIN.)
60 240 180
F ig . 1 . - - I n c o r p o r a t i o n o f l a b e l l e d s u g a r s and s e r i n e i n t o s tr o m a l p r o t e i n .
L O
o\
CPMjML. RETI C U L O C Y T E S
GLUCOSAMINE
SERINE
400C
3 0 0 0 -
2000 -
K O O C
0 3 0 60 9 0 120
TIME CMIN)
F ig , 2 . — I n c o r p o r a t i o n o f s e r i n e - C ^ and g l u c o s a m in e - C ^ i n t o s tro m a l l i p i d s .
CPMjML R E T I C U L O C Y T E S
8 0 0 0 -
6000-j
0 30 60 9 0 120
TIME (MIN)
14 i ii
F ig . 3 . — I n c o r p o r a t i o n o f g lu co sam in e-C and s e r ln e - C
i n t o unwashed rib o s o m a l p e l l e t s .
uo
00
39
l 4
w h i l e g l u c o s a m in e - C i n c o r p o r a t i o n shows a Blow s t e a d y i n
c r e a s e o f l a b e l . R i n s i n g t h e r i b o s o m a l p e l l e t o n c e i n t h e
l y s i n g s o l u t i o n b e f o r e w a s h in g w i t h a c i d rem o v ed much o f
t h e l a b e l , i n d i c a t i n g t h a t i n c o r p o r a t i o n was i n t o s o l u b l e
c o m p o n e n ts . T h is was c o n f i r m e d by d e n s i t y g r a d i e n t a n a l y -
14
s i s . I n c o r p o r a t i o n o f g l u c o s a m i n e - l - C and s e r i n e o r
14
l e u c l n e - C i n t o t h e TCA p r e c i p i t a b l e f r a c t i o n s o f t h e r i
b o so m a l p e l l e t s e p a r a t e d on a s u c r o s e g r a d i e n t a f t e r v a r i
o u s i n c u b a t i o n t i m e s was f o l l o w e d ( P i g . 4 ) . Though much
n e w ly l a b e l l e d p r o t e i n i s a s s o c i a t e d w i t h t h e r i b o s o m e s ,
t h e r e i s v i r t u a l l y no s u g a r i n c o r p o r a t i o n a t t h i s l e v e l .
The l a b e l l e d g l u c o s a m in e a p p e a r s i n t h e s o l u b l e p o r t i o n o f
t h e g r a d i e n t , and i n c r e a s e s w i t h t i m e . I t a p p e a r s c o n c l u
s i v e t h a t g l u c o s a m i n e i s n o t i n c o r p o r a t e d I n t o r i b o s o m e s i n
t h e g e n e r a l r i b o s o m a l p o o l .
14
I n c o r p o r a t i o n o f e i t h e r g lu c o s a m in e - C o r s e r i n e -
14
C I n t o t h e r i b o s o m a l s u p e r n a t a n t b o t h show ed a s t e a d y I n
c r e a s e w i t h tim e ( F i g . 5 ) - H ow ever, b o t h t h e l e v e l o f
l a b e l l i n g o f t h e r i b o s o m e s an d o f t h e r i b o s o m a l s u p e r n a t a n t
w i t h g l u c o s a m in e v a r i e d c o n s i d e r a b l y fro m p r e p a r a t i o n t o
p r e p a r a t i o n , o f t e n n o t b e i n g d e t e c t a b l e . I t seem s p o s s i b l e
t h a t a co m p o n e n t l o o s e l y a s s o c i a t e d w i t h t h e s tr o m a m ig h t be
r e l e a s e d I n t h e l y s i n g p r o c e d u r e . Lo s b o f t h e s t r o m a l g a
l a c t o s e r e c e p t o r by low i o n i c s t r e n g t h w a sh e s was o b s e r v e d
I n l a t e r e x p e r i m e n t s ( T a b l e 8 ) .
3000
2750
2500
2250
2000
7750
1500
1250
1 0 0 0
750
5 00
250
0
Q°260
- -4 GLUCOSAMINE- C ‘4 CPM
. - . . LEUCINE-C1 4 o r SER/NE-C I 4CPmI
8 1 2
FRACTION NO-
,14
F ig . 4 . — S u c ro se g r a d i e n t a n aly sij3 o f g lu co sam in e -C and
s e r in e - C o r le u c in e - C l a b e l l e d rib o s o m e s .
— ° SERINE x 10
• GLUCOSAMINE
90 30 60
iii
F ig . 5 . — I n c o r p o r a t i o n o f g lu co sam in e-C and s e r in e -C
i n t o rib o s o m a l s u p e r n a t a n t p r o t e i n s .
42
R e l e a s e o f P r o d u c t s b y P r o n a s e
T r e a tm e n t o f t h e s tr o m a w i t h p r o n a s e r e l e a s e d t h e
r a d i o a c t i v e p r o d u c t s I n a s o l u b l e fo rm . The e l u t i o n p a t
t e r n s on G-25 S e p h a d ex w e re e s s e n t i a l l y t h e same f o r g l u c o s -
14 14 14 / /- \
am in e-C , g a l a c t o s e - C , and m annose-C ( F i g . o ) . The
r a d i o a c t i v e su g a rB a p p e a r e d b o t h I n t h e am ino a c i d p e a k and
i n t h e s i a l i c a c i d p o s i t i v e p e a k . S in c e r a d i o a c t i v e g l y c o -
p e p t l d e s w ere r e l e a s e d b y p r o n a s e , i t I s e s t a b l i s h e d t h a t
t h e p r o d u c t s i n t o w h ic h t h e s u g a r s w ere i n c o r p o r a t e d i n
c l u d e g l y c o p r o t e i n s .
C e l l F r e e E x p e r i m e n t s
14
The i n c o r p o r a t i o n o f g l u c o s a m i n e - l - C i n t o a TCA
p r e c i p i t a b l e ( n o n - l i p i d e x t r a c t a b l e ) p r o d u c t i n a c e l l f r e e
s y s te m w as e x a m in e d ( T a b le l ) . F o r c o m p a r is o n , t h e r e s u l t s
14
o b t a i n e d f o r t h e i n c o r p o r a t i o n o f U L - s e r in e - C i n t o p r o
t e i n i n a c e l l f r e e s y s te m a r e shown ( T a b le 2 ) . I t can be
s e e n t h a t r i b o s o m e s , w h ich a r e a c t i v e i n p r o t e i n s y n t h e s i s ,
h a v e no e f f e c t on g l u c o s a m in e i n c o r p o r a t i o n i n t o g l y c o p r o
t e i n . S t i m u l a t i o n o f i n c o r p o r a t i o n w o u ld be e x p e c t e d i f
g l u c o s a m in e w ere a d d e d t o t h e n e w ly fo rm ed p r o t e i n b e f o r e
t h e p r o t e i n was r e l e a s e d fro m t h e r i b o s o m e s . The p o s s i b i l
i t y t h a t m em b ran e-b o u n d r i b o s o m e s , n o t i s o l a t e d i n t h e r i -
b o s o m a l f r a c t i o n , may f u n c t i o n i n t h i s r e g a r d was t e s t e d by
a d d i t i o n o f membrane t o t h e p r o t e i n s y n t h e s i z i n g s y s te m .
I n c o r p o r a t i o n o f s e r i n e i n t o p r o t e i n , h o w e v e r, was som ewhat
^3
2 . 0
M A N N O S E -C
3 00
200
1 0 0
0 . 0
2.5
GLUCOSAM INE- C
600
2 0
5 00 ~
4 0 0 ~
gj3 0 0
200
100
0.5
4 0 0
G A L A C T O S E -C
3 0 0
200
1 0 0
Q 0
10 2 0 25 3 0
FRACTION NO.
F i g . 6 . - - E l u t i o n o f l a b e l l e d g l y c o p e p t l d e s fro m
S e p h a d e x - G - 2 5 a f t e r l a b e l l i n g i n i n t a c t c e l l s .
44
TA B L E 1
THE CELL FREE BIOSYNTHESIS OF BABBIT RETICULOCYTE MEM BRANE GLYCOPROTEIN*
S y stem
CPM CPM/mg P r o t e i n
C om plete ( u s in g d ia ly z e d
s u p e r n a ta n t f l u i d )
2 0 ,0 0 0 X g
727 528
C om plete ( u s in g t h e r e d
f r a c t i o n o f t h e 1 5 0 ,0 0 0 X g
sp u n s u p e r n a ta n t f l u i d )
795 57
C o m p le te, u s in g t h e c l e a r
a q u e o u s f r a c t i o n o f th e
1 5 0 ,0 0 0 X g sp u n s u p e r
n a t a n t f l u i d )
655
2 ,4 7 0
l e s s c l e a r s u p e r n a ta n t f l u i d 0
l e s s m embrane
85
p l u s rib o s o m e s 640
p l u s r i b o n u c l e a s e a 720
p lu s C t-am ylase
565
l e s s g a l a c t o s e a n d N - a c e ty l-
n e u ra m ln ic a c i d
255
p l u s CTP, GTP, UTPC 282
d
p l u s g l u t a t h i o n e 255
l e s s m e r c a p to e th a n o l
578
p l u s 6- a z a u r i d i n e e
715
*The c o m p le te s y ste m I n c lu d e s 0 .2 m l o f s u p e r n a ta n t f l u i d ,
0 .1 m l m embrane s u s p e n s io n ( l — 2 m g ), 1 pm e a c h o f g a l a c t o s e an d N -a c e -
ty ln e u r a m in ic a c i d , 1 pC D -g lu c o sa m in e -C 1 4 , 0 .0 0 4 m m e r c a p to e th a n o l,
0.001M M gCIs, 0.05M T ris -H C l b u f f e r , pH 7 . 2 - 7 . 4 , a n d 0.05M KC1. In c u
b a t i o n w a B c a r r i e d o u t f o r o n e h o u r , a f t e r w h ich 10$ TCA w as a d d e d .
The z e r o tim e c o u n ts , 55 CFM, w ere s u b t r a c t e d fro m e ac h r e a d in g .
, a 0 .2 5 mg i n 0 .1 m l. ^ 0 .2 5 mg i n 0 .1 m l. C0 .2 5 p m o le s.
4 X 10“ 3 M. e 4 .5 X 10 M.
45
TABLE 2
THE CELL FREE INCORPORATION OF SERINE-C14 INTO PROTEIN
OF RABBIT RETICULOCYTES*
S y stem
CPM I n c o r p o r a te d
C om plete (u s in g d i a ly z e d
2 0 S s u p e r n a t a n t )
3 ,5 0 0
C om plete ( u s in g th e c l e a r
a q u eo u s f r a c t i o n o f t h e
c e n t r i f u g e d s u p e r n a t a n t )
2 ,3 3 2
C om plete ( u s in g t h e r e d
f r a c t i o n o f t h e c e n t r i
fu g e d s u p e r n a t a n t )
3 ,3 9 2
l e s s s u p e r n a ta n t f r a c t i o n s 170
l e s s ATP, c r e a t i n e p h o s p h a te ,
c r e a t i n e p h o s p h o k in a s e
1 ,1 6 0
p l u s mem brane 1 ,6 2 0
l e s s rib o s o m e s 680
l e s s g l u t a t h i o n e 2 ,2 ^ 0
l e s s CTP, UTP 2 ,4 0 0
l e s s GTP 1 ,0 1 7
p l u s r ib o n u c le a s e
195
p l u s 6— a z a u r i d i n e 3 ,3 0 8
p l u s C t-am ylase
1 ,1 9 5
*The c o m p le te sy ste m I n c lu d e s 0 .2 m l s u p e r n a t a n t f l u i d , 0 .0 5
m l o f th e am ino a c i d m ix tu r e o f L i n g r e l l an d B o rso o k ( 9 9 ) j 120 pg
c r e a t i n e k i n a s e , 10 pM c r e a t i n e p h o s p h a te , 1 pM ATP, 0 .2 5 pM GTP,
20 pM g l u t a t h i o n e , 1 -2 mg rib o s o m e s , JpC s e r in e - C 1 4 , 0.001M M gCle,
0.05M T r is - H C l b u f f e r , a n d 0.05M KC1. I n c u b a tio n w as c a r r i e d o u t f o r
on e h o u r , a f t e r w h ich 10$ TCA w as a d d e d t o t e r m i n a t e th e r e a c t i o n .
The c o u n ts a t 0 t i m e , 176 CPM, w ere s u b t r a c t e d fro m a l l s a m p le s .
46
i n h i b i t e d r a t h e r t h a n e n h a n c e d . N u c l e o s i d e t r i p h o s p h a t e s
an d g l u t a t h i o n e , a t t h e c o n c e n t r a t i o n s u s e d , h a v e o p p o s i t e
e f f e c t s on s e r i n e a n d g l u c o s a m in e i n c o r p o r a t i o n .
I n h i b i t i o n b y a - a m y l a s e , w h ic h i n h i b i t s b o t h g l u
c o s a m in e a n d am ino a c i d i n c o r p o r a t i o n , a p p e a r s t o b e n o n
s p e c i f i c . R i b o n u c l e a s e d r a s t i c a l l y i n h i b i t s p r o t e i n s y n
t h e s i s , b u t h a s no e f f e c t on g l y c o p r o t e i n s y n t h e s i s .
G lu c o s a m in e 1 b i n c o r p o r a t e d i n t o a T C A - p r e c i p i t a b l e
p r o d u c t o f t h e s u p e r n a t a n t f l u i d o b t a i n e d b y c e n t r i f u g a t i o n
a t 2 0 ,0 0 0 x g ( T a b l e l ) . The v a r i o u s s u p e r n a t a n t f r a c
t i o n s , o b t a i n e d b y p r o l o n g e d h i g h s p e e d c e n t r i f u g a t i o n ,
w e re a l s o t e s t e d f o r t h i s a b i l i t y t o i n c o r p o r a t e g l u c o s
am ine i n t o a TCA p r e c i p i t a t e ( T a b l e l ) . W h ile i n c o r p o r a
t i o n i n t o t h e 20S s u p e r n a t a n t f l u i d d o e s n o t d e p e n d on t h e
p r e s e n c e o f s t r o m a , t h e c l e a r s u p e r n a t a n t f l u i d c an no
l o n g e r s u p p o r t g l u c o s a m in e I n c o r p o r a t i o n i n t o a TCA i n s o l u
b l e p r o d u c t i n t h e a b s e n c e o f s t r o m a . I n a d d i t i o n to
s tr o m a i t s e l f , i n c o r p o r a t i o n i n t o s tr o m a i s d e p e n d e n t on
t h e p r e s e n c e o f s o l u b l e enzyme o f t h e c l e a r s u p e r n a t a n t
f l u i d . I t a p p e a r s t h a t t h e r e c e p t o r com p o n en t i n t h e 20S
s u p e r n a t a n t f l u i d , r e s p o n s i b l e f o r t h e I n c o r p o r a t i o n o f
g l u c o s a m in e i n t o a TCA i n s o l u b l e p r o d u c t h a s b eg u n t o s e t
t l e o u t a s t h e f l u f f y l a y e r o v e r t h e p e l l e t .
A t t e m p t s w e re made t o p u r i f y t h e s o l u b l e e n z y m e (s )
b y ammonium B u l f a t e f r a c t i o n s . The e n zy m e(b ) r e m a in e d s o l
u b l e i n u p t o 75$ s a t u r a t i o n w i t h ammonium s u l f a t e th o u g h
47
much p r o t e i n w as p r e c i p i t a t e d .
The p r e s e n c e o f 6 ~ a z a u r i d i n e , a t a c o n c e n t r a t i o n
shown t o i n h i b i t g l y c o p r o t e i n s y n t h e s i s i n s t u d i e s w i t h i n
t a c t E h r l i c h a s c i t e s c e l l s , h a d no e f f e c t on g l u c o s a m i n e
i n c o r p o r a t i o n i n t h i s s y s te m .
I n o r d e r t o a r r i v e a t a c l o s e r i d e n t i t y o f t h e e n
zym es r e s p o n s i b l e f o r t h e a d d i t i o n o f s u g a r m o i e t i e s i n t o
t h e m em brane, two n u c l e o t i d e s u g a r s w e re u s e d i n t h e c e l l
f r e e s y s t e m . T h e r e w as no s i g n i f i c a n t i n c o r p o r a t i o n o f N-
a c e t y l g l u c o B a m i n e fro m U D P - N - a c e t y lg l u c o s a m i n e i n t o s t r o m a
e i t h e r i n t h e p r e s e n c e o r a b s e n c e o f t h e s u p e r n a t a n t f l u i d .
14
On t h e o t h e r h a n d , g a l a c t o s e , f ro m U D P - g a la c t o s e - C was
i n c o r p o r a t e d i n t o t h e s t r o m a i n t h e c e l l f r e e s y s t e m , and
some o f t h e p r o p e r t i e s o f t h e i n c o r p o r a t i n g s y s te m w e re
B t u d i e d ( T a b le 3 )•
U D P - g a l a c t o s e i n c o r p o r a t i o n , i n c o n t r a s t t o g l u c o s
a m in e i n c o r p o r a t i o n , i s c o m p l e t e l y d e p e n d e n t u p o n m em brane
a l o n e ; n o n e o f t h e s o l u b l e enzy m es i n a n y o f t h e s u p e r n a
t a n t f r a c t i o n s t r a n s f e r r e d g a l a c t o s e fro m U D P - g a la c t o s e
i n t o a TCA i n s o l u b l e p r o d u c t w i t h o u t t h e p r e s e n c e o f t h e
m em brane a s a r e c e p t o r . The t r a n s f e r i s e n h a n c e d b y t h e
p r e s e n c e o f t h e s u g a r s a n d t h e m e r c a p t o e t h a n o l .
Some o f t h e p r o p e r t i e s o f t h e U D P - g a la c t o s e i n c o r
p o r a t i n g s y s te m w e re n e x t e x a m in e d . The tim e c o u r s e o f i n
c o r p o r a t i o n , b o t h i n t o g l y c o p r o t e i n a n d i n t o l i p i d f o r com
p a r i s o n , was f o l l o w e d ( P i g . 7)* I n c o r p o r a t i o n i n t o g l y c o -
48
TABLE 3
UDP-GALACTOSE INCORPORATION INTO GLYCOPROTEIN IN A CELL FREE SYSTEM
OF RABBIT RETICULOCYTES*
S y stem CPM I n c o r p o r a te d
C om plete ( u s in g t h e y e llo w
f r a c t i o n o f t h e c e n t r i
fu g e d s u p e r n a t a n t )
499
l e s s m em brane f r a c t i o n
59
l e s s s u g a r s
251
l e s s m e r c a p to e th a n o l 172
p l u s ATP 409
C om plete ( u s in g t h e r e d
f r a c t i o n o f t h e c e n t r i
fu g e d s u p e r n a t a n t )
554
l e s s mem brane 0
C om plete ( u s in g t h e a q u eo u s
f r a c t i o n o f t h e c e n t r i
fu g e d s u p e r n a ta n t)
599
l e s s mem brane 87
*The c o m p le te s y ste m I n c lu d e s 0 .1 m l o f s u p e r n a ta n t f l u i d ,
0 .1 m l o f membrane s u s p e n s io n (1 -2 mg p r o t e i n ) , 0 .5 mg e a c h o f N-
a c e t y l g a l a c t o s a m i n e , an d N - a c e ty ln e u r a m in ic a c i d , 0.004m m e rc a p to
e t h a n o l , 0.001M MgCl2 , O.IM T r is - H C l b u f f e r , pH 7 -2 ~ 7 « 4 , a n d 0 .2 5 p.C
U D P -g a la c to se -C * * . I n c u b a tio n w as c a r r i e d o u t f o r one h o u r , a f t e r
w h ic h 10$ TCA w as ad d ed t o te r m i n a t e t h e r e a c t i o n . The z e r o tim e
sam ple c o u n ts , 80 CPM, w ere s u b t r a c t e d fro m e a c h r e a d in g .
C P M jM G PROTEIN
550
500
450
400
350
300
250
200
150
100
50
0 0 10 20 30 40 50 60 70 80 90
TIME [MINI
lb lb
F ig . 7 - — Time c u rv e : I n c o r p o r a t i o n o f g a la c t o s e - C from U D P -g a la cto se -C i n t o
stro m a i n c e l l f r e e sy ste m . R e a c tio n m ix tu re i n c l u d e s 0 .1 ml s tr o m a l susp en sion ,, 0 .4 nC
U D P - g a la c t o s e - C ^ , 20 nM Mn++, 4x 10“3m m e r c a p to e th a n o l and T ris -H C l b u f f e r , pH 7 . 2 . ^
-~°CPM INTO GLYCOPROTEIN
— *CPM INTO LIPID EXTRACT
50
l i p i d l e v e l s o f f i n 90 m i n u t e s , th o u g h a t a p r o g r e s s i v e l y
d e c r e a s i n g r a t e .
The e n h a n c e m e n t o f t h e t r a n s f e r b y N - a c e t y l g a l a c t o s -
am ine a n d N - a c e t y l n e u r a m i n i c a c i d t o membrane g l y c o p r o t e i n
was ?lm oB t m ax im al a t a c o n c e n t r a t i o n o f 0 , 7 m g/m l ( F i g . 8 } .
The d e p e n d e n c e o f t h e t r a n s f e r t o g l y c o p r o t e i n on
++ -I I
t h e p r e s e n c e an d c o n c e n t r a t i o n o f Mn o r Mg was a l s o
s t u d i e d ( F i g . 9)* F o r c o m p a r is o n , t h e d e p e n d e n c e o f t h e
t r a n s f e r o f g a l a c t o s e t o l i p i d on t h e same m e t a l i o n s i s
shown i n F i g u r e 1 0 . I n a l l c a s e s , t h e o p t i m a l c o n c e n t r a -
++
t i o n i s a p p r o x i m a t e l y 20 i j l M . H ow ever, Mg s u b s t i t u t e s
+ +
p o o r l y f o r Mn i n t h e g l y c o p r o t e i n s y s te m , an d som ew hat
b e t t e r i n t h e t r a n s f e r to l i p i d .
R e l e a s e o f P r o d u c t s fro m S tro m a b y P r o n a s e
I s o l a t i o n o f t h e p r o d u c t s o f t h e t r a n s f e r o f UDP-
14 14
g a l a c t o s e - C and g l u c o s a m i n e - 1-CJ t o t h e s tr o m a w as b e g u n
fro m p r o n a s e d i g e s t s o f t h e l a b e l l e d s t r o m a l p r o d u c t . The
e l u t i o n p a t t e r n fro m t h e G -25 S e p h a d e x colum n ( F i g . l l )
14
show ed t h a t t h e p r o d u c t c o n t a i n i n g g a l a c t o s e - C was i s o
l a t e d a s a s h a r p p e a k , j u s t p r e c e d i n g t h e am ino a c i d p e a k ,
w h i l e t h e p r o d u c t o f g l u c o s a m i n e t r a n s f e r Bhowed t h e same
b r o a d e r , m u l t i - c o m p o n e n t p e a k t h a t was o b s e r v e d a f t e r w h o le
c e l l i n c u b a t i o n s .
100
50 f
0
000 0.75 150 225 ZOO
mg/ml
F ig . 8 . — S u g ar c o n c e n t r a t i o n e f f e c t on g a l a c t o s e
t r a n s f e r to th e s tr o m a l r e c e p t o r . ui
CPM M G P R O T E IN
2000-
1200-
0 9.6 19.2 38.4
METAL ION CONCENTRATION (jjM)
Fig. 9 - — M etal Ion c o n c e n t r a t i o n e f f e c t on th e t r a n s f e r
o f g a l a c t o s e to s tr o m a l p r o t e i n s .
CPMI M G PROTEIN
1 2 0 0
Mn
1000
800
600
200
9.6 38.4
19.2
METAL ION CONCENTRATION LpM]
l2i ■ ) 2;
F ig . 1 0 . — Dependence o f I n c o r p o r a t i o n o f g a la c t o s e - C from U D P -g alacto se-C
i n t o s tr o m a l l i p i d s on m e ta l io n c o n c e n t r a t i o n .
ui
uo
NdO
54
600
GLUCOSAM INE-C
2.5
A O
20 0
g a l a c t o s e - c — 2.5
2 0 0
— v
l-T*-.___1 ________I _____ :
15
FRACTION NO.
2 5
0
b
1 I
i i
i !
? ?
v
F i g . 1 1 . - - E l u t i o n o f l a b e l l e d g l y c o p e p t i d e s from
S e p h a d e x -G -2 5 a f t e r l a b e l l i n g i n a c e l l f r e e s y s te m .
55
F r a c t i o n a t i o n o f S tro m a a n d
S o l u b i l i z a t i o n o f T r a n s f e r a s e
I t seem ed d e s i r a b l e t o f r a c t i o n a t e t h e r e t i c u l o c y t e
s t r o m a i n o r d e r t o s e p a r a t e t h e U D P - g a la c t o s e t r a n s f e r a s e
fro m i t s r e c e p t o r , b o t h o f w h ic h a r e m em brane b o u n d . A t
t e m p t s w e re t h e r e f o r e made to f i n d an a r t i f i c i a l ( e x o g e n o u s )
r e c e p t o r w h ic h w o u ld a c c e p t g a l a c t o s e fro m t h e m em brane-
b o u n d U D P - g a la c t o s e t r a n s f e r a s e . The e x o g e n o u s r e c e p t o r s
t r i e d i n t h e s e e x p e r i m e n t s i n c l u d e d e p i d i d i m a l enzym e-
t r e a t e d ( d e s i a l y z e d ) f e t u i n , a n d a ^ - g l y c o p r o t e i n , d e s i a l y z e d
p o r c i n e s u b m a x i l l a r y m u cin and d e s i a l y z e d p o r c i n e s u b m a x i l -
l a r y m ucin l a c k i n g f u c o s e , a n d h e l l c a s e - t r e a t e d ( d e s i a l y z e d )
f e t u i n an d a ^ - g l y c o p r o t e i n . The c o m p a r a t i v e a c c e p t i n g a b i l
i t i e s p e r mg r e c e p t o r w e re c o m p a red ( T a b l e 4 ) .
R em oval o f s i a l i c a c i d a l o n e d o e s n o t p r o d u c e t h e
r e c e p t o r a b i l i t y o f t h e p o r c i n e s u b m a x i l l a r y g l y c o p r o t e i n .
H ow ever, s u b s e q u e n t r e m o v a l o f t h e f u c o s e m o ie t y d o e s ,
th o u g h t h e r e a c t i v i t y i s r a t h e r lo w . D e s i a l y z e d f e t u i n e x
h i b i t s a v e r y s m a l l r e c e p t o r a b i l i t y , th o u g h n o t a s much a s
h e l l c a s e o r e p i d i m a l - t r e a t e d f e t u i n . B o th h e l i c a s e an d e p i -
d i m a l - t r e a t e d a ^ - g l y c o p r o t e i n a r e v e r y good r e c e p t o r s , I n
te r m s o f i n c o r p o r a t i n g en o u g h l a b e l l e d g a l a c t o s e fro m UDP-
14
g a l a c t o s e - C to a s s a y e a B i l y .
A t t e m p t s w e re a l s o made t o f i n d an e x o g e n o u s r e c e p
t o r f o r t h e l i p i d t r a n s f e r r i n g a c t i v i t y . T h re e r e c e p t o r s
w e re t r i e d : ( l ) Tay S a c h s g a n g l i o s i d e , a g i f t o f D r. J .
O ’B r i e n , (2 ) b r a i n g a n g l i o s l d e s , fro m w h ic h B u g a rs w ere r e -
56
TABLE 4
TRANSFER OF GALACTOSE FROM UDP-GALACTOSE BY MEMBRANE BOUND TRANSFERASE
TO EXOGENOUS RECEPTORS*
S ystem
CPM
I n c o r p o r a te d
CPM/mg E xog en o u s
R e c e p to r ( l e s s
en d o g en o u s c o n t r o l )
C om plete
1 ,1 8 5
p l u s e p id im a l t r e a t e d
( d e s i a l y z e d ) f e t u i n
1 1 ,8 4 0
p l u s e p id im a l t r e a t e d
( d e s i a l y z e d ) a ^ - g l y c o -
p r o t e i n
18 ,9 0 0
p l u s h e l i c a s e t r e a t e d
( d e s i a l y z e d ) f e t u i n
7,337®
—
p l u s h e l i c a s e t r e a t e d
( d e s i a l y z e d ) C ti-g ly c o
p r o t e i n
2 2 ,0 5 0
p l u s d e s i a l y z e d f e t u i n
p l u s d e s i a l y z e d p o r c in e
s u b m a x illa r y m ucin
3 ,6 0 5
27
p lu s d e s i a l y z e d p o r c in e
s u b m a x illa r y m u c in ,
la c k i n g f u c o s e
2 ,1 2 5
*The c o m p le te sy ste m I n c lu d e d MnClg (20(iM ), 0 .1 m l membrane
s u s p e n s io n (1— 2 mg p r o t e i n ) , 0.1M T r is - H C l b u f f e r , pH 0 .2 5 p.C
U D P -g a la c to se — C1 4 , a n d a p p r o x im a te ly 0 .4 mg r e c e p t o r g l y c o p r o t e i n ,
t r e a t e d w ith enzym es a s d e s c r ib e d i n t h e t e x t .
6 1
S o lu t io n w as c lo u d y — e x a c t c o n c e n t r a t i o n n o t know n.
57
moved by m i l d a c i d h y d r o l y s i s , and ( 3 ) c h l o r o f o r m r m e t h a n o l
( 2 : 1 ) e x t r a c t s o f t h e s tr o m a o f r e t i c u l o c y t e s . T h ese r e c e p
t o r s e x h i b i t e d m in im a l a c t i v i t y , and w ere n o t s u f f i c i e n t l y
a c t i v e t o be u s e f u l . The m a jo r p r o b le m was i n o b t a i n i n g
one p h a s e s y s te m w h ic h w o u ld p e r m i t r e a c t i o n . B o th Tw een-
80 an d T r i t o n - X - 1 0 0 , s e p a r a t e l y and t o g e t h e r , w e re u s e d i n
t r y i n g t o m a i n t a i n an a c t i v e s u s p e n s i o n . I t a p p e a r s t h a t
a b r o a d e r s u r v e y o f d e t e r g e n t s w i l l h a v e to be m ade. P e r
h a p s a s y s te m su c h a s t h a t e m p lo y ed by Roseman and h i s c o l
l e a g u e s ( 9 2 ) m ig h t s u f f i c e .
Once an e x o g e n o u s r e c e p t o r was fo u n d , a t t e m p t s w ere
made to s o l u b i l i z e e i t h e r t h e e n d o g e n o u s r e c e p t o r o r t h e
t r a n s f e r a s e enzym e away fro m t h e m em brane, t h e r e b y e f f e c t
i n g t h e i r s e p a r a t i o n . The r e s u l t s o f s o n i c a t i o n and e x
t r a c t i o n w i t h v a r i o u s a g e n t s , i n c l u d i n g b u t a n o l , d i g i t o n l n ,
u r e a , T r i t o n - X - 1 0 0 , an d so d iu m d o d e c y l s u l f a t e a r e shown i n
T a b le 5 . B u t a n o l e x t r a c t i o n i n a c t i v a t e d a l l enzyme a c t i v
i t y . S o n i c a t i o n d i d n o t l i b e r a t e e i t h e r t h e enzyme o r th e
e n d o g e n o u s r e c e p t o r , b u t seem ed t o h a v e a s t i m u l a t o r y e f
f e c t on t h e p a r t i c u l a t e - e n z y m e c a t a l y z e d t r a n s f e r o f g a l a c -
toB e t o membrane l i p i d . D i g i t o n i n h a d som ew hat t h e same e f
f e c t , th o u g h i t i n h i b i t e d t h e t r a n s f e r o f g a l a c t o s e t o g l y
c o p r o t e i n b y a b o u t 6 0 $ . U re a a l s o d i d n o t l i b e r a t e t h e
enzyme o r r e c e p t o r , and h a d an i n h i b i t o r y e f f e c t on b o t h
l i p i d a n d p r o t e i n s y s t e m s . A lth o u g h so d iu m d o d e c y l s u l f a t e
an d T r i t o n - X - 1 0 0 b o t h seem ed p r o m i s i n g , t h e so d iu m d o d e c y l
T A B L E 5
S O L U B IL IZ A T IO N O F S T R O M A L C O M P O N E N T S : E F F E C T S O N U D P -G A L A C T O S E T R A N S F E R A S E S
F O R LIPID A N D G L Y C O P R O T E IN R E C E P T O R S *
Stromal Fractions after Treatment "w ith
Various Reagents or Conditions
C P M Incorporated Into
Stromal Proteins
C P M .Incorporated into
Stromal Lipids
Sonication
150,000 X g pellet 962 3,296
150,000 X g supernatant
25 268
2096 Butanol Extraction
Aqueous phase
Butanol phase All fractions had no activity
Interphase
Dlgltonin
150,000 X g pellet 375 J+,777
150,000 X g supernatant 39 O
6 M Urea
30,900 X g supernatant 58 38 9
30,900 X g pellet 123 1,253
Trlton-X-lOO
150,000 X g pellet 180 1,776
150,000 X g supernatant + 150,000 X g pellet 1,998 1,782
150,000 X g supernatant 0
Sodium Dodecyl Sulfate
150,000 X g pellet lO 0
150,000 X g supernatant 12 310
N o treatment
W hole suspension
913
2,278
*The assay included 20 ( j l M M n , 0.1 m l of each stromal fraction, 0.1 p C UDP-galactose-C14, and. a
final concentration of 0.05M Tris-HCl buffer, p H 7 • 2— 7 - * + , m ade 7 x lO-' * M mercaptoethanol. A 1 3 results are
corrected, to equal original volumes of the stromal suspension.
v_n
CD
59
s u l f a t e t e c h n i q u e h a d two m a jo r d i s a d v a n t a g e s ; t h e r e a c t i o n
iB i n h i b i t e d i n i t s p r e s e n c e , a n d i t l i b e r a t e s n o t o n l y
g l y c o p r o t e i n i n c o r p o r a t i n g a c t i v i t y , b u t a l s o l i p i d i n c o r
p o r a t i n g a c t i v i t y . On t h e o t h e r h a n d , T r i t o n - X - 1 0 0 s t i m u
l a t e d a c t i v i t y , an d l i b e r a t e d o n l y t h e n o n - l i p i d i n c o r p o r a t
i n g a c t i v i t y .
The r e s u l t s o f t e s t i n g t h e a c t i v i t y o f t h e T r i t o n -
X -100 e x t r a c t an d t h e T r i t o n - X - 1 0 0 e x t r a c t e d p e l l e t ( T a b le
6) show t h a t , w h i l e t h e p e l l e t e x h i b i t s v e r y l i t t l e t r a n s
f e r a s e a c t i v i t y , t h e T r i t o n - X - 1 0 0 e x t r a c t c o n t a i n s t h e e n
zyme i n a v e r y h i g h s t a t e o f a c t i v i t y . I t can a l s o be s e e n
t h a t t h e T r i t o n - X - 1 0 0 l i b e r a t e s much o f t h e e n d o g e n o u s a c
t i v i t y .
The l i b e r a t i o n o f t r a n s f e r a s e a c t i v i t y and e n d o g e n
o u s r e c e p t o r a c t i v i t y c an be f o l l o w e d I n T a b le 7* H e re t h e
T r i t o n - X - 1 0 0 c o n c e n t r a t i o n waB v a r i e d , a n d t h e 1 5 0 ,0 0 0 x g
s u p e r was a s s a y e d f o r t r a n s f e r a s e a c t i v i t y b o t h w i t h and
w i t h o u t t h e p r e s e n c e o f a ^ - g l y c o p r o t e i n a s an e x o g e n o u s r e
c e p t o r . W h ile 0 . 7 5 $ ( v / v ) T r i t o n - X - 1 0 0 r e l e a s e d a maximum
o f e n d o g e n o u s a c t i v i t y , t h e maximum enzyme a c t i v i t y to w a rd
t h e a ^ - g l y c o p r o t e i n was a t t a i n e d a t a c o n c e n t r a t i o n o f
0 . 2 5 $ ( v / v ) T r i t o n - X - 1 0 0 .
The p u r i f i c a t i o n o f t h e enzyme was f o l l o w e d u s i n g
t h e a ^ - g l y c o p r o t e i n a s an e x o g e n o u s r e c e p t o r ( T a b le 8 ) .
E l u t i o n o f t h e enzyme fro m t h e S e p h a d e x -G -1 0 0 colum n I s
Bhown i n F i g u r e 12. I n i t i a l a s s a y s w ere done w i t h an d w i t h -
6o
TABLE 6
UDP-GALACTOSE TRANSFERASE ACTIVITY OF TRITON-X-lOO
TREATED STROMAL FRACTIONS*
F r a c t i o n
S tr o m a l s u s p e n s io n i n T r i t o n -
X— 100 "b efo re c e n t r i f u g a t i o n
T r ito n - X - 1 0 0 e x t r a c t e d s tr o m a l
p e l l e t
T r i t o n — X — 100 e x t r a c t e d s tr o m a l
p e l l e t p l u s h e l i c a s e t r e a t e d
Q C jl — g l y c o p r o t e i n a s e x o g en o u s
r e c e p t o r
T r ito n - X - 1 0 0 e x t r a c t o f s tro m a
T r i t o n — X— 100 e x t r a c t o f s tro m a
p l u s h e l i c a s e t r e a t e d ^ - g l y c o
p r o t e i n a s e x o g e n o u s r e c e p t o r
CPM o f G a la c to s e - C 14
I n c o r p o r a t e d
1+73
73
1,277
270
8,600
*The a s s a y in c lu d e d 1 m + iM U D P -g a la c to s e — C^4 (0.01+ jiC ), 20 pM
M nC le, 0 . 1 m l o f t h e T r ito n - X - 1 0 0 e x t r a c t o r o f a s u s p e n s io n o f t h e
T r i t o n e x t r a c t e d p e l l e t , re s u s p e n d e d i n t h e same c o n c e n t r a t i o n o f
T r i t o n , an d w h ere p r e s e n t , 0.1+ mg o f h e l i c a s e t r e a t e d C H ^ -g ly c o p ro te in ,
a l l i n a f i n a l c o n c e n t r a t i o n o f O.O^M T r is - H C l " b u ffe r, pH 7 -2-7.1* a n d
a f i n a l volum e o f 0 .1 6 m l.
61
TABLE 7
RELEASE OF UDP-GALACTOSE TRANSFERASE ACTIVITY FROM TRUON-X-lOO
EXTRACT FROM STROMA*
v / v $ T rito n -X -1 0 0 CPM I n c o r p o r a te d CPM I n c o r p o r a te d I n to
i n T r is - m e r c a p to e th a n o l i n t o E n d o genous R e c e p to r* E x o g en o u s R e c e p to r*
0 .0 0 115 6^3
0 .0 1 110 858
0 .1 0 481 1 3 ,8 5 8
0.2 5
590 20,555
0 .5 0 810
17,815
0.7 5
1 ,0 0 6
1 7 ,7 9 5
1 .0 0 780
1 7 ,7 6 7
3 .0 0 800 17 ,5 0 0
5 .0 0 503
1 5 ,2 9 3
*The s tr o m a l s u s p e n s io n w as d i l u t e d In h a l f w ith v a r io u s con
c e n t r a t i o n s o f T r ito n - X - 1 0 0 , y i e l d i n g t h e f i n a l c o n c e n t r a t i o n s l i s t e d
a h o v e . The d e t e r g e n t s o l u t i o n s a n d s tr o m a l s u s p e n s io n s w ere made up
I n 0.1M T r is - H C l b u f f e r , pH 7*2— '7 .4 , made 7 x 10*”4M i n m e r c a p to e th a n o l.
E ndogenous r e c e p t o r r e f e r s t o t h a t r e c e p t o r p r e s e n t i n th e
s tro m a . E xogenous r e c e p t o r r e f e r s t o h e l i c a s e t r e a t e d CCx-gly c o p r o t e i n .
The a s s a y w as p e rfo rm e d o n 0 . 1 m l o f th e 1 5 0 ,0 0 0 x g s u p e r
n a t a n t f l u i d o f th e T r i t o n s u s p e n s io n , an d in c lu d e d 20 pM Mn , 0 .1 pC
U D P -g a la c to se -C 1 4 , a n d w h ere i n d i c a t e d , a b o u t 4 mg o f t h e h e l i c a s e
t r e a t e d a ^ - g l y c o p r o t e i n .
T A B L E 8
P U R IF IC A T IO N O F U D P -G A L A C T O S E T R A N S F E R A S E A C T I V I T Y *
Sam ple C F M /M g Protein
PurITlaatIon
Factor
Theoretleal
Activity®
Total
Activity®
Stroma, obtained from French press lysate
Zero time
1 hr. incubation
1 hr. incubation plus Triton— X — lO O
1 hr. Incubation plus helicase
treated Qi— glycoprotein
1 hr. incubation plus helicase
treated C £ j _ — glycoprotein
and Triton-X-100
263
1,054
160
2,482
—
—
270,340
44,100
680,364
Stroma, alter vashing in 0.005M Tris
buTTer, three times
Zero time
1 hr. incubation
1 hr. incubation plus Triton— X — lO O
1 hr. incubation plus helicase
treated C f j _ — glycoprotein
1 hr. incubation plus helicase
treated Q ^— glycoprotein
and Triton-X-100
315
6l4
96
2,566
1.2
less
less
1.03 or
16
255,201
41,630
642,264
167,040
26,100
642,000
Triton— X — lO O extracted pellet
zero time
1 hr. incubation
1 hr. Incubation plus helicase
treated ^-glycoprotein
16,700
6.5 or
lo4
167,o4o
606,6l4 36,500
Triton— X — lO O extract
Zero time
1 hr. incubation
1 hr. incubation plus helicase
treated C t^ — glycoprotein
l,46l
16,700
6.5 or
104
157,686
606, 6l4
48,720
484,300
Sephadex G — 100 purified enzym e
Zero time
1 hr. incubation
1 hr. Incubation plus helicase
treated ctx — glycoprotein
33,300 13 or
208
—
—
*The assay consisted, of 20 p M M n , 0.1 m l of each enzym e Traction, 0.02 m l of U D P — galactose— C 14
(0.04 pC), and where indicated, about 0.4 m g o T helicase treated C K j , — glycoprotein, all in a final concentra
tion oT 0.05M Tris-HCl, p H 7-2— 7.4, and a total volume oT 0.2 ml.
aTheoretical activity refers to tlie total calculated yield, allowing Tor aliquots etc. Total ac
tivity reTers to the total actual yield.
CPM
I
o
o o protein
* -------- HEME
* ------ * ENZYME
ACT
10
20
5
05
- 2
0
0 5 15 10
FRACTION NO.
P ig . 1 2 . - - E l u t i o n o f U D P -g alacto se t r a n s f e r a s e
from a Sephadex-G -100 column.
64
o u t t h e p r e s e n c e o f T r i t o n - X - 1 0 0 , The f i r s t o b v i o u s f a c t i s
t h a t t h e low i o n i c s t r e n g t h t r e a t m e n t c a u s e s l o s s o f t h e
e n d o g e n o u s r e c e p t o r , a p p r o x i m a t e l y 35# b e i n g l o s t i n t h i s
s t e p . Of t h e 6 5 $ r e m a i n i n g , a b o u t 3 0$ o f i t i s e x t r a c t a b l e
i n t h e 0 . 5 $ ( v / v ) T r it o n - X - 1 0 0 e x t r a c t ; t h i s i s a b o u t 20$
o f t h e o r i g i n a l am ount p r e s e n t i n t h e s t r o m a . (T h e s e v a l u e s
r e p r e s e n t an a b s o l u t e maximum, s i n c e t h e y a r e d e r i v e d from
a c t i v i t i e s l e s s t h a n t h r e e t i m e s b a c k g r o u n d . ) A maximum o f
6$ o f t h e e x o g e n o u s a c t i v i t y r e m a i n s . Though o n l y one e x
t r a c t i o n was u s e d , n e a r l y a l l o f t h e enzym e a c t i v i t y i s r e
c o v e r e d i n t h e s u p e r n a t a n t f l u i d .
Prom r e p e a t e d p u r i f i c a t i o n e x p e r i m e n t s , i t was s e e n
t h a t b o t h t h e enzyme and r e c e p t o r a r e m em brane b o u n d , w i t h
a c t i v i t y i n t h e a b s e n c e o f T r i t o n - X - 1 0 0 p r o b a b l y b e i n g l o
c a l i z e d a t d i s c r e t e s i t e s i n t h e m em brane. T r i t o n , s o l u b i l
i z i n g t h e enzym e, d e l o c a l i z e s t h e a c t i v i t y , g i v i n g a n e t
o b s e r v e d e f f e c t o f i n c r e a s e d enzym e c o n c e n t r a t i o n . From th e
a c t i v i t y to w a rd a ^ - g l y c o p r o t e i n , i t a p p e a r s t h a t p a r t o f
t h e a c t i v e s i t e s o f t h e enzyme m u st l i e c l o s e t o t h e s u r
f a c e , b u t t h a t t h e m a jo r p a r t o f i t i s n o t a v a i l a b l e f o r
r e a c t i o n w i t h t h e e x o g e n o u s r e c e p t o r , u n t i l o f c o u r s e ,
s o l u b i l i z e d . The f a c t o r by w h ic h s o l u b i l i z a t i o n i n c r e a s e s
t h e enzyme a c t i v i t y v a r i e s w i t h v a r y i n g c e l l p r e p a r a t i o n s .
The e n d o g e n o u s r e c e p t o r i s e a s i l y l o s t fro m t h e m em brane,
w h i l e t h e enzyme a c t i v i t y (m e a s u r e d w i t h t h e e x o g e n o u s r e
c e p t o r ) i s n o t .
65
The S e p h a d e x -G -1 0 0 colum n s e r v e d b o t h t o p u r i f y t h e
enzym e an d t o rem ove t h e T r i t o n - X - 1 0 0 . H ow ever, a n y e n d o g
e n o u s a c t i v i t y p r e s e n t w as n o t s e p a r a t e d . S i n c e T r i t o n - X -
100 was no l o n g e r p r e s e n t i n t h e s o l u t i o n t h e p a r t i a l l y
p u r i f i e d enzyme was r e c e n t r i f u g e d a t 1 5 0 ,0 0 0 x g f o r two
h o u r s . T h is s e r v e d t o r i d t h e enzyme o f a l l e n d o g e n o u s
a c t i v i t y ( T a b l e 9 ) .
A t t e m p t s w ere th e n made to f u r t h e r p u r i f y t h e e n
zyme on DEAE S e p h a d e x . The enzyme fro m S e p h a d e x -G -1 0 0 was
c o n c e n t r a t e d an d d i a l y z e d t o a lo w e r i o n i c s t r e n g t h T r i s
b u f f e r c o n t a i n i n g m e r c a p t o e t h a n o l . The colum n was e q u i l i
b r a t e d w i t h t h e same b u f f e r , and e l u t e d s t e p w i s e w i t h v a r i
o u s c o n c e n t r a t i o n s o f N aC l. The enzyme was e l u t e d from t h e
colum n a t h i g h i o n i c s t r e n g t h (0.5M N aCl) i n a v e r y u n
s t a b l e b u t h i g h l y p u r i f i e d s t a t e . O f te n t h e enzyme a c t i v
i t y was n o t r e c o v e r a b l e fro m t h e colum n e l u a t e . F o r t h i s
r e a s o n , t h e l a s t p u r i f i c a t i o n s t e p was n o t u s e d . When t h e
enzyme was e l u t e d fro m t h e co lu m n , i t r a p i d l y l o s t i t s a c
t i v i t y . T h is l o s s c o u ld n o t b e p r e v e n t e d by t h e a d d i t i o n
o f v a r i o u s p o s s i b l e s t a b i l i z e r s , i n c l u d i n g a lb u m i n , Mn+ + ,
a n d t h e a - ^ - g l y c o p r o t e i n r e c e p t o r .
P u r i f i c a t i o n o f e n d o g e n o u s r e c e p t o r . A t t e m p t s w ere
a l s o made t o p r e p a r e t h e e n d o g e n o u s r e c e p t o r o f r e t i c u l o
c y t e s tr o m a i n a p u r i f i e d form by p h e n o l e x t r a c t i o n o f
s t r o m a l s u s p e n s i o n s ( T a b le 1 0 ) . The T r i t o n - X - 1 0 0 e x t r a c t
66
TABLE 9
SEPARATION OF ENDOCENOUS RECEPTOR OF GALACTOSE FROM THE
UDP-GALACTOSE TRANSFERASE
CPM o f G a la c to s e - C 14
F r a c t i o n I n c o r p o r a t e d
Enzyme f r a c t i o n fro m S e p h a d ex G-100
colum n b e f o r e r e c e n t r i f u g a t i o n
Enzyme f r a c t i o n fro m S e p h a d ex G-lOO
colum n,, b e f o r e r e c e n t r i f u g a t i o n p l u s
h e l i c a s e t r e a t e d C t^ - g ly c o p r o te ln
Enzyme f r a c t i o n fro m S ep h a d ex colum n
a f t e r r e c e n t r i f u g a t i o n
Enzyme f r a c t i o n fro m S e p h a d ex colum n
a f t e r r e c e n t r i f u g a t i o n p l u s h e l i c a s e
t r e a t e d C t^ - g ly c o p r o te in
1,578
7,330
o
7,1^3
67
TABLE 10
TRANSFER OF GALACTOSE FROM UUP-GALACTOSE TO STROMAL RECEPTOR
ISOLATED AFTER PHENOL EXTRACTION*
S y stem
Q
CPM I n c o r p o r a te d
One h o u r I n c u b a tio n o f
4 ,0 5 5
543
s tr o m a l s u s p e n s io n
T r ito n -X -1 0 0 e x t r a c t e d
p e l l e t o f s tro m a p l u s
p u r i f i e d r e c e p t o r
fro m stro m a
T r ito n -X -1 0 0 e x t r a c t o f
stro m a p l u s p u r i f i e d
r e c e p t o r fro m stro m a
1 ,5 4 3
*The s tr o m a l r e c e p t o r w as p r e p a r e d a s I n M eth o d s.
A z e ro tim e w as 119 CPM. The a s s a y in c lu d e d 20 nM Mn++,
0.05M T ris -H C l b u f f e r , pH 7 * 2 -7 * 4 , 7 x 10- 3 M m e r c a p to e th a n o l,
ljiC U D P -g a la c to se -C 1 4 , an d 0 .1 m l o f e a c h s tr o m a l f r a c t i o n .
8 1
The CPM a r e r e p o r t e d f o r e q u i v a l e n t am o u n ts o f
enzym e, a n d th e p u r i f i e d r e c e p t o r i n t h e 0 .1 m l u s e d h e r e i s
p u r i f i e d fro m 1 .0 m l o f o r i g i n a l s tr o m a l s u s p e n s io n . The
c o n t r o l v a lu e s f o r t h e T r ito n -X -1 0 0 e x t r a c t and e x t r a c t e d
p e l l e t h a v e b e e n s u b t r a c t e d .
68
o f s t r o m a w i l l t r a n s f e r g a l a c t o s e fro m U D P - g a l a c t o s e t o t h e
g l y c o p r o t e i n o f t h e d i a l y z e d a q u e o u s p h a s e a f t e r p h e n o l e x -
14
t r a c t i o n . None o f t h e g a l a c t o s e - C was f o u n d i n t h e l i p i d
e x t r a c t o f t h i s f r a c t i o n a f t e r i n c u b a t i o n . L y o p h i l i z a t i o n
o f t h e d i a l y z e d g l y c o p r o t e i n f r a c t i o n c a u s e d a p p a r e n t d e -
n a t u r a t i o n , s i n c e i t becam e d i f f i c u l t to r e d i s s o l v e .
S t a b i l i t y o f t h e t r a n s f e r a s e . E f f e c t s o f s t o r a g e
i n v a r i o u s s t a t e s , a n d l y o p h i l i z a t i o n w e re s t u d i e d ( T a b l e
1 1 ) . The t r a n s f e r a s e i s v e r y s e n s i t i v e t o f r e e z e - d r y i n g ,
w i t h o r w i t h o u t t h e p r e s e n c e o f T r i t o n - X - 1 0 0 . P r e s e n c e o f
e i t h e r e n d o g e n o u s r e c e p t o r o r t o t a l T r i t o n - X - 1 0 0 e x t r a c t
a l l o w s p r e s e r v a t i o n o f t h e enzym e f o r a t l e a s t two w e ek s a t
4 ° , w h i l e t h e p u r i f i e d f r a c t i o n i s h a l f a c t i v e a t two w e e k s .
S p e c i f i c i t y o f t h e t r a n s f e r a s e . The U D P - g a l a c t o s e
14
t r a n s f e r a s e w o u ld n o t t r a n s f e r N - a c e t y l g a l a c t o s a m i n e - C
14
fro m U D P - N - a c e t y l g a l a c t o s a m i n e - C e i t h e r t o t h e s t r o m a l
r e c e p t o r i n t h e c r u d e p r e p a r a t i o n o r t o t h e h e l i c a s e
t r e a t e d a ^ - g l y c o p r o t e i n i n t h e p u r i f i e d enzym e d e v o i d o f
a n y e n d o g e n o u s r e c e p t o r a b i l i t i e s .
P r o p e r t i e s o f t h e p u r i f i e d t r a n s f e r a s e s y s t e m . The
p u r i f i e d t r a n s f e r a s e a c t i v i t y , u s i n g t h e a ^ - g l y c o p r o t e i n a s
a r e c e p t o r , w as m ax im al when t h e Mn++ c o n c e n t r a t i o n was
20 jiM, a s i n t h e c r u d e s y s te m . H ow ever, i n c o n t r a s t t o t h e
+ + + +
c r u d e s y s te m , Mg c o u l d no l o n g e r s u b s t i t u t e f o r t h e Mn
i o n ( F i g . 1 3 ) .
69
TABLE 11
STABILITY OF THE UDP-GALACTO SE TRANSFERASE IN VARIOUS STATES*
Enzyme F r a c t i o n A c t i v i t y (CPM)
F r e s h l y p r e p a r e d T r i t o n — X -100
2 .2 4 0
e x t r a c t o f stro m a
L y o p h iliz e d T rito n -X -1 0 0 ex
t r a c t o f s tro m a , r e d i s s o l v e d 523
t e s t e d im m e d ia te ly
T r ito n -X -1 0 0 e x t r a c t o f s tro m a ,
s t o r e d 8, a t 4 ° , 14 d a y s
2 ,2 1 8
G-100 p u r i f i e d enzym e f r a c t i o n ,
f r e s h l y p r e p a r e d
5 .5 3 3
G-100 p u r i f i e d enzym e f r a c t i o n
l y o p h i l i z e d , r e d i s s o l v e d t o 84o
same v o lu m e , t e s t e d Im m e d ia te ly
G-100 p u r i f i e d enzyme f r a c t i o n ,
2 ,4 9 0
s t o r e d a t 4 ° , l 4 d a y s
*The enzym e w as a s s a y e d b y i t s a b i l i t y t o t r a n s f e r
g a la c to s e - C i 4 fro m U D P -g a la c to se -C 14 t o h e l l c a s e t r e a t e d a x-
g l ^ c o p r o t e i n . The a s s a y m ix tu r e in c lu d e d 20 pM Mn++., 7 x
10 ^ m e r c a p to e th a n o l, 4 mg o f t h e g l y c o p r o t e i n r e c e p t o r ,
0 .0 4 pC U D P -g a la c to s e -C 14, a l l i n a f i n a l c o n c e n t r a t i o n o f
0.05M T r is - H C l b u f f e r , pH r[.2 -r J,kJ i n a f i n a l volum e o f 0 .1 6
m l. I n c u b a tio n w as f o r one h o u r a t 3 7 °•
Q >
The enzyme w as r e d i s s o l v e d i n 0.1M T r i s a n d 7 *
10”* ^ m e r c a p to e th a n o l; i t w as s t o r e d a t 4 ° , i n th e same b u f
f e r , i n t i g h t l y cap p e d h y d r o l y s i s t u b e s .
C P M INCORPORATED I N 1 5 MIN.
1400 —
4 0 0
200
CONCEN TRATI ON [/v
P ig . 1 3 . --D ependence o f p u r i f i e d U D P -g a la cto se t r a n s f e r a s e on m e ta l io n c o n c e n t r a
t i o n . R e a c tio n m ix tu re c o n t a i n s 0 .0 4 mg enzyme s o l u t i o n 0 .4 txC U D P -g a la c to se -C 1^ , 0 .4 mg
a ]_- g l y c o p r o t e i n r e c e p t o r , 4 x 10“3m m e r c a p to e th a n o l and T ris -H C l b u f f e r , pH 7 . 2 . ^
71
T ra n sfe r* o f t h e g a l a c t o s e w as f o l l o w e d a s a f u n c t i o n
o f enzym e c o n c e n t r a t i o n u n d e r t h e c o n d i t i o n s o f a s s a y ( F i g .
1 4 ) . F i f t e e n m in u t e i n c u b a t i o n s w e re r u n a t 3 7 ° . L i n e a r
c o r r e l a t i o n w as o b t a i n e d b e tw e e n i n c o r p o r a t i o n a n d enzym e
c o n c e n t r a t i o n .
T r a n s f e r o f t h e g a l a c t o s e w as f o l l o w e d a t v a r i o u s
t e m p e r a t u r e s u n d e r t h e c o n d i t i o n s o f a B s a y . F i f t e e n m in u t e
i n c u b a t i o n s , a s w e l l a s a z e r o tim e w e re r u n a t e a c h te m
p e r a t u r e . The enzym e h a s go od a c t i v i t y a t lo w e r t e m p e r a
t u r e s , an o ptim um a t a b o u t 37° j a n d i s c o m p l e t e l y i n a c t i
v a t e d a t 52° ( F i g . 1 5 ) .
A t t e m p t s w e re made t o s a t u r a t e t h e t h e o r e t i c a l r e
c e p t o r s i t e s a v a i l a b l e on t h e a ^ - g l y c o p r o t e i n ( T a b l e 1 2 ) ,
a n d , u s i n g an e x c e s s o f r e c e p t o r , t o t r a n s f e r a s much g a
l a c t o s e fro m U D P - g a l a c t o s e a s p o s s i b l e . I n t h e p r e s e n c e o f
e x c e s s r e c e p t o r , 7 3 $ o f t h e t o t a l g a l a c t o s e p r e s e n t a s UDP-
g a l a c t o s e was a d d e d t o t h e g l y c o p r o t e i n i n 24 h o u r s . U s in g
a l a r g e e x c e s s o f U D P - g a l a c t o s e ( i l l , T a b l e 1 2 ) , I n c o r p o r a
t i o n waB l i n e a r u p t o 24 h o u r s , b u t a t t h e e n d o f t h i s i n
c u b a t i o n t i m e , l e s s t h a n 20$ o f t h e t h e o r e t i c a l r e c e p t o r
s i t e s h a d r e a c t e d . W ith a s l i g h t e x c e s s o f r e c e p t o r s i t e s ,
t h e i n c o r p o r a t i o n w as n o t l i n e a r w i t h t i m e , b u t e x p o n e n t i a l
( I I , T a b le 12) i n t h e t h r e e h o u r s r u n .
The r a t e o f i n c o r p o r a t i o n o f g a l a c t o s e w as f o l l o w e d
a s a f u n c t i o n o f t h e c o n c e n t r a t i o n o f t h e a ^ - g l y c o p r o t e i n
t h e o r e t i c a l r e c e p t o r s i t e s ( F i g . 1 6 ) . B e c a u s e o f t h e v e r y
2400ft
4
2200 f t
o /O O O f t
o 8 0 0 -
- 6 0 0 —
ft 400 -
200
0 0.0/002
020
035 0.10 0 0 5
ENZYME A D D E D \m l\
F ig . 1 4 . — E f f e c t o f enzyme c o n c e n t r a t i o n on g a l a c t o s e t r a n s f e r . R e a c tio n m ix tu re
c o n t a i n s 0 . 4 mg a ^ - g l y c o p r o t e i n r e c e p t o r , 0 .4 U D P -g a la c to s e -C ^ ft 20 jaM Mn++, 4x lO “3 ^
m e r c a p to e th a n o l and T ris -H C l b u f f e r , pH 7 . 2 . Enzyme s o l u t i o n i s 0 .4 m g/m l i n T r i s b u f f e r .
C P M TRANSFERRED
4000
3 0 0 0 -
2000 -
1 0 0 0 -
F ig . 15*— T r a n s f e r o f g a l a c t o s e - C ^ from U B P -galactose-C ^* t o O i- g ly c o p r o te ln a t v a r io u s te m p e ra-
temperature , c
ttT y D « « 1 n a + ■ < - > rv
t u r e s . A ssay (15 m in .) in c lu d e s 20 nM M n++, 0 .4 |aC U D P -galactose-C 14, 0 .1 4 mg 0 ^- g ly c o p r o te in , 0.05M T r is
(pH 7 .2 ) and 4 X 10 3 m e rc a p to e th a n o l.
74
TABLE 12
INCORPORATION WITH TIME OF UDP-GALACTOSE-C14 INTO GLYCOPROTEIN RECEPTOR*
H ours
I I I I I I
CPM
pM G a la c to s e
X 10 5 CPM
pM G a la c to s e
X 10 3 CPM
pM G a la c to s e
X 10
0
0 .5
988 6 . 4 l 28 4 6 .4
175 5 4 .3
1 .0 2 ,3 9 0 1 5 -4 8 68 1 1 2 .8
555
1 3 2 .4
2 .0
3 ,1 1 3
2 0 .1 6 205 3 4 0 .4
733
1 7 4 .4
3 .0 3 ,6 9 0 2 3 .9 0 428 71 0 .2 1 ,4 5 8
3 4 7 .5
24 4 ,2 4 0 27*50 — —
7 ,7 8 5
2 ,4 1 3 .0
*The enzyme c o n c e n t r a t i o n s / m l in c u b a t io n m edium a r e e q u a l. The
a s s a y in c lu d e d 20 pM Mn++, U D P -g a la c to se -C 1 4 , h e l i c a s e t r e a t e d Q ^ - g ly -
c o p r o t e i n , w hose c o n c e n t r a t i o n i s c a l c u l a t e d a s t h e t h e o r e t i c a l r e c e p
t o r s i t e s made a v a i l a b l e b y r e l e a s e o f g a l a c t o s e , 7 x 10 m e rc a p to
e t h a n o l , an d 0.05M T r is - H C l b u f f e r , pH 7 * 2 -7 * 4 .
m 4
I . E x c e s s r e c e p t o r : 3*7 x 10 pM U D P -g a la c to se
0 .1 3 pM t h e o r e t i c a l r e c e p t o r s i t e s
0 .2 1 mg enzyme
I I . E x c e s s U D P -g a la c to se : 1 .0 1 pM U D P -g a la c to se
0 .7 5 M M r e c e p t o r s i t e s t h e o r e t i c a l
0 .3 4 5 mg enzyme
I I I . E x c e s s U D P -g a la c to s e : 0 .4 pM U D P -g a la c to se
0 .1 5 pM t h e o r e t i c a l r e c e p t o r s i t e s
0 .3 4 5 mg enzyme
4 8 0 0 -
Q
k)
£ 360
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&
8
0
i
Uj
1
o
5
o
1 I 1 I I
2 4 0 0 -
1200 -
mjjM THEORETICAL RECEPTOR SITES (X 10 )
F ig . l b . —E f f e c t o f a ^ - g l y c o p r o t e i n t h e o r e t i c a l r e c e p t o r s i t e B on th e t r a n s f e r o f
g a l a c t o s e - C 1^ from U D P -g a la c to se -C 1^ a t v a r i o u s t im e s . A ssay i n c l u d e s 0 .4 U D P -galac-
t o s e - C - ^ , 20 nM Mn, 0.05M T r i s (pH 7 .2 ) and 0.004M m e r c a p to e th a n o l.
76
low i n c o r p o r a t i o n a t e a r l i e r times., p r a c t i c a l l i m i t a t i o n s
n e c e s s i t a t e d u s i n g 5 m in u te i n c u b a t i o n p e r i o d s I n c a l c u l a t
i n g an a p p ro x im a te Km ( F i g . 1 7 ). A f t e r a p p a r e n t s a t u r a t i o n
o f t h e enzyme w i t h t h i s s u b s t r a t e , t h e v e l o c i t y a p p e a r e d to
i n c r e a s e a g a i n , when t h e c o n c e n t r a t i o n was a b o u t 15 tim e s
t h e loweBt c o n c e n t r a t i o n s t u d i e d . T h is c o u ld r e s u l t from
more s i t e B b e i n g made a v a i l a b l e by t h e a d d i t i o n o f g a l a c
t o s e , o r by a d d i t i o n to g a l a c t o s e i t s e l f .
The i n i t i a l v e l o c i t y o f t r a n s f e r o f g a l a c t o s e to
a ^ - g l y c o p r o t e i n r e c e p t o r was t h e n f o l l o w e d a s a f u n c t i o n o f
tim e ( F i g . 1 8 ) . A p p r o x im a te ly 14 m^M o f t h e o r e t i c a l r e c e p
t o r s i t e s w ere u s e d . P r a c t i c a l l i m i t a t i o n s n e c e s s i t a t e d
u s i n g 5 m in u te i n c u b a t i o n tim e s ( F i g . 1 9 ) . Tlie tim e c o u r s e
o f i n c o r p o r a t i o n o f g a l a c t o s e shows t h a t a t l a t e r t i m e s , a s
t h e c o n c e n t r a t i o n o f s u b s t r a t e i s d e c r e a s i n g , t h e r a t e i s
i n c r e a s i n g . T h is c o u ld b e due to a d d i t i o n a l r e c e p t o r s i t e s
b e i n g made a v a i l a b l e by a d d i t i o n o f t h e g a l a c t o s e , o r , by
a d d i t i o n to g a l a c t o s e i t s e l f .
w oo
.400
.200
.0750 000 .100 .600 .400 .700 .500
F ig . 1 7 . - - C a l c u l a t i o n o f an approxim ate Km v alu e f o r
a ^ - g l y c o p r o t e i n t h e o r e t i c a l r e c e p t o r s i t e s .
-vi
-<]
5 0 0 -
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o
100
.015 .030 .120 .150 .090 .060
m jjM UDP-GALACTOSE
Fig. 1 8 .—E f f e c t o f U D P-galactose-C 1^ on th e t r a n s f e r o f g a la c t o s e - C 1^ to om-
g l y c o p r o t e l n a t v a r io u s tim e s. Assays were run a t s a t u r a t i n g c o n c e n tr a ti o n o f c u - g l y c o
p r o t e i n s i t e s and under same c o n d it i o n s . --3
oo
140 —
80 —
60 —
40
20 —
-20 -10 10 30 80 20 40 50 60
ijS
iii
F ig . 1 9 . - - C a l c u l a t i o n o f an approxim ate Km v a lu e f o r UDP-galactose-C
vo
V. DISCUSSION AND CONCLUSIONS
One o f t h e m ost d i f f i c u l t p r o b le m s f a c i n g b io c h e m
i s t s i n t e r e s t e d i n m o l e c u l a r b i o l o g y i s t h e c o r r e l a t i o n o f
b i o c h e m i c a l m echanism s w i t h t h e i r c y t o l o g i c a l s i t e s . F r a c
t i o n a t i o n o f c e l l u l a r com ponents s u f f i c i e n t to d e te r m i n e r e
a c t i o n p ath w ay s haB t h e i n h e r e n t d i s a d v a n t a g e o f d e s t r o y i n g
t h e f u n c t i o n a l i n t e g r i t y o f t h e c e l l a s a w h o le . F u r t h e r
m ore, su c h d i s r u p t i o n g e n e r a t e s d i f f i c u l t i e s i n p r e c i s e l y
d e f i n i n g th e s u b c e l l u l a r f r a g m e n t s ; w hat i s i s o l a t e d a s
p a r t o f a p a r t i c u l a t e c e l l u l a r o r g a n e l l e i n one f r a c t i o n a
t i o n , may be i s o l a t e d a s a s o l u b l e com ponent i n a n o t h e r .
The t a s k o f d e t e r m i n i n g i n w hat form i t e x i s t s i n v iv o i s
o f t e n a f o r m id a b le o n e . A c u r r e n t c a s e I n p o i n t I s th e
p r o b le m o f t h e b i o s y n t h e s i s o f g l y c o p r o t e i n s .
G l y c o p r o t e i n s a r e c h a r a c t e r i s t i c a l l y e x t r a c e l l u l a r
( 6 5 ), b e i n g e i t h e r s e c r e t i o n s o f c e l l s o r c o n s t i t u t i n g p a r t
o f th e c e l l u l a r m em branes. The p ro b le m o f t h e i r s y n t h e s i s ,
t h e n , n e c e s s i t a t e s c o n s i d e r a t i o n o f t h e c l o s e l y r e l a t e d c e l
l u l a r p r o c e s s e s o f p r o t e i n s e c r e t i o n , and t h e m e t a b o l i c d y
nam ics and s y n t h e s i s o f mem branes. F u r t h e r , s i n c e g l y c o
p r o t e i n s a r e c o n j u g a t e d p r o t e i n s , th e r e l a t i o n s h i p s o f t h e i r
s y n t h e s i s to n o n - c o n j u g a t e d p r o t e i n s y n t h e s i s m ust be e s
t a b l i s h e d .
80
8 1
From a l l t h e a v a i l a b l e d a t a , I t a p p e a r s c l e a r t h a t
a d d i t i o n o f t h e c a r b o h y d r a t e to p r o t e i n p r o b a b l y d oes n o t
t a k e p l a c e s i m u l t a n e o u s l y w ith th e i n t e g r a t i o n o f amino
a c i d s i n t o t h e p o l y p e p t i d e c h a i n , b u t r a t h e r i s added to
p r o t e i n s t h a t e x i s t i n a p r e - f o r m e d p o o l . In v a r i o u s c e l l
s y s te m s , th e m a jo r s i t e o f t h i s a d d i t i o n a p p e a r s to be th e
membranes o f th e e n d o p la s m ic r e t i c u l u m . V a r i o u s t r a n s f e r
a se r e a c t i o n s have b een d e s c r i b e d w hich add s i n g l e s u g a r s
t o t h e g ro w ing c a r b o h y d r a t e c h a i n . N o n e t h e l e s s , no one haB
y e t s u c c e e d e d I n p i n p o i n t i n g th e a d d i t i o n r e a c t i o n o f th e
f i r s t c a r b o h y d r a t e o n to t h e amino a c i d o f t h e p r o t e i n m o i
e t y . C o n f l i c t i n g e v id e n c e p l a c e s th iB r e a c t i o n e i t h e r d i
r e c t l y on t h e r ib o s o m e s o r w i t h i n th e n e tw o rk o f th e e n d o
p l a s m i c r e t i c u l u m , b u t i n d e p e n d e n t o f rib o so m e bound p r o
t e i n .
S tu d y o f th e b i o s y n t h e s i s o f t h e g l y c o p r o t e i n o f
r e t i c u l o c y t e s tro m a o f f e r s s e v e r a l a d v a n t a g e s o v e r more
c o m p l i c a t e d Bystem s. T h is c e l l c o n t a i n s no endoplaB m lc
r e t i c u l u m , and d o es n o t s e c r e t e p r o t e i n s . I t s membrane I s
e a s i l y I s o l a t e d . From whole c e l l s t u d i e s u s i n g r e t i c u l o
c y t e s and r e d b l o o d c e l l s , i t was d e t e r m i n e d t h a t r e t l c u l o -
14
c y t e s a r e c a p a b l e o f a c t i v e l y i n c o r p o r a t i n g g lu c o s a m in e -C
I n t o g l y c o p r o t e i n , w h i l e r e d c e l l s were v i r t u a l l y i n a c t i v e
i n t h i s r e g a r d . F u r t h e r m o r e , from i n c o r p o r a t i o n s t u d i e s
■\h 14
w i t h g lu c o s a m in e - C , g a l a c t o s e - G , s e r i n e - C , and l e u -
c i n e - C 1^ , i t i s o b v i o u s t h a t t h e r e t i c u l o c y t e s tro m a i s a c -
t i v e l y i n c o r p o r a t i n g s u g a r s and amino a c i d s i n t o n o t o n l y
g l y c o p r o t e i n b u t p r o t e i n and l i p i d s a s w e l l .
L a b e l l e d amino a c i d s c o u ld be r e c o v e r e d i n th e TCA-
p r e c i p i t a b l e f r a c t i o n s o f r ib o s o m e s s e p a r a t e d on a sucroB e
g r a d i e n t , w h e rea s l a b e l l e d g lu c o s a m in e c o u ld n o t , w h e th e r
o r n o t th e r ib o s o m e s were p r e t r e a t e d w i t h d e s o x y c h o l a t e .
T h is i s c o n c l u s i v e e v id e n c e t h a t c a r b o h y d r a t e i s n o t added
to t h e p r o t e i n on r ib o s o m e s i n t h e g e n e r a l r l b o s o m a l p o o l .
14
The i n c o r p o r a t i o n o f g lu c o s a m in e -C i n t o TCA p r e -
c i p i t a b l e p r o d u c t o f stro m a i n t h e c e l l f r e e s y s te m d e
p e n d ed on t h e p r e s e n c e o f s o l u b l e enzymeB. RibosomeB d id
n o t s t i m u l a t e th e i n c o r p o r a t i o n . I n c o r p o r a t i o n o f amino
a c i d i n t o p r o t e i n was n o t s t i m u l a t e d by th e p r e s e n c e o f
s t r o m a . Thus, t h e g e n e r a l r i b o s o m a l p o o l can b e d i s m i s s e d
a s a p o s s i b l e s i t e o f g lu c o s a m in e i n c o r p o r a t i o n i n t o p r o
t e i n . I t i s c o n c e i v a b l e t h a t t h e r e i s a n o t h e r r l b o s o m a l
p o o l , a s s o c i a t e d w i t h th e membrane, t h a t i s r e s p o n s i b l e
f o r t h e I n c o r p o r a t i o n o f amino a c i d s i n t o Btroma, and t h a t
g lu c o s a m in e i s i n c o r p o r a t e d a t t h i s l e v e l . N o tin g t h e h i g h
a c t i v i t y o f amino a c i d s and s u g a r s found i n t h e stro m a i n
whole c e l l s t u d i e s , one would e x p e c t , i f t h i s se co n d p o o l
e x i s t e d , t h a t amino a c i d i n c o r p o r a t i o n i n a c e l l f r e e s y s
tem would be s t i m u l a t e d by th e a d d i t i o n o f s tro m a . W hile
g lu c o s a m in e i n c o r p o r a t i o n i s i n c r e a s e d by s t r o m a l a d d i t i o n ,
no s t i m u l a t i o n was o b s e r v e d i n th e c a s e o f amino a c i d i n
c o r p o r a t i o n . Thus, even i f t h e r e a r e membrane bound r i b o
83
somes t h a t I n c o r p o r a t e amino a c i d s . I n c o r p o r a t i o n o f g l u c o s
amine a p p e a r s to be a s e p a r a t e p r o c e s s . F u r t h e r s u p p o r t
f o r t h i s c o n c l u s i o n comes from t h e f a c t t h a t r i b o n u c l e a s e
d o e s n o t i n h i b i t i n c o r p o r a t i o n o f g l u c o s a m in e i n t o th e g l y
c o p r o t e i n , w h i l e I t I n h i b i t s amino a c i d i n c o r p o r a t i o n d r a s
t i c a l l y . P r o t e i n s y n t h e s i s by m em brane-bound RNA o f th e
b a c t e r i a l s y s te m was shown t o be RNAase s e n s i t i v e (9 3 )•
T h is i n i t B e l f I s n o t c o n c l u s i v e p r o o f , s i n c e t h e RNA
c l o s e l y a s s o c i a t e d w i t h t h e membrane was somewhat more r e
s i s t a n t to r i b o n u c l e a B e th a n n o n -b o u n d RNA. mRNA t i g h t l y
bound to r ib o s o m e s h a s a l s o b een shown to be r e s i s t a n t to
breakdow n by a p h o s p h o d i e s t e r a s e (9^0 •
I s o l a t i o n o f th e U D P - g a la c to s e t r a n s f e r a s e r e a c t i o n ,
w hich o c c u r s I n d e p e n d e n t l y o f any o t h e r s u g a r n u c l e o t i d e ,
i n d i c a t e s t h a t s u g a r s a r e p r o b a b l y a d d ed one a t a tim e to
th e g l y c o p r o t e i n . The p u r i f i c a t i o n and s o l u b i l i z a t i o n
s t u d i e s show t h a t b o t h t h e enzyme and r e c e p t o r a r e membrane
bo u n d , and th e r e a c t i o n i s c o m p l e t e l y I n d e p e n d e n t o f any
s o l u b l e enzym es. I t a p p e a r s , h o w e v er, t h a t i n t e g r a t i o n o f
th e g l y c o p r o t e i n r e c e p t o r i n t o s tro m a i s n o t a s f i r m a s f o r
t h e enzyme. E ndogenous r e c e p t o r iB s l o w l y l o s t w i t h s u c
c e s s i v e w ash es a t low I o n i c s t r e n g t h . I t h a s lo n g been
known t h a t low i o n i c s t r e n g t h damages m i t o c h o n d r i a l mem
b r a n e s ( 9 5 , 9 6 ) . I t h a s a l s o b e en more r e c e n t l y shown (97)
t h a t s t r o m a l bound enzymes o f r e d c e l l s a r e l o s t on low
I o n i c s t r e n g t h w a s h in g . The t r a n s f e r a s e o f t h e r e t i c u l o -
8 h
c y t e s tr o m a s t u d i e d h e r e was n o t l o s t a t a l l , r e m a in i n g
f u l l y a c t i v e and a t t a c h e d to t h e membrane.
T r ito n - X - 1 0 0 e x t r a c t i o n o f t h e enzyme, a lo n g w ith
u s e o f ex og en o u s r e c e p t o r B , r e v e a l s s e v e r a l i n t e r e s t i n g
p o i n t s . B e f o r e s o l u b i l i z a t i o n , o n l y a Bmall p o r t i o n o f t h e
t o t a l enzyme a c t i v i t y i s a v a i l a b l e f o r r e a c t i o n w i t h e x t r a -
s t r o m a l s o l u b l e r e c e p t o r s . Upon s o l u b i l i z a t i o n o f t h e
enzyme, t h e a c t i v i t y i s g r e a t l y i n c r e a s e d . The r a t i o o f
a c t i v i t y b e f o r e s o l u b i l i z a t i o n to w a rd e x o g en o u s r e c e p t o r s ,
t o t h a t a f t e r s o l u b i l i z a t i o n , v a r i e d g r e a t l y from p r e p a r a
t i o n to p r e p a r a t i o n , and may be r e l a t e d to t h e s t a g e o f
m a t u r a t i o n o f th e r e t i c u l o c y t e . T r it o n - X - 1 0 0 e x t r a c t i o n
a l s o i n c r e a s e d th e a c t i v i t y o f t h e enzyme to w a rd endogenous
r e c e p t o r . T h is c o u ld b e e x p l a i n e d a s an e f f e c t i v e i n c r e a s e
i n e i t h e r t h e enzyme c o n c e n t r a t i o n o r r e c e p t o r , i . e . , s o l u
b i l i z a t i o n p e r m i t s an i n c r e a s e d i n t e r a c t i o n o f enzyme and
r e c e p t o r upon r e l e a s e from t h e s t r o m a l m a t r i x .
The enzyme re m a in e d s o l u b l e and a c t i v e , even a f t e r
r e m o v a l o f t h e T r i t o n , r e v e a l i n g t h a t i t i s t r u l y s o l u b l e ,
b u t more I m p o r t a n t , t h a t i n t e g r a t i o n w i t h o t h e r s t r o m a l
com ponents iB n o t a n e c e s s a r y r e s t r i c t i o n f o r a c t i v i t y .
I t i s i n t e r e s t i n g to compare t h i s r e t i c u l o c y t e
t r a n s f e r a s e w i t h t h e s o l u b l e t r a n s f e r a s e i s o l a t e d from g o a t
c o lo B tru m by Roseman and h i s c o - w o r k e r s ( ^ 6 ) . B oth th e
c o l o s t r u m t r a n s f e r a s e and t h e p a r t i c u l a t e t r a n s f e r a s e a r e
d e p e n d e n t upon Mn++, a l t h o u g h Mg++ w i l l p a r t i a l l y s u b s t l -
85
t u t e . Once t h e r e t i c u l o c y t e enzyme i s s o l u b i l i z e d and p u r i
f i e d however,, i t s d e p e n d e n c e on Mn++ i s a b s o l u t e . The c o
l o s t r u m t r a n s f e r a s e i s r e t a i n e d b y Sep had ex-G -10 0 g e l ,
w h i l e th e s o l u b i l i z e d enzyme from r e t i c u l o c y t e s i s n o t .
E i t h e r i t i s a l a r g e r m o l e c u l e , o r e x i s t s i n a g g r e g a t e s o f
enzyme m o l e c u l e s , o r ev en a s h e t e r o g e n e o u s m o l e c u l a r a g g r e
g a t e s s t i l l I n t i m a t e l y a s s o c i a t e d a s i n t h e s t r o m a . The
enzyme from r e t i c u l o c y t e s i s s p e c i f i c f o r t h e t r a n s f e r o f
g a l a c t o s e from U D P - g a la c t o s e ; U D P - N - a c e t y lg a la c t o s a m in e
w ould n o t t r a n s f e r to e i t h e r en d o g en o u s o r e x o g e n o u s r e c e p
t o r . The c o l o s t r u m enzyme on th e o t h e r h a n d , u t i l i z e d
b o t h s u b s t r a t e s . The Km f o r U D P -g a la c to s e was s m a l l e r f o r
t h e r e t i c u l o c y t e enzyme by a power o f 10.
Exogenous r e c e p t o r s were p r e p a r e d by s e q u e n t i a l r e
m oval o f s i a l i c a c i d and t h e n g a l a c t o s e from t h e a ^ - g l y c o -
p r o t e i n and f e t u i n . The t r a n s f e r o f g a l a c t o s e by t h e r e
t i c u l o c y t e enzyme to t h e s e r e c e p t o r s d e m o n s t r a t e s t h a t th e
enzyme r e c o g n i z e s N - a c e t y l g l u c o s a m i n e a s a r e c e p t o r s i t e ,
s i n c e t h i s s u g a r f o l l o w s g a l a c t o s e i n t h e c a r b o h y d r a t e s e
q u e n c e . The f a c t t h a t p o r c i n e s u b m a x i l l a r y mucin l a c k i n g
s i a l i c a c i d and f u c o s e c o u ld a c t a s a r e c e p t o r f o r th e
g a l a c t o s e , I n d i c a t e s t h a t t h e g a l a c t o s e m ig h t be added to
a n o t h e r g a l a c t o s e m o ie ty l i n k e d to t h e g l y c o p r o t e i n . K a t z -
man and E y l a r (97) have shown t h a t a g a l a c t o s e r e s i d u e f o l
lows a f u c o s e r e s i d u e i n a t r i s a c c h a r i d e t h e y h a v e i s o l a t e d
from t h i s g l y c o p r o t e i n . T h a t a d d i t i o n to g a l a c t o s e m ig h t
86
b e o c c u r r i n g i s a l s o s u p p o r t e d by t h e f a c t t h a t aB g a l a c t o s e
i s a dd ed to t h e a - ^ - g l y c o p r o t e i n from U D P - g a la c t o s e , a se co n d
r e a c t i o n becom es e v i d e n t a t l a t e r t i m e s . I t i s c o n c e i v a b l e
t h a t a s g a l a c t o s e i s a d ded to t h e g l y c o p r o t e i n , i t becomes
a new r e c e p t o r e nd , to w hich a d d i t i o n a l g a l a c t o s e can be
a d d e d . T h is d o es n o t im p ly t h a t su ch an a d d i t i o n t a k e s
p l a c e i n v iv o w i t h t h e e n d o g en o u s r e c e p t o r .
The c o l o s t r u m t r a n s f e r a s e c o u l d r e p l a c e m ost o f t h e
g a l a c t o B e removed from o r o s o m u c o id , w h i l e t h e r e t i c u l o c y t e
enzyme c o u ld n o t . T h is c o u ld be due to d i f f e r e n c e s i n t h e
r e s i d u e s a d j a c e n t t o g a l a c t o s e i n th e c a r b o h y d r a t e s e
q u e n c e s o f t h e en d o g en o u s and exo g en o u s r e c e p t o r s , w i t h a
r e s u l t a n t s p e c i f i c i t y o f th e t r a n s f e r a s e . I t m ig h t a l s o be
due to a l a c k o f p r o p e r c o n d i t i o n s to d r i v e th e r e a c t i o n
f o r w a r d i n th e p u r i f i e d s y s te m . F or e x am p le , i n th e r e
a c t i o n :
U D P -g a la c to se + G ly c o p r o te in -* • G l y c o p r o te in - g a la c to s e + UDP
a membrane bound UDPase, n o t I s o l a t e d w i t h t h e t r a n s f e r a s e ,
m ig h t b r e a k down t h e UDP to d r i v e t h e r e a c t i o n f o r w a r d .
UDPase i s known to be h i g h l y a c t i v e i n G o lg i membranes,
w hich may be i m p l i c a t e d i n g l y c o p r o t e i n s y n t h e s i s . A n o th e r
f a c t o r t h a t m ust be c o n s i d e r e d i n t h e i n v i t r o r e a c t i o n i s
t h e i n t e r f e r i n g s e c o n d a r y r e a c t i o n t h a t becomes e v i d e n t a s
t h e r e a c t i o n p r o g r e s s e s .
87
The a b i l i t y o f v a r i o u s r e a g e n t s to e x t r a c t t h e e n
zyme r e v e a l e d s e v e r a l i n t e r e s t i n g f a c t s . T r ito n - X - 1 0 0
s o l u b i l i z e s t h e enzyme t h a t t r a n s f e r s g a l a c t o s e to g l y c o
p r o t e i n , b u t l e a v e s th e t r a n s f e r a s e t h a t t r a n s f e r s g a l a c t o s e
to l i p i d i n t h e s tr o m a . E v i d e n t l y , a t l e a s t two d i f f e r e n t
t r a n s f e r a s e s f o r g a l a c t o s e a r e p r e s e n t , and th e two enzymes
a r e c o m p l e t e l y i n d e p e n d e n t o f one a n o t h e r . I t i s a l s o o b
v i o u s t h a t t r e a t m e n t w i t h d e s o x y c h o l a t e e x t r a c t s b o t h th e
en d o g en o u s r e c e p t o r and th e enzyme away from t h e membrane.
T h is p o i n t s o u t a s e r i o u s p i t f a l l i n u s i n g d e s o x y c h o l a t e to
s e p a r a t e r ib o s o m e s from membranes i n o t h e r s y s te m s . I t
makes i t v i r t u a l l y i m p o s s i b l e to d i s t i n g u i s h w hat i s t r u l y
membrane bound and w hat i s s o l u b l e o r d e p e n d e n t on r i b o
som es. I t i s p o s s i b l e t h a t th e i n v iv o i n t e g r i t y haB b e en
c o m p l e t e l y d e s t r o y e d .
S o l u b i l i z a t i o n o r re m o v a l o f some s t r o m a l compon
e n t s , w i t h a c o n c o m it a n t i n c r e a s e i n a c t i v i t y i n th e Bys
tem s r e m a in i n g i n th e s t r o m a , such a s was o b s e r v e d w i t h th e
l i p l d - g a l a c t o s e sy s te m , shows a g a i n t h a t c o m p le te i n t e g r i t y
o f t h e membrane i s n o t n e c e s s a r y f o r m e ta b o lis m o f i t s i n
d i v i d u a l p a r t s . At th e same tim e i t i n d i c a t e s t h a t i n t e
g r a t i o n i n t o th e membrane p l a c e s some r e s t r i c t i o n o r c o n
t r o l on th e m e t a b o l i c a c t i v i t y o f a membrane com ponent.
I n c o n c l u s i o n , s e v e r a l a s p e c t s o f th e m e t a b o lis m o f
r e t i c u l o c y t e stro m a have b e en r e v e a l e d and a r e sum m arized
i n F i g u r e 20. A c t i v a t i o n o f sugarB by s o l u b l e enzymes o c -
88
I . GLUCOSAMINE + X S o lu b le E n z^ » GLUCOSAMINE-X
I I . GLUCOSAMINE-X + MEMBRANE ENZYME
| > MEMBRANE----- ENZYMEj.
RECEPTOR |
GLUCO SAMINE-RECEPTOR
I I I . UDP-GALACTOSE + MEMBRANE-----ENZYME2 ++
| — > MEMBRANE ENZYME^
GLYCOPROTEIN RECEPTOR |
GLYCOPROTEIN-( GALACTOSE)
IV . UDP-GALACTOSE + MEM BRANE------ ENZYME 3 ++
| j m — > MEMBRANE ENZYME3
GLYCOLIPID RECEPTOR |
GLYCOLIPID-(GALACTOSE)
F ig . 2 0 . — Summary o f s tr o m a l r e a c t i o n s
89
c u r s , and a t l e a s t two s t r o m a l t r a n s f e r a s e s have b e e n i d e n
t i f i e d . The s tro m a m ust e x i s t i n i o n i c a l l y o r h y d r o p h o b i c -
a l l y l i n k e d s u b - u n i t s , s i n c e t h e g l y c o l i p i d s y n t h e s i z i n g
s y s te m h a s b e en s e p a r a t e d from t h e g l y c o p r o t e i n s y n t h e s i z i n g
Bystem by d e t e r g e n t s . W ith in e a c h s u b - u n i t , th e t r a n B f e r a s e
enzymes m ust be i n t i m a t e l y a s s o c i a t e d w i t h t h e i r e n do gen ou s
r e c e p t o r s . I n t h e c a s e o f th e g l y c o p r o t e i n a t l e a s t , w here
theB e h av e b e en s e p a r a t e d , t h e enzyme and i t s r e c e p t o r m ust
a l s o be i o n i c a l l y o r h y d r o p h o b i c a l l y l i n k e d .
I t I s hoped t h a t t h i s r e s e a r c h h a s op ened t h e way
f o r f u t u r e s t u d i e s t h a t w i l l r e v e a l th e c o m p le te s y n t h e s i s
and i n t e g r a t i o n o f s t r o m a l p a r t s .
V I . SUM MARY
Whole c e l l i n c u b a t i o n s t u d i e s have shown t h a t w h ile
r ed c e l l strom a i s no l o n g e r a c t i v e l y i n c o r p o r a t i n g s u g a r s
i n t o g l y c o p r o t e i n , r e t i c u l o c y t e strom a i s B t i l l m e t a b o l i c -
a l l y a c t i v e i n t h i s r e g a r d . L i n e a r i t y w i t h tim e i s o b s e r v e d
l4 14
f o r t h e i n c o r p o r a t i o n o f g a l a c t o B e - C and g lu c o s a m in e -C
i n t o s t r o m a l g l y c o p r o t e i n . No l a b e l l i n g o f r i b o s o m e s ,
s e p a r a t e d on a s u c r o s e g r a d i e n t , b e f o r e o r a f t e r t r e a t m e n t
w i t h d e s o x y c h o l a t e , was o b s e r v e d i n s t u d i e s w i t h g l u c o s -
14 14-
am ine-C , i n c o n t r a s t to s t u d i e s w i t h s e r i n e - C . Thus,
su g a r a d d i t i o n to g l y c o p r o t e i n doeB n o t depend on th e g e n e r
a l r lb o s o m a l p o o l .
I n a c e l l f r e e s y s te m , g l u c o s a m in e i n c o r p o r a t i o n
i n t o s tro m a d ep en d ed upon t h e p r e s e n c e o f s o l u b l e enzym es,
b u t was i n d e p e n d e n t o f t h e a d d i t i o n o f r i b o s o m e s t h a t a c
t i v e l y i n c o r p o r a t e d amino a c i d s . N u c l e o t i d e t r i p h o s p h a t e s ,
g l u t a t h i o n e , and r i b o n u c l e a s e h a d o p p o s i t e e f f e c t s on
g l u c o s a m i n e - C 1^ and s e r i n e - C 1^ I n c o r p o r a t i o n . The s o l u b l e
enzymes were p a r t i a l l y p u r i f i e d by h i g h sp e e d c e n t r i f u g a t i o n
t o s e d im e n t h em o g lo b in and o t h e r p r o t e i n s , o r b y ammonium
s u l f a t e p r e c i p i t a t i o n . O th e r p r o p e r t i e s o f g l u c o s a m in e i n
c o r p o r a t i o n were com pared w i t h s e r i n e i n c o r p o r a t i o n . N-
90
91
l4
a c e t y l g l u c o s a m l n e - C from i t B UDP d e r i v a t i v e was n o t i n c o r
p o r a t e d . G a l a c t o s e , on th e o t h e r h an d , c o u ld be i n c o r p o r
a t e d from i t s U D P - d e r i v a t i v e b o t h i n t o l i p i d s and i n t o g l y
c o p r o t e i n . T h is i n c o r p o r a t i o n d e p en d e d on t h e p r e s e n c e o f
s tr o m a a l o n e , and d i d n o t r e q u i r e s o l u b l e enzym es. Good
a c t i v i t y r e q u i r e d t h e p r e s e n c e o f Mn o r Mg . The p r o p
e r t i e s a t th iB p a r t i c u l a t e s y s te m were s t u d i e d .
A tte m p ts w ere made t o f r a c t i o n a t e t h e s tr o m a i n
Buch a way a s to s e p a r a t e th e l i p i d i n c o r p o r a t i n g a c t i v i t y
from t h e g l y c o p r o t e i n i n c o r p o r a t i n g a c t i v i t y , and to s e p a
r a t e t h e g l y c o p r o t e i n r e c e p t o r from th e t r a n s f e r a s e enzyme.
S e p a r a t i o n o f th e l i p i d and g l y c o p r o t e i n a c t i v i t y was
a c h i e v e d by e x t r a c t i o n o f s tro m a w i t h T r it o n - X - 1 0 0 . T h is
e x t r a c t i o n e f f e c t e d t h e t r u e s o l u b i l i z a t i o n o f t h e t r a n s
f e r a s e enzyme i n a h i g h l y a c t i v e s t a t e .
At t h i s p o i n t , s e v e r a l m a c r o m o le c u la r r e c e p t o r s
were s u c c e s s f u l l y s u b s t i t u t e d f o r th e e n do gen ou s r e c e p t o r ;
such an ex og en o u s r e c e p t o r was n e c e s s a r y to f o l l o w s e p a r a
t i o n o f th e enzyme and en do gen ou s r e c e p t o r . E p i d i d i m a l
enzyme t r e a t e d d e s i a l y z e d a ^ - g l y c o p r o t e i n and f e t u i n ,
h e l i c a s e t r e a t e d d e s i a l y z e d a ^ - g l y c o p r o t e i n and f e t u i n ,
a ^ - g a l a c t o s i d a s e t r e a t e d d e s i a l y z e d a ^ - g l y c o p r o t e i n , and
d e s i a l y z e d p o r c i n e s u b m a x i l l a r y mucin ( l e s s f u c o s e ) c o u ld
a l l a c t a s r e c e p t o r s .
A f t e r p a r t i a l p u r i f i c a t i o n o f t h e enzyme a c t i v i t y
and re m o v a l o f T r it o n - X - 1 0 0 on S e p h a d ex -G -1 0 0 , th e en d o g -
92
e n o u s r e c e p t o r was s e p a r a t e d by r e c e n t r i f u g a t i o n o f t h e
a c t i v e p e a k s . The enzyme a c t i v i t y to w a rd e x o g en o u s r e c e p
t o r was p u r i f i e d a b o u t 180 f o l d .
Some p r o p e r t i e s o f t h e p u r i f i e d enzyme s y s te m were
s t u d i e d . The r e q u i r e m e n t f o r Mn++ was a b s o l u t e . The r e
q u i r e m e n t f o r U D P - g a la c to s e a s a s u b s t r a t e was s p e c i f i c .
N e i t h e r U D P - N - a c e t y lg a la c t o s a m in e n o r U D P - N - a c e t y lg l u c o s -
amine w ould s u b s t i t u t e . A t t e m p t s to s a t u r a t e th e t h e o r e t i
c a l r e c e p t o r s i t e s o f t h e a ^ - g l y c o p r o t e i n w ere n o t s u c c e s s
f u l . A p p ro x im ate v a l u e s w ere c a l c u l a t e d f o r b o t h s u b
s t r a t e s .
ThuB i t was shown t h a t c a r b o h y d r a t e i n c o r p o r a t i o n
i n t o g l y c o p r o t e i n d o e s n o t o c c u r a t t h e l e v e l o f p r o t e i n
s y n t h e s i s i n r a b b i t r e t i c u l o c y t e s . S o l u b i l i z a t i o n o f a
s t r o m a l U D P - g a la c to s e t r a n s f e r a s e was a c h i e v e d . G e n t le
f r a c t i o n a t i o n s o f th e s tr o m a , p r e s e r v i n g enzyme a c t i v i t i e s ,
h a s open ed t h e way f o r f u r t h e r s t u d i e s o f t h e s y n t h e s i s and
i n t e g r a t i o n o f i t s co m p o n e n ts.
A P P E N D I X
93
APPENDIX
P r e p a r a t i o n o f h e l i c a s e t r e a t e d a ^ - g l y c o p r o t e i n .
S i a l i c a c i d was rem oved from a ^ - g l y c o p r o t e i n by h y d r o l y s i s
i n 0 . 1 N HgSO^ a t 80° f o r 1 h o u r .
H e l i c a s e was d i s s o l v e d i n 0.02M sodium c i t r a t e b u f
f e r , pH 4 . 3 ; and d l a l y z e d v e r s u s t h e same b u f f e r f o r t h r e e
h o u r s .
2 4 .3 mg d l a l y z e d h e l i c a s e i n 1 .8 ml o f 0.02M sodium
c i t r a t e b u f f e r , pH 4 . 3 , was add ed to 1 0 0 .0 2 mg o f d e s i a l y z e d
a - g l y c o p r o t e i n i n 1 ml o f t h e same b u f f e r . A m i x t u r e o f
p e n i c i l l i n and s t r e p t o m y c i n ( 0 . 1 m l, c o n t a i n i n g 0 .0 0 5 u n i t s
o f e a c h a n t i b i o t i c ) was a d d e d , a s w e l l a s 2 d r o p s o f t o l u
e n e . The m i x t u r e was i n c u b a t e d a t 37° f o r a p p r o x i m a t e l y
40 h o u r s .
A f t e r i n c u b a t i o n t h e m i x t u r e was n e u t r a l i z e d w i t h
1 N KOH, t h e enzyme p r o t e i n was p r e c i p i t a t e d w i t h 1 N HCIO^,
and th e g l y c o p r o t e i n c o n t a i n i n g s u p e r n a t a n t f l u i d was d i -
a l y z e d v e r s u s w a t e r and l y o p h i l i z e d .
G a l a c t o s e r e l e a s e was m e a su re d w i t h a G a l a c t o s t a t
k i t ; h e l i c a s e c o n t r o l s were ru n a t a l l t i m e s . D u rin g i n
c u b a t i o n no hexo sam ine was r e l e a s e d .
94
95
P r e p a r a t i o n o f h e l i c a s e t r e a t e d f e t u i n . T h is was
p r e p a r e d i n e x a c t l y t h e same manner a s t h e a ^ - g l y c o p r o t e i n
e x c e p t t h a t th e h e l i c a s e was i n c u b a t e d w i t h 8 0 .0 mg o f d e
s i a l y z e d f e t u i n . A g a in , no h e x o sa m in e was r e l e a s e d d u r i n g
i n c u b a t i o n .
P r e p a r a t i o n o f d e s i a l y z e d p o r c i n e s u b m a x l l l a r y mu
c i n , and d e s i a l y z e d f e t u i n . The sa m p le s were d i s s o l v e d i n
0 . 1 0 N HgSOj^ and h y d r o l y z e d a t 80° f o r one h o u r .
P r e p a r a t i o n o f d e s i a l y z e d p o r c i n e B u b m a x illa r y mu
c i n l a c k i n g f u c o s e . 4 2 9 .4 mg o f th e sam ple was d i s s o l v e d
i n 25 ml o f 1 .0 N HgSO^ and h y d r o l y z e d a t J0° f o r 3 h o u r s .
P r e p a r a t i o n o f e p i d i d i m a l enzyme t r e a t e d a ^ - g l y c o -
p r o t e i n . Enzymes e x t r a c t e d from sh e e p e p i d i d i m a l g l a n d s
were d i s s o l v e d i n 0.02M sodium c i t r a t e b u f f e r , pH 4 . 3
( 1 5 .5 m g /m l), and 2 ml was mixed w i t h 96 mg d e s i a l y z e d 0^ -
g l y c o p r o t e i n / 0 .9 6 ml w a t e r . I n c u b a t i o n , p r e c i p i t a t i o n , and
n e u t r a l i z a t i o n w ere c a r r i e d o u t e x a c t l y a s f o r t h e h e l i c a s e
t r e a t e d a - ^ - g l y c o p r o t e i n . D u rin g i n c u b a t i o n a p p r o x i m a t e l y
4 . 4 mg h e x o s e were rem oved, a s w e l l a s 1 3 .5 mg h ex o sam in e
a s N - a c e t y l - g a l a c t o s a m i n e and 4 . 5 mg o f h ex o sam in e a s N-
a c e t y l - g l u c o s a m i n e . E p i d i d i m a l enzyme c o n t r o l s w ere ru n ■
t h r o u g h o u t .
P r e p a r a t i o n o f e p i d i d i m a l enzyme t r e a t e d a ^ - g l y c o -
p r o t e i n . 8 3 . 7 mg o f d e s i a l y z e d f e t u i n was t r e a t e d e x a c t l y
i n t h e same m anner a s th e a ^ - g l y c o p r o t e i n a b o v e . A f t e r i n
c u b a t i o n , 3 . 7 mg o f h e x o se had b een r e l e a s e d , a s w e l l a s
5 .4 6 mg o f h e x o sam in e a s N - a c e t y l - g a l a c t o s a m i n e , and 1 .8 mg
o f h e x o sam in e a s N - a c e t y l - g l u c o s a m i n e .
P r e p a r a t i o n o f U D P - N - a c e t y lg a la c t o s a m in e and UDP-N-
a c e t y l g l u c o s a m i n e . The h e x o s a m in e - l- P O ^ was a c e t y l a t e d i n
l4
t h e c o l d w i t h a c e t i c a n h y d r i d e - C i n t h e p r e s e n c e o f w a t e r ,
m e t h a n o l , and t r i e t h y l a m i n e b i c a r b o n a t e . The pH was m a in
t a i n e d b e tw ee n 6 . 5 and 8 . 0 w i t h NaHCO^ d u r i n g t h e one h o u r
r e a c t i o n .
The sam ple was l y o p h i l i z e d , d i s s o l v e d i n w a t e r , and
p l a c e d on a p y r i d i n e form AG50 WX8 colum n. A f t e r e l u t i o n ,
th e sam ple was l y o p h i l i z e d and d i s s o l v e d i n p y r i d i n e . T r i -
N - o c ty la m in e was a d d ed , and th e m i x t u r e e v a p o r a t e d to d r y
n e s s s e v e r a l t i m e s .
Dry u r i d i n e m on ophosphate m o r p h o l i d a t e was added
i n v a c u o , and t h e e n t i r e r e a c t i o n m i x t u r e r e p e a t e d l y d i s
s o l v e d i n p y r i d i n e , w hich was th e n e v a p o r a t e d o f f . F i
n a l l y , t h e m i x t u r e was d i s s o l v e d i n p y r i d i n e and m a i n t a i n e d
a t 6 0° f o r 18 h o u r s .
At th e end o f th e r e a c t i o n t im e , t h e s o l v e n t was
e v a p o r a t e d ( i n v a c u o ) and sodium a c e t a t e i n w a t e r was a d d e d .
The m i x t u r e was e x t r a c t e d w ith e t h e r t h r e e t i m e s , and th e
97
e t h e r l a y e r r e - e x t r a c t e d w i t h w a t e r .
The a q u e o u s l a y e r s w ere c o n c e n t r a t e d i n vacuo and
p l a c e d on a D E A E - c e llu lo s e colum n. The e l u t i n g s o l v e n t was
t r i e t h y l a m i n e b i c a r b o n a t e , pH 7.5* The sa m p le s were l y o
p h i l i z e d and f u r t h e r p u r i f i e d on p a p e r c h ro m a to g ra p h y i n
i s o b u t y r i c acldiNH^OHrHgO ( 5 7 : ^ * 3 9 ) . The b a n d s were c u t
o u t , e l u t e d from t h e p a p e r , and r e c h r o m a t o g r a p h e d to check
p u r i t y .
L I T E R A T U R E C I T E D
98
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This dissertation has been
microfilmed exactly as received 67-13,751
LAICO, M arion T heresa, 1938-
STUDIES ON THE BIOSYNTHESIS OF GLYCOPROTEIN
IN RETICULOCYTE STROMA.
U niversity of Southern C alifornia, P!h.D., 1967
B iochem istry
University Microfilms, Inc., A nn Arbor, M ichigan

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Creator Laico, Marion Theresa, 1938- (author)

Core Title Studies on the biosynthesis of glycoprotein in reticulocyte stroma

School Graduate School

Degree Doctor of Philosophy

Degree Program Biochemistry

Degree Conferral Date 1967-06

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 Eylar, Edwin H. (committee chair), Marx, Walter G. (committee member), McNall, Earl (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c18-147137

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