A guanosine diphosphate-L-fucose glycoprotein fucosyltransferase from Corynebacterium insidiosum
by Peter Ludwig Sadowski
A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE in Microbiology
Montana State University
© Copyright by Peter Ludwig Sadowski (1972)
Abstract:
Since Corynebacterium insidiosum produces a high molecular weight phytotoxic glycoprotein of which
38.7% is fucose, the enzyme catalyzed transfer of fucose to glycoproteins was investigated.
Fucosyltransferase activity was demonstrated using a crude cell membrane preparation from C,
insidiosum. Guanosine diphosphate-L-fucose was used as the sugar nucleotide glycosyl donor. Two
glycoproteins were used as fucosyl acceptors: a phytotoxic glycoprotein from C. insidiosum subjected
to mild acid hydrolysis, and a glycosidase-treated αracid glycoprotein obtained from pooled human
plasma. The cell membrane preparation demonstrated a pH optimum of 7.5, and the incorporation
appeared to be linear for 1+5 minutes. When the bacterial glycoprotein was used, concentrations in
excess of 0.2 μg per μ1 of reaction mixture appeared to be saturating. The addition of chelating agents
and nonionic detergents inhibited transferase activity. The presence of MnCl2 in the reaction mixture
appeared to enhance the incorporation of fucose. The fucosyltransferase could be demonstrated in the
soluble fraction. STATEM ENT
OF P E R M IS SIO N
TO C O PY
I n / p r e s e n t i n g th is t h e s i s in p a r t i a l fu lfillm en t of th e
r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t M o n ta n a S t a t e
U n iv e rsity ,
I a g r e e th a t th e L i b r a r y ' s h a l l m a k e it f r e e l y
a v ailab le f o r in s p e c tio n .
I f u r t h e r a g r e e th a t p e r m i s s i o n f o r
e x t e n s i v e c o p y in g of th is t h e s i s f o r s c h o l a r l y p u r p o s e s m a y b e
g ra n te d by my m a jo r p r o f e s s o r ,
D i r e c t o r of L i b r a r i e s .
o r,
in h i s a b s e n c e ,
b y th e
T t is u n d e r s t o o d th a t a n y c o p y in g o r
p u b l i c a t i o n of th is t h e s i s f o r f in a n c ia l g a i n s h a l l n o t b e a l l o w e d
w ith o u t m y w r i t t e n p e r m i s s i o n .
k.
'
C
■
i>
V \
S
.nx
J?0
Ti 1 1 2 0
176
A ' G U A N O S IN E D IP H O S P H A T E -L -F U C O S E G L Y C O
P R O T E IN F U C O S Y L T R A N S F E R A S E FROM
C O R Y N E B A C T E R I UM I N S I D I O S U M
by
PETER
L U D W IG S A D O W S K I
A t h e s i s s u b m i t t e d to t h e G r a d u a t e F a c u l t y in p a r t i a l
fu lfillm en t of th e r e q u i r e m e n t s f o r th e d e g r e e
of
M ASTER
OF
S C IE N C E
in
M icro b io lo g y
A p p ro v ed :
H ead,
M a jo r D e p a rtm e n t
C h airm a n /,
E x a m i n i n g C o m m itte e
G ra d u ate D ean
M O N TA N A S T A T E U N IV E R S IT Y
B o z e m a n , M o n ta n a
D e c e m b e r , 1972
iii
ACKNOW LEDGEM ENTS
I w o u l d lik e to e x p r e s s m y d e e p a p p r e c i a t i o n to D r .
A.
G ary
S t r o b e l f o r h is h e lp a n d g u id a n c e th ro u g h o u t m y g r a d u a te
s t u d i e s a t M o n ta n a S t a t e U n i v e r s i t y .
I w o u l d lik e to t h a n k D r .
D r.
S tro b e l, D r.
R eed,
and
H a p n e r f o r t h e i r h e l p in p r e p a r i n g th is m a n u s c r i p t .
I a m i n d e b t e d to D r .
U n iv e rsity ,
W illiam H e s s , B r i g h a m
Young
f o r th e e l e c t r o n m i c r o g r a p h a n d to D r .
H arry
S c h a c h t e r , U n i v e r s i t y of T o r o n t o , f o r th e g l y c o s i d a s e tre a te d
of - a c i d g ly c o p ro te in .
I a l s o w i s h to t h a n k D r .
P e te r
I
A l b e r s h e i m , U n i v e r s i t y of C o l o r a d o ,
f o r th e g a s c h r o m a t o g r a p h y
d a ta .
I w o u l d lik e to t h a n k D r .
K e n t,
J r.,
A rth u r K a r r , J r . ,
and D r .
f o r t h e i r a s s i s t a n c e in e d itin g th is m a n u s c r i p t a n d
f o r t h e i r h e l p a n d s u g g e s t i o n s in th e l a b o r a t o r y .
I a m v e r y g r a t e f u l to m y w if e ,
m a n u sc rip t.
M a rc ia ,
w h o t y p e d th is
Sam
iv
TABLE
OF
CONTENTS
Page
V IT A
................................................ ■
.......................................................... •
ii
A C K N O W L E D G M E N T S ............................................................................... .
iii
TABLE
C O N T E N T S ..........................................................................
iv
vi
OF
L IS T
OF
T A B L E S .....................................................................................
L IS T
OF
F IG U R E S .
................................................................
vii
A B S T R A C T ..........................
viii
IN T R O D U C T IO N
. . . . .
................................................................
E X P E R IM E N T A L
P R O C E D U R E ...............................................
I
.
9
C u l t u r i n g .....................................................................................................
9
P r e p a r a t i o n of c e ll f r e e
9
e x t r a c t ................................................
P r o t e i n d e t e r m i n a t i o n ..........................................
P r e p a r a t i o n of p r i m e r
S u b stra te
.
.
9
.. . . . . . . . . . .
....................................................................
11
F u c o s y l t r a n s f e r a s e a s s a y ..........................................................
V e r i f i c a t i o n of i n c o r p o r a t i o n of f u c o s e
M a te ria ls
RESU LTS.
11
.
. . . . . . .
11
13
.....................................................................................................
13
..........................................................................................................
15
P r o d u c t s of t h e r e a c t i o n ....................................
15
L o c a t i o n of t r a n s f e r a s e a c ti v ity in th e s u b c e l l u l a r
fra c tio n s. . .
15
V
Page
R e a c t i o n p r o p e r t i e s ...............................................................................
21
A c c e p t o r s p e c i f i c i t y s t u d i e s ...............................
31
D IS C U S S IO N .
...............................................................
37
S U M M A R Y ...............................................................................................................
43
L IT E R A T U R E
44
C I T E D ...............................................................................
vi
L IS T
OF
TABLES
Page
T a b le I
T h e f r a c t i o n a t i o n s c h e m e f o r C_. i n s i d i o s u m
T a b le 2
T h e l o c a t i o n of G D F - f u c o s y l t r a n s f e r a s e
a c ti v ity in th e v a r i o u s s u b c e l l u l a r f r a c t i o n s
of C_. i n s i d i o s u m . ..................................... .....
18
T h e e ffe c t of v a r i o u s a g e n t s o n f u c o s y l t r a n s f e r a s e a c t i v i t y .......................... .
28
T h e r e l a t i v e i n c o r p o r a t i o n of f u c o s e - ^ C
a c h i e v e d w ith v a r i o u s p r i m e r s . . . . .
36
T a b le 3
T a b l e J+
.
.
10
vii
L IS T
OF
F IG U R E S Page
F ig u re
I.
F ig u re 2.
F ig u re 3 .
S c h e m e of g u a n o s i n e d i p h o s p h a t e - L f u c o s e f u c o s y l t r a n s f e r a s e a c tiv ity f r o m
C . in sid io su m u s in g p a rtia lly h y d r o ly z e d
p h y to to x ic g l y c o p r o t e i n a s th e p r i m e r .....................
T r a c i n g of r a d i o c h r o m a t o g r a m s of s u g a r
r e s i d u e s f o u n d in u n h y d r o l y z e d ( X )
a n d h y d r o l y z e d ( Y ) p h y to to x ic g l y c o ­
p r o t e i n p r i m e r s .........................................
8
17
A n e l e c t r o n m i c r o g r a p h of th e c e ll
m e m b r a n e f r a c t i o n .......................................................... . .
.
20
F i g u r e 2+.
T h e p H d e p e n d e n c e of f u c o s e i n c o r p o r a t i o n
.
23
F ig u re
T h e d e p e n d e n c e of f u c o s e i n c o r p o r a t i o n o n
t i m e ....................................................................................................
25
T h e d e p e n d e n c e of f u c o s e i n c o r p o r a t i o n o n
t h e c o n c e n t r a t i o n of th e p r i m e r ....................................
2?
5.
F ig u re 6 .
F ig u re 7.
F ig u re
8.
F ig u re 9.
.
T h e c o l u m n c h r o m a t o g r a p h y of r e a c t i o n m i x - ■
t u r e s o n S e p h a d e x G - 2 5 . ...................................................
30
T h e a f f e c t of a c i d h y d r o l y s i s o n th e a b ility
of th e p h y to to x ic g l y c o p r o t e i n to s e r v e a s a
f u c o s y l a c c e p t o r ................................................................................ . 3 3
T h e u s e of f u c o s y l t r a n s f e r a s e to i n c o r p o r a t e
f u c o s e in to v a r i o u s d e r i v a t i v e s of h u m a n
p l a s m a O ^ - a c id g l y c o p r o t e i n .........................................
35
viii
ABSTRACT
S i n c e C o r y n e b a c t e r i u m i n s i d i o s u m p r o d u c e s a h ig h
m o l e c u l a r w e i g h t p h y to to x ic g l y c o p r o t e i n of w h i c h 3 8 .7 % is
f u c o s e , t h e e n z y m e c a t a l y z e d t r a n s f e r of f u c o s e to g l y c o p r o t e i n s
w a s in v e stig a te d .
F u c o s y l t r a n s f e r a s e a c tiv ity w a s d e m o n s t r a t e d
u s i n g a c r u d e c e ll m e m b r a n e p r e p a r a t i o n f r o m C , i n s i d i o s u m .
G u a n o s in e d ip h o s p h a te - B -f u c o s e w a s u s e d a s th e s u g a r n u c le o ­
tid e g l y c o s y l d o n o r . T w o g l y c o p r o t e i n s w e r e u s e d .as f u c o s y l
a c c e p to rs:
a p h y to to x ic g l y c o p r o t e i n f r o m C . i n s i d i o s u m
s u b j e c t e d to m ild a c i d h y d r o l y s i s , a n d a g l y c o s i d a s e - t r e a t e d
<Xr a c i d g l y c o p r o t e i n o b t a i n e d f r o m p o o l e d h u m a n p l a s m a .
The
c e ll m e m b r a n e p r e p a r a t i o n d e m o n s t r a t e d a p H o p tim u m of 7 * 5 , ■
a n d t h e i n c o r p o r a t i o n a p p e a r e d to b e l i n e a r f o r 4 5 m i n u t e s .
W h e n th e b a c t e r i a l g l y c o p r o t e i n w a s u s e d , c o n c e n t r a t i o n s in
e x c e s s of 0 . 2 pig p e r p.1 of r e a c t i o n m i x t u r e a p p e a r e d to b e
sa tu ra tin g .
T h e a d d i t i o n of c h e l a t i n g a g e n t s a n d n o n io n ic
d e t e r g e n t s in h ib ite d t r a n s f e r a s e a c t i v i t y .
T h e p r e s e n c e of
M n C lg in t h e r e a c t i o n m i x t u r e a p p e a r e d to e n h a n c e th e
i n c o r p o r a t i o n of f u c o s e .
T h e f u c o s y l t r a n s f e r a s e c o u ld b e
d e m o n s t r a t e d in th e s o l u b l e f r a c t i o n .
IN T R O D U C T IO N
I n th e p a s t d e c a d e ,
m e m b ra n e a sso c ia te d en zy m e sy ste m s
h a v e b e e n d e s c r i b e d a n d i m p l i c a t e d in th e s y n t h e s i s of m a c r o - •
m o l e c u l e s in g e n e r a l , a n d g l y c o p r o t e i n s in p a r t i c u l a r .
w o r k h a s b e e n r e v ie w e d ex ten siv e ly
T h is
( 1 0 ,-2 3 , 2 8 ) .
T h e 6 - d e o x y - D - g a l a c t o s e , D - f u c o s e , is fo u n d a s a
c o n s t i t u e n t of n u m e r o u s g l y c o p r o t e i n s .
p la s m a g ly c o p ro te in s
( 7 , 2 7 ),
I t h a s b e e n f o u n d in
b lo o d g r o u p s u b s t a n c e s
( 3 2 ),
g l y c o p r o t e i n s of w h o l e b r a i n f r a c t i o n s a n d n e r v e e n d i n g s
p h y to h e m a g g lu tin s fro m
p ro te in s
(I),
seeds
( 8 ),
(1 8 ,3 3 ),
p la sm a m e m b ra n e g ly c o ­
a n d p h y to to x ic g l y c o p r o t e i n s
(2 5 , 2 9 ).
T h e t r a n s f e r of f u c o s e into g l y c o p r o t e i n s is c a t a l y z e d b y a
c l a s s of e n z y m e s k n o w n a s I u c o s y l t r a n s f e r a s e s .
G u a n o sin e
d i p h o s p h a t e f u c o s e s e r v e s a s t h e g l y c o s y l d o n o r in t h e s e
re a c tio n s.
T h e i m p o r t a n c e of t h i s s u g a r n u c l e o t i d e a s th e
g l y c o s y l d o n o r w a s f i r s t d e m o n s t r a t e d in v iv o b y K o r n f i e l d
e t a l _ . '( 2 0 ) .
T h e y h a d a t t e m p t e d to d e t e c t a n u c l e o t i d e l i n k e d ,
fu c o s e c o n tain in g ,
o l i g o s a c c h a r i d e i n t e r m e d i a t e in th e s y n t h e s i s •
of th e h e t e r o s a c c h a r i d e
c h a i n s of b lo o d g r o u p s u b s t a n c e s .
T h e i r a t t e m p t to d e m o n s t r a t e t h e o l i g o s a c c h a r i d e n u c l e o t i d e w a s
u n su c c e ssfu l.
T h e y d id fin d l a r g e a m o u n t s of g u a n o s i n e
d i p h o s p h a t e f u c o s e in t h e c e ll f r a c t i o n ,
w h i c h w a s s h o w n to
c o n ta in th e n e w ly s y n th e s iz e d b lo o d g r o u p s u b s t a n c e s .
T h e f i r s t w o r k e r s to d e m o n s t r a t e a n e n z y m a t i c t r a n s f e r
2
of L - f u c o s e f r o m g u a n o s i n e d i p h o s p h a t e - L - f u c o s e u s e d l a c t o s e a s
th e fu c o sy l a c c e p to r
trisa c c h a rid e ,
(i2 ).
T h e p r o d u c t of th e r e a c t i o n w a s t h e
f u c o s y l l a c t o s e , w h i c h is th e s i m p l e s t f u c o s e
c o n ta i n in g o l i g o s a c c h a r i d e f o u n d in m ilk .
T h e e n z y m e w h ic h
c a t a l y z e d t h i s r e a c t i o n w a s t h e p a r t i c u l a t e c e ll f r a c t i o n of
la c ta tin g c a n in e m a m m a r y t i s s u e .
T h is p a rtic u la te p re p a ra tio n
w o u l d a l s o c a t a l y z e t h e f o r m a t i o n of t h e t r i s a c c h a r i d e
i n c u b a t e d w ith D - g l u c o s e ,
w hen
u rid in e d ip h o s p h a te - D - g a la c to s e , an d
g u a n o sin e d ip h o s p h a te -L -fu c o s e .
T h e in v iv o i n c o r p o r a t i o n of f u c o s e into th e g l y c o p r o t e i n s of
a d u lt m a l e r a t s
in 1 9 6 7 .
w a s s t u d i e d a t le n g th b y B e k e s i a n d W i n z l e r
T h e y f o u n d th a t L - f u c o s e - 1
(3)
w a s a su itab le
p r e c u r s o r in t h e i r e x a m i n a t i o n of t h e b i o s y n t h e s i s of t h e g l y c o ­
p r o t e i n a s t h e f u c o s e w a s n o t r a p i d l y c o n v e r t e d in to o t h e r s u g a r s
o r C O g . ■ B y s t u d y i n g th e t r i c h l o r o a c e t i c a c i d s o l u b l e a n d
in so lu b le ra d io a c tiv e p r o d u c ts fro m
a n im a ls s a c r if ic e d at v a r io u s
t i m e s , t h e f o llo w in g s e q u e n c e of r e a c t i o n s w a s p o s t u l a t e d :
F u c o s e -------> F u c o s e - I - P ------- > G D P - F u c o s e ------ > G l y c o p r o t e i n s
M p re re c e n tly ,
T ru jillo a n d G a n
(3 1 )
h a v e s h o w n t h a t th e
s e q u e n c e of r e a c t i o n s p r o p o s e d b y B e k e s i a n d W i n z l e r c a n a l s o
b e p r e d i c t e d f r o m in v i t r o s t u d i e s u s i n g b o v in e t h y r o i d g l a n d
slic e s.
T h e in v i t r o i n c o r p o r a t i o n of f u c o s e - l - ^ C
w as
3
s i m i l a r l y f o ll o w e d b y c h r o m a t o g r a p h i c a n a l y s i s of th e t r i c h l o r o ­
a c e t i c a c i d s o l u b l e a n d in s o l u b l e r a d i o a c t i v e p r o d u c t s f o u n d a t
v a r io u s tim e s d u rin g in c u b atio n .
G ro llm a n and M a rc u s
(13)
d e m o n s t r a t e d th e in v itr o
i n c o r p o r a t i o n of f u c o s e into s o l u b l e b lo o d g r o u p H s u b s t a n c e .
T h e g ly c o sy l d o n o r w a s g u a n o s in e d i p h o s p h a t e - L - f u c o s e , and
th e e n z y m e p r e p a r a t i o n w a s o b ta in e d fro m h o g g a s tr ic
m ucosa.
A n im m u n o l o g i c a l t e c h n i q u e w a s a v a i l a b l e f o r th e id e n tif ic a tio n of
th e r a d i o a c t i v e e n d p r o d u c t a s t h e r e c e p t o r p l u s i n c o r p o r a t e d
f u c o s e w a s i d e n t i c a l to th e b lo o d g r o u p H s u b s t a n c e ,
th e H
s p e c i f i c i t y b e i n g d e t e r m i n e d b y th e t e r m i n a l 0 < - L - f u c o s y l (I
> 2) - ^ - D - g a la c to s y l lin k a g e .
T h e a n t i g e n i c d e t e r m i n a n t s of t h e A ,
h av e fu co se
H o w ev er,
B , O (H )
sy ste m
<Y- ( I — > 2 ) lin k e d to a D - g a l a c t o s y l r e s i d u e
all
(26).
t h e d i s t i n g u i s h i n g f e a t u r e b e t w e e n th e L e w i s b lo o d
g r o u p ty p e a
( L e a ) a n d th e L e w i s b lo o d g r o u p t y p e b ( L e ^ )
i s t h a t th e L e *3 g l y c o p r o t e i n h a s a f u c o s e
( I —> 2 ) l i n k a g e
w h e r e a s t h e L e a g l y c o p r o t e i n d o e s n o t (11+).
U s i n g th is i n f o r m a t i o n a n d t h e f a c t th a t " s e c r e t o r s "
A,
B,
O (H )
se c re te
s o l u b l e b lo o d g r o u p s u b s t a n c e s a n d " n o n s e c r e t o r s 11
s e c r e t e L e a g ly c o p ro te in ,
S h e n et a l .
(26)
d e m o n s t r a t e d th at
b lo o d g r o u p s u b s t a n c e s p e c if ic ity c a n b e a c c o u n t e d f o r b y a
sp e c ific f u c o s y l t r a n s f e r a s e .
F ro m
t h e i n f o r m a t i o n a b o u t th e
4
f u c o s y l l i n k a g e s in a n t i g e n i c d e t e r m i n a n t s of th e v a r i o u s b l o o d '
gro u p s,
it c o u ld b e p r e d i c t e d t h a t o n e s h o u l d fin d a s p e c if ic
0 ( - ( I — >-2 ) - f u c o s y l t r a n s f e r a s e in " s e c r e t o r s "
a n d a n a b s e n c e of
t h i s t r a n s f e r a s e in " n o n s e c r e t o r s " . • T h i s w a s e s t a b l i s h e d to b e
th e c a s e .
F u r t h e r w o r k o n CX - L - f u c o s y l t r a n s f e r a s e s a n d t h e i r
r e l a t i o n s h i p s to b lo o d g r o u p c h a r a c t e r s w a s d o n e b y C h e s t e r
a n d W a tk i n s
( 6 ) a n d b y G ro llm a n et a l .
form er p aper , 2-,
3-,
(12+) in 1 9 6 9 .
I n th e
a n d 2+ - ( X ' - F - f u c o s y l t r a n s f e r a s e s w e r e
d e m o n s t r a t e d in p e r s o n s w h o a r e
" se c re to rs"
g ro u p su b sta n c e s;
w e r e f o u n d to h a v e 3 - a n d
" n o n se c re to rs"
2+-( X - L - f u c o s y l t r a n s f e r a s e s .
T h e la tte r p a p e r d istin g u ish e s
e n z y m a tic a lly b e tw e e n w o m e n w ho a r e
w om en w ho a re
L ea (-)
m i n a n t s of L e a a n d L e ^
N -a c e ty lg lu c o sa m in e .
of s o l u b l e b lo o d
or L e^ (-) .
Le
ci
(+) or L e
b
(+ )
and
T h e stru c tu ra l d e te r­
c o n t a i n f u c o s e lin k e d D<f - ( I — > 2+) to
An
- ( I —> 2+ ) - h i c o s y l t r a n s f e r a s e w a s
d e m o n s t r a t e d in t h e m ilk of w o m e n w h o h a d L e w i s t y p e a n t i g e n s
M o re re c e n tly ,
Jarkovsky
(I?)
a l s o d id s o m e w o r k o n f u c o s y l -
t r a n s f e r a s e s in h u m a n m ilk w h i c h w e r e s p e c i f i e d b y t h e L e w i s
b lo o d ty p e . ■
D u r i n g th is t i m e , n o t all w o r k o n " f u c o s y l t r a n s f e r a s e s w a s
d o n e u s i n g t h e b lo o d g r o u p s u b s t a n c e s .
a l.
In 1968,
B o s m a n n et
( 5 ) f o u n d t w o f u c o s y l t r a n s f e r a s e s in H e L a c e l l s .
The
5
o b j e c t i o n s to s o m e of t h e p r e v i o u s w o r k o n t r a n s f e r a s e s u s i n g
p a r t i c u l a t e e n z y m e p r e p a r a t i o n s w a s a v o i d e d in t h i s w o r k b y
s o l u b i l i z i n g th e t r a n s f e r a s e s
X -1 0 0 .
T rito n
T h e e v i d e n c e f o r tw o f u c o s y l t r a n s f e r a s e s is b a s e d o n
sp ec ific ity .
fu co se:
w ith th e n o n io n ic d e t e r g e n t ,
T w o p r i m e r s s h o w e d s u c c e s s f u l i n c o r p o r a t i o n of
fe tu in , fro m
w h ic h sialic a c id a n d g a la c to s e w e r e
re m o v e d e x p o sin g a te rm in a l N -a c e ty !g lu c o s a m in e , an d p o rc in e
su b m a x illa ry g ly c o p ro te in from
w h ic h sialic a c id a n d f u c o s e w e r e
re m o v e d e x p o sin g a te rm in a l g a la c to s e .
G ly c o p ro te in fu c o sy l-
t r a n s f e r a s e a c ti v ity w a s s o u g h t in H e L a c e l l s s i n c e 94% of th e
f u c o s e in th e c e ll i s n o t e x t r a c t a b l e b y o r g a n i c s o l v e n t s a n d
t h e r e f o r e p r e s u m a b l y b o u n d in g l y c o p r o t e i n s .
I n th e e a r l y 1 9 7 0 's , f u c o s y l t r a n s f e r a s e s r e s p o n s i b l e f o r th e
i n c o r p o r a t i o n of f u c o s e into g l y c o p r o t e i n s h a v e b e e n f o u n d in
m any s y ste m s.
fu c o sy ltra n sfe r a s e
G rim e s and B u rg e
( 1 1 ) h a v e id e n tifie d a
in c h i c k e m b r y o f i b r o b l a s t c u l t u r e 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 of f u c o s e in to t h e m e m b r a n e g l y c o p r o t e i n
of S i n d b i s v i r u s .
T h i s a c tiv ity c a n b e d e m o n s t r a t e d in i n f e c te d
a n d u n i n f e c t e d c e l l s im p ly in g s o m e s o r t of h o s t m o d if ic a tio n .
A fu c o sy ltra n sf e r a s e
h a s b e e n i s o l a t e d f r o m r a t s m a l l i n te s tin e
m u c o sa re sp o n sib le fo r
(4).
o( - ( I —> 2 ) l i n k a g e s in b lo o d g r o u p s
Jabbal and S c h a c h te r
f e r a s e s in p o r k l i v e r .
(1 6 )
They a re
h a v e found tw o f u c o s y l t r a n s ­
d i f f e r e n t i a t e d n o t o n ly b y
6
p r i m e r sp ec ific ity ,
M e lc h e rs
ferase
(2 4 )
b u t a l s o b y k in e tic d a t a .
and M elc h e rs
(22)
P a rk h o u s e and
h a v e found a f u c o s y l t r a n s -
r e s p o n s i b l e f o r th e s y n t h e s i s o f .th e c a r b o h y d r a t e p o r t i o n
of i m m u n o g lo b u l in M ( I g M )
tu m o r M O P C
21.
o b ta in e d fro m
m ouse p la sm a -c e ll
T h e i n c o r p o r a t i o n of f u c o s e w a s n e c e s s a r y
f o r t h e s e c r e t i o n of I g M .
Z a tz and B a ro n d e s
(3 3 ,3 4 )
have
id e n tifie d f u c o s y l t r a n s f e r a s e s in r a t b r a i n w h i c h w ill i n c o r p o r a t e
f u c o s e in to p o r c i n e p l a s m a g l y c o p r o t e i n s a f t e r t h e y h a v e b e e n
s u b j e c t e d to m ild a c i d h y d r o l y s i s a s w e ll a s into e n d o g e n o u s
re c e p to rs.
N o b a cte ria l fu c o s y ltra n sfe ra s e s have b e en re p o rte d even
t h o u g h G i n s b u r g h a s id e n tifie d a n e n z y m e f r o m A e r o b a c t e r
a e r o g e n e s r e s p o n s i b l e f o r t h e f o r m a t i o n of g u a n o s i n e d i p h o s p h a t e
fu co se
( 9 ).
S in c e
C o ry n e b a c te riu m
i n s i d i b s u m p r o d u c e s a h ig h
m o l e c u l a r w e i g h t p h y to to x ic g l y c o p r o t e i n of w h i c h 3 8 . 7 $ is f u c o s e
(2 5 ),
it w o u l d s e e m
r e a s o n a b l e th a t th is b a c t e r i u m
possesses
g u a n o s in e d i p h o s p h a t e - L - f u c o s e f u c o s y ltr a n s f e r a s e activ ity .
T h erefo re,
t h e p u r p o s e of th is r e p o r t i s tw o f o ld :
s t r a t e f u c o s y l t r a n s f e r a s e a c tiv ity
(F ig .
s o m e of th e p a r a m e t e r s of th is a c t i v i t y .
I),
I)
to d e m o n ­
a n d 2 ) to e s t a b l i s h
F ig u re I .
S c h e m e of g u a n o s i n e d i p h o s p h a t e - L - f u c o s e f u c o s y l t r a n s f e r a s e a c tiv ity f r o m
C_. i n s i d i o s u m u s i n g
p a r t i a l l y h y d r o l y z e d p h y to to x ic g l y c o p r o t e i n a s th e
p rim e r.
8
GDP- L-FUCOSE+PRiMER+
CEII MEMBRANE ENZYME
PRIMER-L-FUCOSE + GDP +
CEII MEMBRANE ENZYME
E X P E R IM E N T A L
C u ltu rin g :
Jensen
PROCEDURE
T h e c u l t u r e of C o r y n e b a c t e r i u m
( c o u r t e s y of F ' . I .
in sid io su m
(M c C u ll. )
F r o s h e i s e r , U n i v e r s i t y of M i n n e s o t a )
w a s m a in ta in ed a n d c u ltu re d a s d e s c r ib e d b y , R ie s a n d S tr o b e l
( 2 5 ) e x c e p t th a t s u c r o s e w a s s u b s t i t u t e d f o r g l u c o s e a s th e
carbon s o u rc e .
P r e p a r a t i o n of c e ll f r e e e x t r a c t :
s p e c ifie d t i m e s .
frac tio n s w e r e
w ere
The
T h e c e l l s w e r e h a r v e s t e d at,
c e ll m e m b r a n e s a n d o t h e r s u b c e l l u l a r
o b t a i n e d a s d e s c r i b e d in T a b l e I .
T h e c e ll s
c o l l e c t e d b y s u b j e c t i n g t h e s u s p e n s i o n to c e n t r i f u g a t i o n a t
2 2 , OOOxg a n d 1+0C .
S u b s e q u e n t s t e p s w e r e c o n d u c t e d "at 4 ° S
u n le s s o th e r w is e sp ec ifie d .
T h e c e lls w e r e w a s h e d 3 - 5 tim e s
w ith a b u f f e r w h i c h c o n s i s t e d of T r i s - H C l , 0 . 0 5 M ,
0.1+M s u c r o s e .
M elsu n g e n
The
c e l l s w e r e d i s r u p t e d in a n A p p a r a t e b a u
( r e f e r r e d to a s M . S . K . )
( G la sp e rle n ,
pH 7 .5 and
0 .1 1 -0 .1 2
mm)
u sin g g la s s b e a d s
p r e v i o u s l y w a s h e d in b u f f e r .
The ■
c e l l s w e r e d i s r u p t e d in 20 s e c o n d b u r s t s to a to ta l of 2 m in u te s
to a v o i d o v e r h e a t i n g .
T h e c e ll m e m b r a n e p e l l e t ,
o b ta in e d by
s u b j e c t i n g th e c e ll d e b r i s s u p e r n a t a n t liq u id to c e n t r i f u g a t i o n a t
4 8 ., 5 0 Oxg f o r 1 2 0 m i n u t e s ,
c o n s i s t i n g of 0 . 0 5 M
w a s r e s u s p e n d e d in a s o lu tio n
T r i s - H C l , a n d 0 . 4M s u c r o s e , p H 7 . 5 ,
u s i n g a 15 ml P y r e x g l a s s h o m o g e n i z e r .
P r o te in d e te rm in a tio n :
P r o t e i n w a s ' d e t e r m i n e d b y th e m e th o d of
T a b le L
T h e fr a c tio n a tio n s c h e m e f o r C . in sid io su m
C u ltu re
2 2 , 0 0 0 x g , 10 m in .
P e l l e t , c e l l s a n d CaCOo
w a s h w ith T r i s - H C l b u f f e r ___________
C aC O ^
C e lls
S u p e r n a t a n t liq u id
(d isc a rd )
S u p e r n a t a n t liq u id
(d isc a rd )
D i s r u p t i o n in M . S . K . , 2 m in .
c e l l s a fte r, f r a c t i o n a t i o n
________' tSqOOxe:. 3 0 m in .________
C e ll d e b r i s p ellet
(d isc a rd )
C ell d e b r is
s u p e r n a t a n t liq u id
4 8 , 5 0 0 x g , 120 m in .
C ell m e m b ra n e s
. H ig h S p e e d
s u p e r n a t a n t liq u id
(d is c a rd )
K
O
11
L ow ry
e t al_. ( 2 1 ) .
B o v in e s e r u m
a l b u m in w a s u s e d a s a
sta n d a rd .
P r e p a r a t i o n of p r i m e r :
in sid io su m
T h e p h y to to x ic g l y c o p r o t e i n f r o m
C .
w a s o b ta in e d a s d e s c r i b e d by P i e s a n d S tr o b e l
T h e to x in w a s s t o r e d a t - 2 0 0C until u s e d .
(2 5 ).
The. p rim e r w as
r o u t i n e l y p r e p a r e d b y d i s s o l v i n g 1 0 0 m g lo ts of g l y c o p r o t e i n in
2N H C l, 3 -5
m l, in a s e a l e d t e s t t u b e a n d h e a t i n g th e s o lu tio n
in a n . o v e n a t IO O 0C f o r 3 0 . m i n u t e s .
T h e p ro d u c t w a s d ia ly z e d
a g a i n s t f r e q u e n t c h a n g e s of d is tille d d e i o n i z e d w a t e r a t 1+°C f o r a
m in im u n of 7 2 h o u r s .
T h e g l y c o p r o t e i n w h i c h c a m e o u t of
so lu tio n w a s r e m o v e d by c e n trifu g a tio n a n d th e r e s u ltin g s u p e r ­
n a tan t d r i e d u s in g hot a i r .
VThen . p r i m e r w a s n e e d e d ,
s u p e r n a t a n t w a s r e s u s p e n d e d in d is tille d
g ly c o sid a se -tre a te d
th e d r i e d
d e io n iz e d w a te r .
The
C ^ - a c i d g l y c o p r o t e i n w a s a gift f r o m D r .
H „
S c h a c h t e r , D e p a r t m e n t of B i o c h e m i s t r y , " U n i v e r s i t y of T o r o n t o .
■j l
S u b s tra te :
fu co se-^ C ),
G u a n o sin e d ip h o s p h a te -L -fu c o s e a m m o n iu m
salt,
195 m C i/m m o l,
C(U),
(G D P -
w a s o b tain ed fro m
A m e r s h a m / S e a r l e a n d s t o r e d a t - 2 0 ° C until u s e d .
T h e s o lu tio n
u s e d f o r e n z y m e a s s a y w a s p r e p a r e d b y d i s s o l v i n g th e G D P f u c o s e - 4 C in d is tille d w a t e r
F u c o sy ltra n sfe ra se
assay:
( 5 5 ,5 0 0
c p m /p l;
128 p m o le s ).
T h e enzym e p re p a ra tio n ,
p rim e r,
12
and su b stra te w e re
c o m b in e d o n a p a ra film
s trip a n d th e n
i n c u b a t e d in a s m a l l d i a m e t e r g l a s s tu b e a t 20 0C .
T h e co n cen -'
t r a t i o n of th e v a r i o u s r e a c t a n t s w a s a s s p e c i f i e d .
T h e re a ctio n s
w e r e t e r m i n a t e d b y s p o ttin g t h e r e a c t i o n m i x t u r e s o n W h a tm a n
no. T p a p e r.
T h e u n r e a c t e d s u b s t r a t e w a s r e m o v e d b y h ig h
v o l t a g e p a p e r e l e c t r o p h o r e s i s a t Zhr v / c m
b o ra te ,
pH
9 .1 .'
U n d e r th e se
in 0 . 0 5 M s o d i u m t e t r a ­
c o n d itio n s ,' th e s u b s tr a te
(an d
b r e a k d o w n p r o d u c t s of th e s u b s t r a t e ) will m i g r a t e b u t p o l y m e r i c
p r o d u c t s w ill r e m a i n a t th e o r i g i n .
T h e a m o u n t of f u c o s e
i n c o r p o r a t e d in to g l y c o p r o t e i n w a s d e t e r m i n e d b y c u ttin g o u t th e
o r i g i n a n d m e a s u r i n g t h e r a d i o a c t i v i t y in a P a c k a r d L i q u i d
S c in tilla tio n S p e c t r o m e t e r
(M odel 3 3 2 0 ) .
T h e o rig in s trip w a s
i m m e r s e d in a liq u id s c in tilla tio n c o c k t a i l c o n s i s t i n g of 1 . 5 ml
of a b s o l u t e m e t h a n o l a n d 1 3 . 5 ml of s c in t illa tio n s o lu tio n
c o n t a i n i n g 5 . 0 g of 2 , 5 - d i p t i e n y l o x a z o l e a n d 1 0 0 m g of p - b i s 2 ( 5 - p h e n y l o x a z o l y l ) - b e n z e n e p e r l i t e r of t o l u e n e .
T h e c h an n e ls
r a t i o m e t h o d w a s u s e d to c o r r e c t f o r q u e n c h i n g .
O n e e x p e r im e n t w a s c o n d u c te d u sin g a S e p h a d e x 0 - 2 5
c o l u m n to s e p a r a t e t h e u n r e a c t e d s u b s t r a t e f r o m th e r e a c t i o n
m ix tu re .
T h e r e a c t i o n w a s i n c u b a t e d in a s m a l l t e s t tu b e a t
2 0 0C a n d 3 5 r e v o l u t i o n s p e r m i n u t e .
T h e re a c tio n w as t e r ­
m i n a t e d b y p l a c i n g t h e t e s t t u b e in a b o ilin g w a t e r b a t h a n d
d r y i n g th e r e a c t i o n m i x t u r e w ith a s t r e a m
of n i t r o g e n g a s .
The
13
p r o d u c t m i x t u r e w a s r e s u s p e n d e d in 4 0 0 p.1 of d is tille d w a t e r
an d frac tio n a ted by c h ro m a to g ra p h y on a S e p h a d e x G -2 5
c o lu m n ,
31 cm x 1 . 9 c m .
The
0 .5
ml f r a c t i o n s w e r e c o l l e c t e d ,
a n d t h e n p l a c e d in c o u n tin g v i a l s c o n ta i n in g 15 ml of A q u a s o l
u n i v e r s a l liq u id s c i n t illa tio n c o c k ta il
( N ew E n g lan d N u c le a r) .
V e r i f i c a t i o n of i n c o r p o r a t i o n of f u c o s e :
T h e re a c tio n p ro d u c ts
w e r e e l u t e d f r o m t h e o r i g i n w ith 10 ml of w a t e r p e r o r i g i n a f t e r
e l e c t r o p h o r e s i s of 13 s t a n d a r d r e a c t i o n m i x t u r e s .
T h e e lu a n t
w a s c o lle c te d a n d d r ie d u sin g hot a i r , an d th e n f u r th e r d rie d
u n d e r vacuum
over
at ro o m te m p e ra tu re .
T h e ra d io -
l a b e l e d g l y c o p r o t e i n w a s d i s s o l v e d in d is tille d w a t e r to g iv e a
fin a l c o n c e n t r a t i o n of 3 6 4 0 d p m in a 100 p i.
A 3 0 p.1 a liq u o t of
g l y c o p r o t e i n w a s d r i e d in a t e s t tu b e a n d r e s u s p e n d e d i n 2 . 0
of I N
TEA
(triflu o ro a c e tic a c id )
ml
a n d s u b j e c t e d to h y d r o l y s i s in
a s e a l e d t e s t tu b e f o r 2 h o u r s a t 1 2 0 ° C .
S u g ars
w e r e id e n tifie d b y d e s c e n d i n g p a p e r c h r o m a t o ­
g r a p h y in e th y l a c e t a t e - a c e t a t e - f o r m i c a c i d - H ^ O
v /v /v /v .
T h e s u g a r sta n d a rd s
of T r e v e l y a n
(3 0 ).
( 1 8 : 3 : 1 =4)
w e r e l o c a t e d u s i n g th e m e t h o d s
T h e ra d io la b e le d p rim e r and m o n o s a c ­
c h a r id e s w e r e lo c a te d u sin g a P a c k a r d R a d io c h ro m a to g r a m
Scanner
M ate ria ls :
(M odel 3 8 5 ) .
T r i t o n X - 1 0 0 a n d t r i s ( h y d r o x y m e t h y l ) a m in o m e t h a n e
14
(T riz m a )
so d iu m
w e r e o b ta in e d fro m
c a c o d y l a t e a n d th e E D T A
L a b o ra to rie s,
g rad e.
S ig m a C h e m ic a l C o m p a n y ,
In c.
w e r e o b ta in e d fro m
K&K
A ll o t h e r , c h e m i c a l s u s e d w e r e r e a g e n t
The
RESULTS
P ro d u c ts
of th e r e a c t i o n :
E l e c t r o p h o r e s i s of a n y r e a c t i o n
m i x t u r e c o n t a i n i n g p r i m e r a n d a c t i v e e n z y m e y i e l d e d tw o r a d i o ­
activ e p e a k s .
O n e w a s a t. th e o r i g i n a n d t h e o t h e r w a s a b r o a d
p e a k e x t e n d i n g 10 c m to 16 cm off th e o r i g i n r e l a t i v e to th e
m o v e m e n t of t h e p i c r i c a c i d m a r k e r a t 20 c m .
w h ic h r e m a in e d at th e o rig in w e r e
s u b j e c t e d to d e s c e n d i n g ■
p a p e r c h r o m a t o g r a p h y fo llo w in g e l u tio n .
in F i g u r e 2 .
T h e co m p o n en ts
T h e re su lts a re
show n
C h r o m a t o g r a p h y of th e u n h y d r o l y z e d p r o d u c t
s h o w e d all th e r a d i o a c t i v i t y a t t h e o r i g i n .
re a c tio n p ro d u ct sh o w e d a tra c e
T h e h y d ro ly z e d
of r a d i o a c t i v i t y a t th e o r i g i n
a n d ta m a j o r i t y of t h e r a d i o a c t i v i t y a p p r o x i m a t e l y f o u r i n c h e s
f r o m th e o r i g i n .
O n ly t r a c e a m o u n t s of th e r a d i o a c t i v i t y i n c o r ­
p o r a t e d a p p e a r e d to b e s o l u b l e in th e liq u id s c in t illa tio n c o c k t a i l .
L o c a t i o n of t r a n s f e r a s e a c tiv ity in t h e s u b c e l l u l a r f r a c t i o n s :
S u b c e l l u l a r f r a c tio n s w e r e a s s a y e d f o r f u c o s y l t r a n s f e r a s e activ ity .
T h e re s u lts from
s h o w n in T a b l e 2 .
a t y p i c a l c e ll p r e p a r a t i o n p r o c e d u r e a r e
T h e h ig h s p e e d s u p e r n a t a n t liq u id d e m o n ­
s t r a t e d a h i g h e r s p e c i f i c a c tiv ity t h a n t h e c e ll m e m b r a n e p r e c i p ­
ita te , h o w e v e r ,
i n a s m u c h a s t h e a c tiv ity a s s o c i a t e d w ith th e
m e m b r a n e f r a c t i o n c o u ld b e e a s i l y c o n c e n t r a t e d b y p e lle tin g a t
4 8 , 5 0 0 x g , it w a s u s e d a s a s u b j e c t of th is s t u d y .
D r.
H e s s k in d ly s u p p l i e d t h e e l e c t r o n m i c r o g r a p h
3)
(F ig .
W illiam
of th e c e ll
F ig u r e 2 .
T r a c i n g of r a d i o c h r o m a t o g r a m s of s u g a r r e s i d u e s
f o u n d in u n h y d r o l y z e d
(X )
and h y d ro ly z e d
p h y to to x ic g l y c o p r o t e i n p r i m e r s .
r e a c tio n m ix tu re c o n ta in e d :
(Y )
E a c h c o m p le te
p h y to to x ic g l y c o p r o t e i n
s u b j e c t e d to m ild a c i d h y d r o l y s i s , 3 . 7 T S 5 Q D P f u c o s e - ^ C , 2 6 .2 p m o le s;
m ic ro m o le s;
T r i s , pH 7 .5 ,
s u c r o s e , 10 m i c r o m o l e s ;
1 .2 5
a n d 20 pig
of e n z y m e p r o t e i n in a to ta l v o l u m e of . 0 3 5 m l .
T h e c ells w e r e
b e in g h a r v e s t e d .
c u l t u r e d f o r 50 h o u r s p r i o r to
.
c o u n t s / min
c o u n t s / m IN
17
o-
Y
4
INCHES
5
18
T a b le 2.
T h e l o c a t i o n of G D P - f u c o s y l t r a n s f e r a s e a c tiv ity
in th e v a r i o u s s u b c e l l u l a r f r a c t i o n s of C . i n s i d i o s u m
S u b c e llu la r
fra c tio n
V o lu m e
ml
T o tal a
a c tiv ity
S p e c ific k
a c tiv ity
C e lls a fte r
frac tio n a tio n
( c ru d e p re p a ra tio n )
245
4900
8 .0
C ell d e b r is
( s u p e r n a t a n t liq u id )
a f te r 5 9 0 0 xg
150
8550
4 7 .5
45
4320
1 6 .0
115
2783
2 2 .0
8
.270
1 3 .0
C ell d e b r is
(p re c ip ita te )
H ig h s p e e d
s u p e r n a t a n t liq u id
a f t e r !+S5SOOxg
C ell m e m b ra n e s
a
T h e n u m b e r of p m o l e s of f u c o s e i n c o r p o r a t e d p e r ml of
su b c e llu la r fra c tio n .
k
T h e n u m b e r of p m o l e s of f u c o s e i n c o r p o r a t e d p e r m g of
p ro te in .
t
19
'I
Vl
%
F ig u r e 3 .
A n e l e c t r o n m i c r o g r a p h of th e c e ll m e m b r a n e
fra c tio n .
T h e fie ld i s m a g n if ie d 1 0 , 2 0 0 X ,
20
■%
,
%
TM *
■ <
r X y
/f
*
£
£
.
^r
W%i
a
. A XjS S r
, j-i'1,
.
*X
21
m e m b ra n e fra c tio n .
R ea c tio n p r o p e r tie s :
pH
o p tim u m a t p H 7 . 5
T h e f u c o s y ltr a n s f e r a s e a ctiv itie s h a v e a
( F ig .
4 ).
T h e tim e c o u r s e of f u c o s e
i n c o r p o r a t i o n a p p e a r e d to b e l i n e a r a f t e r th e f i r s t t w o m in u te s
(F ig .
5)
a n d c o n t i n u e d to be. l i n e a r u p to 4 5 m i n u t e s .
p o in ts w e r e
Two
o b t a i n e d a t 3 0 a n d 4 5 m i n u t e s u s i n g a c e ll m e m ­
b r a n e p r e p a r a t i o n o b ta in e d by s o n ic a tio n a n d a r e th e r e f o r e not
show n.
C o n c e n t r a t i o n of t h e p r i m e r in t h e r e a c t i o n m i x t u r e s
d id a f f e c t th e i n c o r p o r a t i o n of f u c o s e
(F ig .
6)
w ith t h e s a t u r a t i n g
a m o u n t of p r i m e r n e e d e d f o r m a x i m u m a c tiv ity a t 7 -1 0 p g
p g p e r p i of r e a c t i o n m i x t u r e ) .
(0 .2
T h e a d d itio n of n o n io n ic , d e t e r s
g e n t s a n d c h e l a t i n g a g e n t s d id n o t e n h a n c e t r a n s f e r a s e a c tiv ity
(T a b le 3 ) .
T h e p r e s e n c e of M n C lg a t h ig h c o n c e n t r a t i o n s
a p p e a r e d to i n c r e a s e t h e a m o u n t of f u c o s e i n c o r p o r a t i o n b u t th e
a d d i t i o n of M g C lg c o m p l e t e l y in h ib ite d t h e r e a c t i o n ,
c o n c e n tra tio n s
(T a b le 3 ) .
e v e n a t lo w
G u a n o s i n e t r i p h o s p h a t e d id n o t e n ­
h a n c e i n c o r p o r a t i o n of f u c o s e
( T a b le 3 ) .
S e v e r a l c h a n g e s w e r e m a d e in t h e r e a c t i o n m i x t u r e , i n c u ­
b a t i o n p r o c e d u r e , a n d m e th o d o f s e p a r a t i n g t h e r e a c t i o n p r o d u c t
fro m u n r e a c t e d s u b s t r a t e y ie ld e d a c o n s id e ra b ly h ig h e r am ount
of i n c o r p o r a t i o n
(F ig .
7 ).
T h e d iffe re n c e b e tw e e n th e c o n tro l
a n d t h e a c t i v e p r e p a r a t i o n of 5 0 , 0 0 0
c o u n t s is e q u i v a l e n t to 1 1 6
22
%
■. t.
F i g u r e . if.
T h e p H d e p e n d e n c e of f u c o s e i n c o r p o r a t i o n .
p H 's d e n o te d a r e
of t h e r e a c t i o n m i x t u r e .
The
The
t h r e e r e a c t i o n s h a v i n g a c i d p H c o n t a i n s o d iu m
c a c o d y l a t e b u f f e r ; t h e r e m a i n i n g fiv e c o n ta i n T r i s
b u ffer.
E a c h co m p le te r e a c tio n m ix tu re c o n tain ed :
p h y to to x ic g l y c o p r o t e i n s u b j e c t e d to m ild a c i d
h y d r o ly s is , 3 . 7 TSJ
G D P - f u c o s e - ^ C , 2 6 .2 p
m o l e s ; b u f f e r , 1 .2 5 m i c r o m o l e s ; s u c r o s e ,
m ic ro m o le s;
of . 0 3 5 m l.
10
a n d e n z y m e p r o t e i n in a to ta l v o lu m e
T h e c e l l s w e r e c u l t u r e d f o r 50 h o u r s
p r i o r to b e i n g h a r v e s t e d .
FUCOSE INCORPORATED
( p MOLESzZ m g OF PROTEIN )
23
IO O -
50 —
F ig u r e 5.
T h e d e p e n d e n c e of f u c o s e i n c o r p o r a t i o n o n t i m e .
E a c h c o m p le te r e a c t i o n m ix tu re c o n ta in e d :
p h y to ­
to x ic g l y c o p r o t e i n s u b j e c t e d to m ild a c i d h y d r o l y s i s ,
3 . 7 P-S > G D P - f u c o s e - ^ C , 2 6 . 2 p m o l e s ;
pH 7*5,
m o le s;
1 .2 5
m ic ro m o le s; s u c r o s e ,
T ris ,
10 m i c r o ­
a n d 20 u g of e n z y m e p r o t e i n in a to ta l
v o l u m e of . 0 3 5 m l.
T h e c ells w e r e
50 h o u r s p r i o r to b e i n g h a r v e s t e d .
c u ltu re d fo r
FUCOSE INCORPORATED ( p MOLES )
25
IOOf
TIME ( min )
26
F ig u re 6 .
T h e d e p e n d e n c e of f u c o s e i n c o r p o r a t i o n o n th e
c o n c e n t r a t i o n of th e p r i m e r .
r e a c tio n m ix tu re c o n ta in e d :
E a c h c o m p le te
G D P - f u c o s e ^-C ,
2 6 .2 p m o le s ; T r i s , pH 7 .5 ,
su cro se,
10 m i c r o m o l e s ;
1 .2 5 m ic ro m o le s;
enzym e p ro te in ,
20 T S
a n d t h e p h y to to x ic g l y c o p r o t e i n s u b j e c t e d to m ild
a c i d h y d r o l y s i s in a to ta l v o lu m e of .0 5 0
T h e cells w e r e
b e in g h a r v e s t e d .
m l.
c u l t u r e d f o r 50 h o u r s p r i o r to
FUCOSE INCORPORATED ( p MOLES)
27
.500
—
.2 5 0 -
yU-g OF PRIMER
T a b le 3 .
T h e e ffe c t .of v a r i o u s a g e n t s o n f u c o s y l t r a n s f e r a s e activity-
c h a n g e s in the- n o r m a l
in c u b a tio n m ix tu re
R elativ e f u c o s y ltr a n s f e r a s e
a c tiv ity
%
None
100
I j i l of T r i t o n X - 1 0 0 , 1.0%
6
. 0.1 %
67
EDTA,
1 .0 mM
0 . 1 ' mM
22
6?
M g C l g , 10 j i m d l e s
5 jim d le s
2 jim o le s
0
0
0
M n C l g , 10 j i m o l e s
5 jim o le s
2 jim o le s
306
0
0
G T P , 0 .0 2 5 jim o le s
^
N o n e , e n z y m e p r e p a r a t io n fro m c ells
f r a c t i o n a t e d in p r e s e n c e of I m M E D T A
N o n e , e n z y m e p r e p a r a t io n fro m c ells
f r a c t i o n a t e d in p r e s e n c e of 0 .1 % T r i t o n
X -1 0 0
33
50
0
ro
CO.
29
.
"i -
•
• •
':
F ig u r e 7 .
T h e c o l u m n c h r o m a t o g r a p h y of r e a c t i o n m i x t u r e s
on S ep h ad ex G -2 5 .
tu re
E a c h c o m p l e t e r e a c t i o n m ix ­
c o n tain ed :
p h y to to x ic g l y c o p r o t e i n s u b j e c t e d
14to m ild a c i d h y d r o l y s i s , 20 p g ; G D P - f u c o s e C ,
1920 p m o le s ; T r i s , pH 7 .5 ,
sucrose,
22 m ic ro m o le s;
2 .7 5 m ic ro m o le s;
a n d 2 4 7 . 5 P-g of e n z y m e
p r o t e i n in a to ta l v o l u m e of . 1 0 0 m l.
w ere
T h e c ells
c u l t u r e d f o r 6 5 h o u r s p r i o r to b e i n g
h a rv e ste d .
150
-
@
Reaction
□
Heat Inactivated
Control
ioo-
FRACTlON (ml.)
31
p m o l e s of f u c o s e i n c o r p o r a t e d .
A c c e p t o r s p e c if ic ity s t u d i e s :
C . in sid io su m
T h e p h y to to x ic g l y c o p r o t e i n f r o m
w a s h y d r o l y z e d f o r v a r y i n g a m o n t s of tim e
a c c o r d i n g to th e p r o c e d u r e d e s c r i b e d p r e v i o u s l y .
T h e to x in
h y d r o ly z e d f o r 30 m in u te s s e r v e d a s th e m o s t su ita b le a c c e p t o r
fo r fu co se
(F ig .
8 ).
T r a n s f e r a s e a c tiv ity c o u ld b e s u c c e s s f u l l y
d e m o n s tr a te d u sin g e n zy m a tic a lly tr e a te d
a p rim e r
( F ig .
9 ).
Cf - a c i d g l y c o p r o t e i n a s
I
T h e a m o u n t of f u c o s e i n c o r p o r a t e d w a s
i n v e r s e l y p r o p o r t i o n a l to th e a m o u n t of p r i m e r p r e s e n t u p to a
c o n c e n t r a t i o n of 0 . 8 p.g of p r i m e r p e r jj.1 of r e a c t i o n m i x t u r e .
The
D ^ -a c id g l y c o p r o t e i n t r e a t e d w ith s i a l i d a s e a p p e a r e d to b e
t h e b e s t p r i m e r f o r t h e e n z y m a t i c i n c o r p o r a t i o n of f u c o s e
(T a b le 4 ) .
t
F ig u re 8.
T h e a ffe c t of a c i d h y d r o l y s i s o n th e a b ility of th e
p h y to to x ic g l y c o p r o t e i n to s e r v e a s a f u c o s y l
a cc e p to r.
ta in e d :
E a c h co m p le te re a c tio n m ix tu re c o n ­
p h y to to x ic g l y c o p r o t e i n s u b j e c t e d to m ild
a c i d h y d r o l y s i s , I ycg
G - D P - f u c o s e - ^ C , 128 p
m o le s;
.2 5 m ic ro m o le s ;
2 .0
T r i s , pH 7 .5 ,
m ic ro m o le s;
su cro se,
a n d 13 T S of . e n z y m e p r o t e i n in
a to ta l v o l u m e of . 0 1 0 m l.
T h e c e lls w e r e c u l­
t u r e d f o r 6 4 h o u r s p r i o r to b e in g h a r v e s t e d .
33
FUCOSE INCORPORATED (p MOLES)
.3 0 0 F
15
30
60
120
TIME OF HYDROLYSIS
OF INTACT TOXIN
(PRIMER), min.
F ig u re 9.
T h e u s e of f u c o s y l t r a n s f e r a s e to i n c o r p o r a t e
f u c o s e into v a r i o u s d e r i v a t i v e s of h u m a n p l a s m a
c ^ -a c id g ly c o p ro te in .
tu re
c o n tain ed :
E a c h c o m p l e t e r e a c t i o n m ix ­
p h y to to x ic g l y c o p r o t e i n s u b j e c t e d
to m ild a c i d h y d r o l y s i s , I ^ ig ;
128 p m o le s ; T r i s , pH
sucrose,
2 .0
7 ‘. 5 ,
m ic ro m o le s;
GDP - f u c o s e - ,
.2 5 m ic ro m o le s ;
a n d 13 / i g of e n z y m e
p r o t e i n in a to ta l v o l u m e of . 0 10 m l.
w ere
T h e c e ll s
c u l t u r e d f o r 6 4 h o u r s p r i o r to b e i n g
h a rv e ste d .
35
FUCOSE INCORPORATED (p MOLES)
.SO O f
a, Glycoprotein, treated with
.7 0 0 .6 0 0 —
.5 0 0 .4 0 0 .3 0 0 —
. 200 -
.IOO
4
8
12
16
/xg OF PRIMER
20
36
T a b le 4.
T h e r e l a t i v e ■i n c o r p o r a t i o n of f u c o s e - ^ C
a c h i e v e d w ith v a r i o u s p r i m e r s
F u c o sy l a c c e p to r
R e la tiv e f u c o s y ltr a n s f e r a s e
a c tiv ity
%
P h y to to x ic g ly c o p ro te in
15 m i n . h y d r o l y s i s
30 m in . h y d r o ly s is
60 m in . h y d r o l y s i s
120 m in . h y d r o ly s is
15
36
24
23
E n d o g e n o u s p r i m e r 3.
23
OtJ-acid g l y c o p r o t e i n ,
t r e a t e d w ith S i a l i d a s e b
100
o f ,- a c id g l y c o p r o t e i n ,
t r e a t e d w ith s i a l i d a s e :
^ -g a la c to sid a se c
61
N a t i v e g l y c o p r o t e i n in th e c e ll m e m b r a n e p r e p a r a t i o n
6
O p tim u m
c o n c e n t r a t i o n of 4 p g / p l of r e a c t i o n
c
O p tim u m
c o n c e n t r a t i o n of 2 ' p g / p l of r e a c t i o n
D IS C U S S IO N
I n t h e s t a n d a r d a s s a y f o r e n z y m e a c ti v ity , t h e a m o u n t of
f u c o s e i n c o r p o r a t e d in to th e g l y c o p r o t e i n p r i m e r w a s c o n s i d e r e d
to b e t h e a m o u n t of r a d i o a c t i v i t y a t th e o r i g i n a f t e r e l e c t r o p h o ­
r e s i s a n d s u b t r a c t i o n of th e h e a t i n a c t i v a t e d c o n t r o l .
S tro b e l
R ie s and
( 2 5 ) f o u n d t h a t th e p h y to to x ic g l y c o p r o t e i n f r o m
in sid io su m
C_.
w o u l d n o t m i g r a t e in a n e l e c t r o p h o r e t i c f i e l d .
The'
r a d i o a c t i v i t y f o u n d in th e p e a k w h i c h h a d m i g r a t e d off th e o r i g i n
w a s p r e s u m e d to c o n t a i n u n r e a c t e d s u b s t r a t e a n d s u b s t r a t e
b re a k d o w n p ro d u c ts.
T h i s h e t e r o g e n e i t y a n d th e r e v e r s e
o sm o t­
ic flo w a s a r e s u l t of s u b j e c t i n g th e p a p e r to e l e c t r o p h o r e s i s f o r
fo u r h o u rs
w o u l d ' a c c o u n t f o r t h e b r o a d n e s s of th e p e a k .
T h e d a t a o b t a i n e d b y e lu tin g t h e r e a c t i o n p r o d u c t w h ic h
r e m a i n e d a t t h e o r i g i n a n d t h e n s u b j e c t i n g it to d e s c e n d i n g p a p e r
c h r o m a t o g r a p h y i n d i c a t e d th a t t h e p r i m e r w h i c h b o u n d th e f u c o s e
w a s h o m o g e n o u s in r e s p e c t to its t e n d e n c y to r e m a i n a t th e
o rig in .
T h e c h r o m a t o g r a p h y of t h e r e a c t i o n p r o d u c t w h ic h w a s
h y d r o l y z e d i n d i c a t e d t h a t th e r a d i o a c t i v i t y w a s i n c o r p o r a t e d in a n
a c i d l a b ile b o n d ,
a n d t h a t th is r a d i o a c t i v i t y d e m o n s t r a t e d a s i m i ­
l a r R g i a c o s e 3-s f u c o s e .
I t w a s e s ta b lis h e d th a t th e ra d io a ctiv ity
w h ic h r e m a in e d at th e o rig in a fte r e le c tr o p h o r e s is w a s in c o r p o ­
r a t e d a n d t h a t th is r a d i o a c t i v i t y ■c o r r e s p o n d e d to f u c o s e .
A n y f u c o s e r e m a i n i n g a t' t h e o r i g i n in th e s t a n d a r d r e ­
a c t i o n m i x t u r e s a p p a r e n t l y is n o t t h e r e s u l t of a f u c o lip id i n t e r -
38
m e d i a t e s i n c e o n ly t r a c e s of t h e r a d i o a c t i v i t y b e c a m e s o l u b l e in
t h e t o l u e n e liq u id s c i n t illa tio n c o c k t a i l .
T he tra n sfe ra se
a c tiv ity w a s f o u n d to b e c o n c e n t r a t e d in
t h e m e m b r a n e s a n d h ig h s p e e d s u p e r n a t a n t liq u id c e ll f r a c t i o n s .
T he tra n sfe ra se
w a s n o t c o n s i s t e n t l y fo u n d in t h e s o l u b l e f r a c t i o n .
B a u e r _et a l ., ( 2 ) f o u n d th a t m a n n o s y l t r a n s f e r a s e c o u l d o n ly b e
d e m o n s t r a t e d in D i c t y o s t e l i u m d i s c o i d e u m
c e r t a i n t i m e s in its g r o w t h c y c l e .
so lu b le f r a c tio n s at
T h e c e ll m e m b r a n e s w e r e
u s e d t h r o u g h o u t th e w o r k a s t h e p r e l i m i n a r y p a r a m e t e r s f o r
tra n sfe ra se
a c tiv ity w e r e e s t a b l i s h e d w ith th a t f r a c t i o n .
The
a c ti v ity w a s m o r e c o n c e n t r a t e d in th e c e ll m e m b r a n e p r e p a r a t i o n ,
a n d t h i s w a s a n i m p o r t a n t c o n s i d e r a t i o n in r e g a r d to r e a c t i o n
v o lu m e s.
N o r e a d y t e c h n i q u e w a s a v a i l a b l e f o r r e d u c t i o n of t h e
h ig h s p e e d s u p e r n a t a n t liq u id v o l u m e .
T h e f u c o s y l t r a n s f e r a s e ' a c ti v ity a p p e a r e d to h a v e a p H
o p tim u m of 7 . 5
w h i c h is c o n s i s t e n t w ith th e o p tim u m o b s e r v e d
by o th e r in v e stig a to rs
(6 ,1 3 ,1 6 ).
T h e r e a c t i o n a p p e a r e d to
d e m o n s t r a t e a l i n e a r tim e c o u r s e u p to 4 5 m i n u t e s .
T h e a m o u n t of p r i m e r th a t w ill s a t u r a t e t h e r e a c t i o n is
q u ite lo w c o m p a r e d to w h a t o t h e r i n v e s t i g a t o r s h a v e o b s e r v e d .
F o r ex am p le,
100 p g of
Jab b al and S c h a c h te r
(16) r o u t i n e l y e m p l o y e d
o < - a c id g l y c o p r o t e i n in a 10 p.1 r e a c t i o n m i x t u r e .
T h e i r r e a c tio n s a lso
c o n t a i n e d 5 . 2 pig of p r o t e i n a n d 2 4 0 0
39
p m o l e s of s u b s t r a t e p e r pi of r e a c t i o n m i x t u r e .
in p r o te in c o n c e n tr a tio n
T h e d isp a rity
( a p p r o x i m a t e l y 2 . 0 p.g/p.1 in m y r e a c t i o n s )
i s n o t a s g r e a t a s t h e v a r i a n c e in s u b s t r a t e l e v e l s
p e r ;ul in m y r e a c t i o n s ) .
(1 2 .6 p m o le s
T h e c o m p a r a t i v e l y s m a l l a m o u n t of
p r i m e r n e c e s s a r y to a c h i e v e s a t u r a t i o n ' m ig h t b e r e l a t e d to t h e
lo w s u b s t r a t e le v e ls e m p lo y e d .
Jab b al and S c h a c h te r
c h ela tin g a g e n t E D T A
ever,
(16)
c a u s e d a 65% d e c r e a s e
H u d g in a n d S c h a c h t e r
t h e a c ti v ity of s i a l y l t r a n s f e r a s e
a d d i t o n of
s h o w e d th a t t h e p r e s e n c e of t h e
in a c t i v i t y .
H ow ­
(15) f o u n d o n ly a 10% d e c r e a s e in
w ith th e a d d itio n of E D T A .
E D T A to t h e r e a c t i o n m i x t u r e ,
The
o r t h e d i s r u p t i o n of
t h e c e l l s in t h e p r e s e n c e of th e c h e l a t i n g a g e n t w a s f o u n d to b e
d e l e t e r i o u s to t h e a c ti v ity of th e t r a n s f e r a s e
T he p resen ce
(T a b le 3 ) .
of T r i t o n X - 1 0 0 in s o m e p a r t i c u l a t e p r e p a r a ­
t i o n s h a s b e e n f o u n d to e n h a n c e , e n z y m e a c tiv ity
( I f , 1 1 ,1 6 ) .
T h i s w a s n o t t r u e in th e c a s e of t h i s e n z y m e s i n c e i n c r e a s i n g
t h e a m o u n t ' of d e t e r g e n t in t h e r e a c t i o n m i x t u r e c a u s e d a d e ­
c r e a s e in th e a c ti v ity
and S c h a c h te r
(16)
(T a b le 3 ) .
T h i s i s in c o n t r a s t to J a b b a l
w h o f o u n d t h a t T r i t o n X^-IOO w a s d e s i r a b l e
in t h e r e a c t i o n m i x t u r e a n d t h a t a d d itio n of th e d e t e r g e n t b e y o n d
o p tim u m c o n c e n t r a t i o n d id n o t a d v e r s e l y a ffe c t t h e a c t i v i t y ; th e
o p tim u m c o n c e n t r a t i o n in t h e i r w o r k w a s 0.1% a n d t h e y i n c r e a s e d
t h e c o n c e n t r a t i o n u p to 2 .0 % .
T h e d e te rg e n t a p p a re n tly a c ts on
40
t h e lip id c o n s t i t u e n t s of th e m e m b r a n e c a u s i n g it t o ' u n fo ld w ith
t h e r e s u l t th a t m o r e e n z y m e is e x p o s e d .
O th e r w o r k e r s have
e m p l o y e d T r i t o n X - 1 0 0 to s o l u b l i z e m e m b r a n e b o u n d f u c o s y l tra n sfe ra se s.
B o s m a n n e t al_. ( 5 )
d e s c r i b e d th e c h a r a c t e r i z a ­
t io n of t w o s u c h t r a n s f e r a s e s in H e B a c e l l s .
C . in sid io su m
T h e d i s r u p t i o n of
c e l l s in th e p r e s e n c e of th e d e t e r g e n t fa ile d to
y ie ld a d e te c ta b le s o lu b le f u c o s y ltr a n s f e r a s e .
T h e a d d itio n of M n C l^ to t h e r e a c t i o n m i x t u r e p r o d u c e d a
r a t h e r d r a m a t i c i n c r e a s e in t h e a c t i v i t y .
to to ta lly in h ib it th e r e a c t i o n .
are
B u t,
T h e s e d a ta ,
M g C l^ a p p e a r e d
a lt h o u g h r e p r o d u c i b l e ,
s u s p e c t d u e to th e v e r y h i g h b a c k g r o u n d l e v e l s of i n c o r p o ­
r a t i o n e n c o u n t e r e d in th e h e a t i n a c t i v i a t e d c o n t r o l s .
A
s ig n if ic a n t i n c r e a s e in t h e a m o u n t of d e t e c t a b l e f u c o s e
in c o r p o r a tio n w a s o b ta in e d by c h a n g in g th e r e a c tio n m ix tu r e ,
in c u b a tio n p r o c e d u r e ,
s u b s tr a te from
a n d t h e m e t h o d of s e p a r a t i n g u n r e a c t e d
re a c te d s u b stra te
(F ig .
7 ).
No la rg e changes
i n th e r e l a t i v e c o n c e n t r a t i o n of t h e i n g r e d i e n t s in a t y p ic a l
re a c tio n w e r e
m ad e-.
a m o u n t of p r i m e r ,
c e n tra tio n .
T h ere
w a s a tw o fo ld i n c r e a s e in th e
a n d a 1 .5 fo ld i n c r e a s e in t h e s u b s t r a t e c o n ­
T h e to ta l v o lu m e w a s i n c r e a s e d t o '100 pd a n d th is
m a d e it p o s s i b l e to a g g i t a t e th e r e a c t i o n m i x t u r e d u r i n g i n c u b a ­
tio n .
T h e s ig n if ic a n t d i f f e r e n c e n e c e s s a r y to a c c o u n t f o r a n a p ­
p r o x i m a t e l y 2 5 0 fo ld i n c r e a s e in d e m o n s t r a b l e f u c o s y l t r a n s f e r a s e
41
a c ti v ity is d ifficult to e x p l a i n .
T h e c o n s t a n t m ix in g of th e r e ­
a c t i o n i n g r e d i e n t s s e e m s to b e w o r t h y of n o t i c e „
T h e m e th o d of
s e p a r a t i n g t h e r e a c t e d s u b s t r a t e f r o m th e i n c o r p o r a t e d f u c o s e
sh o u ld a ls o be c o n s id e r e d .
T h e co n sta n t s tir r in g m ay h av e
s i g n if ic a n tly i n c r e a s e d th e c h a n c e c o l l i s i o n s b e t w e e n s u b s t r a t e
and en zy m e.
P o s s i b l y , a g l y c o p r o t e i n f r a g m e n t c a p a b l e of
b in d in g f u c o s e w h i c h e lu t e d a t t h e v o id v o lu m e of th e S e p h a d e x
G --25 c o l u m n ,
w ill m i g r a t e f r o m th e o r i g i n o n t h e e l e c t r o p h o r e s i s .
M a y b e l o n g e x p o s u r e to t h e s o d i u m b o r a t e b u f f e r c a u s e d i n c o r p o ­
r a t e d f u c o s e to e v e n t u a l l y m i g r a t e f r o m th e o r i g i n .
T h e h ig h
a m o u n t of i n c o r p o r a t i o n o b t a i n e d in t h e h e a t i n a c t i v a t e d c o n t r o l
i s h a r d to e x p l a i n b u t n o t u n u s u a l in t h i s t y p e of s y s t e m .
O t h e r m e t h o d s a r e g e n e r a l l y f o u n d b e f o r e th e i n f o r m a t i o n is
p u b lish e d
( 19) .
T h e p h y to to x ic g l y c o p r o t e i n c h a r a c t e r i z e d b y R i e s a n d
S tro b e l
(2 5 )
p r o v e d to b e a s u i t a b l e a c c e p t o r f o r f u c o s e a f t e r
m ild a c i d h y d r o l y s i s .
c h a rid e s :
T h e g l y c o p r o t e i n c o n t a i n s fiv e m o n o s a c ­
L -fu c o se , m annose,
n o s e in a r a t i o of 1 0 : 1 : 5 : 5 ;
a m o u n ts.
g lu c o se ,
g a la c to se ,
and rh a m -
r h a m n o s e w a s o n ly p r e s e n t in t r a c e
T h e r e m o v a l of f u c o s e a s
w e ll a s t h e o t h e r s u g a r s
h a s b e e n v e rifie d by p a p e r c h ro m a to g ra p h y an d by g a s-liq u id
c h ro m a to g ra p h y .
T h e to x in h y d r o l y z e d f o r 3 0 m i n u t e s s h o w e d
t h e o p tim u m a b ility to i n c o r p o r a t e f u c o s e .
H y d r o l y s i s f o r th a t
42
l e n g t h of tim e r e m o v e d th e o p tim u m a m o u n t of f u c o s e w ith o u t
d e s t r o y i n g s p e c i f i c b in d in g s i t e s f o r t h e s u g a r a n d t h e e n z y m e .
A lth o u g h th e
o < -a c id g l y c o p r o t e i n k in d ly s u p p l i e d b y D o c t o r
S c h a c h t e r s h o w e d a g r e a t e r a m o u n t of i n c o r p o r a t i o n ,
a com ­
p a r i s o n of b in d in g e f f i c i e n c i e s is n o t a p p r o p r i a t e a s t h e p l a s m a
g l y c o p r o t e i n ! s b in d in g s i t e s w e r e p r e p a r e d e n z y m a t i c a l l y w h i c h is
a m o r e sp ec ific a n d g e n tle r te c h n iq u e .
i n c o r p o r a t i o n into t h e s i a l i d a s e t r e a t e d
T h e h i g h e r a m o u n t of
O f-a c id g l y c o p r o t e i n m ig h t
J
im p ly t h e p r e d o m i n a n c e of a f u c o s y l t r a n s f e r a s e
° f - ( l —^ 3 ) - g a l a c t o s e l i n k a g e s .
H o w ev er,
sp ec ific fo r fu c o s e
in c o rp o ra tio n w a s a lso
s e e n in t h e s i a l i d a s e - ^ - g a l a c t o s i d a s e t r e a t e d g l y c o p r o t e i n i m ­
p licatin g a t r a n s f e r a s e
lin k a g e s;
sp ec ific f o r f u c o s e : N -a c e ty lg lu c b s a m in e
t h e r e is n o N - a c e t y l g l u c o s a m i n e p r e s e n t in t h e p h y t o -
t o x ic g l y c o p r o t e i n .
A lso ,
th e d e c r e a s e in th e a m o u n t of f u c o s e
i n c o r p o r a t e d w ith a n i n c r e a s e of t h e
o (-a c id g ly c o p ro te in c o n ­
c e n tra tio n n e e d s s o m e c la rific a tio n .
P o ssib ly ,
t h e r e a r e b in d in g
s i t e s a v a i l a b l e f o r t h e e n z y m e w h e r e n o b in d in g s i t e s f o r th e
s u g a r e x ist.
th e c o n d itio n .
H e r e a g a in ,
lo w s u b s t r a t e l e v e l s w o u l d a g g r a v a t e
SUM M ARY
G u a n o s i n e d i p h o s p h a t e - L - f u c o s e f u c o s y l t r a n s f e r a s e a c tiv ity
h a s b e e n f o u n d in a c r u d e
C o ry n e b a c te riu m
c e ll m e m b r a n e p r e p a r a t i o n f r o m
in sid io su m .
T h e i m p o r t a n c e of th is o b s e r v a ­
tio n l i e s in t h a t n o p r e v i o u s r e p o r t of a f u c o s y l t r a n s f e r a s e in
b a c te ria h a s b een m a d e .
T h e f u c o s y l a c c e p t o r u tiliz e d to
e s t a b l i s h s o m e of t h e p a r a m e t e r s
of t h e r e a c t i o n w a s t h e p h y t o ­
t o x ic g l y c o p r o t e i n p r o d u c e d b y t h e o r g a n i s m .
T h e g ly c o p ro te in
w a s f i r s t s u b j e c t e d to m ild a c i d h y d r o l y s i s a n d t h e n u s e d a s a n
a c c e p to r.
I n c o r p o r a t i o n of f u c o s e into h u m a n p l a s m a
c^-acid
g l y c o p r o t e i n s b y th is f u c o s y l t r a n s f e r a s e im p l i e s a s p e c if ic ity
d e te r m in e d b y te rm in a l a n d p e n u ltim a te s u g a r s .
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73
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