81 P
Medical Research Society Communications
222
EFFECPS OF C O " A A G I N A ON LWPfECYE
S'MICLEOTIDME IN CoPlTwlL SWJECI'S AND PATIENTS
WrPB PRImnRY -BLmINAeufA
S.
BARWICK, T. SHAE, A.D.B. WEBSTER AND T.J.
PETERS
Divisions of C l i n i c a l C e l l Biology and
Inmunological Medicine, MRC C l i n i c a l Reeeazch
Centre, Watford Road, Blurar, Mid&
(XLE)
Lymphocytes from p a t i e n t s w i t h X - l i n k e d
or
COnmOn
variable
(CVE)
primary
hyposamaglobulinaemia have low a c t i v i t i e s of
the plasma membrane ectoenzyme S'nucleotidaae
(S'NT), b u t other plasma membrane enzymes eg
y-glutamyl t r a n s f e r a a e , l e u c i n e aminopeptidaae
and adenosine
diphosphataae are no&
(T.
Shah, A.D.B.
W b s t e r and T.J. Peters, Clin.
Exp. I m u n o l . ,
1983,
53,
413).
Lymphocyte
from
these
patients
is
S'nucleotidase
f u n c t i o n a l l y abnormal w i t h a decreased a f f i n i t y
for its s u b s t r a t e ?@
and
I!?
€or a oompetitive
inhibitor a-p-methylene
ADP (T. Shah, A.D.B.
Webster and T.J. Peters, C l i n . Exp. I m u n o l . ,
1984, 57, 149).
5'Nucleotidase is inhibited by the l e c t i n
concanavalin A (Con A) and t h e r e f o r e a s t u d y of
the kinetics o f Con A i n h i b i t i o n was undertaken
with CVE and c o n t r o l lymphocytes t o f u r t h e r
i n v e s t i g a t e the enzyme abnormality i n them
patients.
Con A was found t o be a p a r t i a l l y
non-competitive inhibitor, i n d i c a t i n g that it
had DD effect on substrate binding and that Con
A and s u b s t r a t e bind independently t o d i f f e r e n t
sites
on
the
eneyme.
The
partial
non-conpetitive i n h i b i t i o n was c h a r a c t e r i s e d by
p l o t t i n g V vs ( I ) at fixed ( S ) .
Intercepts
f r a r t h e d i r e c t l i n e a r p l o t vis., 1/(I ) p l o t s
w e r e t h e n used
t o c a l c u l a t e the i n h i b i t o r
constanks for lymphocyte 5'm f o r both p a t i e n t s
and c o n t r o l s .
values for control(0.55pM f 0.21
(mean f SD) n = 5) and CVE ( 0 . 6 1 MI4
0.18, n =
7 ) lymphocytes w e r e similar.
I t is concluded
that the defect i n CVE does n o t affect the
glycan m i e t y of S'NT or alter the d i s s o c i a t i o n
of the enzyme-substrate complex.
The
223
Ki
POLYAMINES I N STIMULUS : RESPONSE COUPLING
D.W. Howat, E.N.
J. Chayen
Chayen, L. Bitensky and
Division of C e l l u l a r Biology, Kennedy
I n s t i t u t e of Rheumatology, London W6 7DW
Very rapid s t i m u l a t i o n of glucose 6-phosphate
dehydrogenase (G6PD) a c t i v i t y i s a common biochemical i n d i c a t o r of c e l l u l a r response t o
various s t i m u l i . I t i s assumed t h a t t h e s e
s t i m u l i generate a chemical messenger t h a t conv e r t s G6PD from i t s l e s s to i t s more a c t i v e fom.
The f a c t t h a t many of these s t i m u l i 'also a c t i v a t e o r n i t h i n e decarboxylase (ODC) a c t i v i t y , and
polyamine s y n t h e s i s , suggested t h a t a polyamine
could take t h e p l a c e of t h e p o s i t i v e l y charged
molecule normally used for t h e i n v i t r o a c t i v a t i o n of G6PD. To t e s t t h i s stimulus : response
coupling we have used s t r i p s of g a s t r i c fundus
muscle o f f a s t e d male W i s t a r a l b i n o r a t s (2033oOg) which, when stimulated by 5-hydroxytryptamine (5HT; 10-4M) i n T8 c u l t u r e m e d i u m , c o n t r a c t
r a p i d l y , due t o t h e i n f l u x of calcium (Weinstock
& Weiss, Br.J.Pharmaco1,
1979 E, 593-599); t h i s
i s a s s o c i a t e d with a concomitant r i s e i n G6PD
2.5 min). The
a c t i v i t y (peak a c t i v i t y w i t h i n 1
a c t i v i t y has been measured cytochemically, by
microdensitometry. The a c t i v i t y i n t h e muscle
of such s t r i p s r o s e to 175% (f15; mean f SEM;
n = 7 r a t s ) of t h e b a s a l a c t i v i t y . I n s t r i p s of
t h e same t i s s u e , exposed t o 1 0 - 3 M p u t r e s c i n e ,
b u t no 5-HT, t h e peak a c t i v i t y was delayed
(2-7 min) and was 161% (f13.8; n = 6 r a t s ) .
Exposure t o t h e T8 m e d i u m alone, f o r up t o 7 min,
gave no change i n a c t i v i t y . Spermidine and
spermine had l i t t l e , or only v a r i a b l e e f f e c t .
Unlike p u t r e s c i n e , n e i t h e r caused measurable
c o n t r a c t i o n . In t h e muscle of t h r e e r a t s maint a i n e d on a vitamin Bg-deficient d i e t , t o
d e p l e t e pyridoxal phosphate ( t h e c o f a c t o r f o r
O D C ) , t h e G6PD response t o 5HT was 137%, 100%
and 100%of b a s a l l e v e l , a s a g a i n s t 163% i n r a t s
f e d t h e same d i e t supplemented w i t h vitamin Bg.
Thus p u t r e s c i n e , t h e immediate product of ODC
a c t i v i t y , might mediate t h e a c t i v a t i o n of GGPD
i n stimulus : response coupling.
-
~
224
-.
LEUCOCYTE ZINC AND PROSTAGLANDIN
PRODUCTION IN PREGNANCY
K. SIMMER, N .PUNCHARD, G.MURPHY*
and R.P.H.THOMPSON
Gastrointestinal Laboratory, Rayne Institute,
St. Thomas' Hospital , London and
Department of Biochemistry*,
Guy's Hospital Medical School, London
Intrauterine growth retardation (IUGR) is
a major cause of perinatal morbidity and mortality. Zinc is important for the structure and
function of membranes, the metabolism of essential fatty acids and protein accumulation and
growth. An association between zinc depletion
and IUGR might occur through disturbed prostaglandin(PG) synthesis. The zinc content and PG
metabolism of leucocytes from 16 mothers 24-48h
after delivery and from 15 control women were
investigated and related to fetal growth and
smoking.
Polymorphonuclear (PMN) cells and mononuclear (MN) cells were separated on FicollPaque. Cells were incubated for 2h in TC 199
medium, with zymosan as stimulant, and the PG
production analysed by radioinnnunoassay. The
zinc content of the remaining cells was measured
by atomic absorption spectrophotometry.
Mothers of small -for-gestational -age(SGA)
babies (n=6) had lower zinc levels than nonpregnant controls: 5% v 75*6pg/1010PMN, K0.05
and 120t11 v 167*13/10fOMN, pC0.025. PMN and MN
cells were correlated, ~ 0 . 0 2 5 .