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OJ Shellfish
REGULA TIO
(l.
1. 159-163.
1998.
OF MYTILUS GALLOPROVI
CIALIS GL YCOGE PHOSPHORYLASE BY
GLUCOSE A D GL COSE-6-PHOSPHATE
J A SERRA O, PILAR S AREZ ALO SO,
eo LOPEZ AND L. ose R GARCIA MARTIN*
F E el L
. L CA BL
Myli/lIs
galloprovincialis.
glycogcn phosphorylase.
I TROD CTIO
regulation.
glucose,
glucose--phosphate
Correspondence 10 F. San Juan errano. Departamento de Biologfa Fundamental. Facultad de Ciencia .. Uni er idad de Vigo. Apdo. 784. 36200
Vigo. Spain.
159
160
SA
MATERIALS
JUAN
D METHODS
Animals
o/ Phosphorylase
Extracts
Glyeogen pho phorylase aetivity wa mea ured spectrophotornetrically in the direetion of glyeogen breakdown, a de eribed in
San Juan el al. (1991). The reaction mixture for phosphoryla e b
contained 40 mM tris-acetate buffer pH 7.0, 5 rnM irnidazole, 2
mM EOTA, lA mM 2-ereaptoelhanol. 5 mM acetate-Mg, 5 ~M
glueose-I-6-diphosphate,
0.6 mM
AOP, 80 mM pho phate
buffer, pH 7.0, 1.6 mM AMP, 16 mg/mL glycogen, 0.5 I.U. of
glueo e-6-phosphate dehydrogena e. 0.1 J.U. of pho phoglucornula e. and enzyme preparation in a total volume of 1.0 mL. The
reaction mixture for pho phoryla. e a lacked EOTA and AMP. and
the glycogen and pho phate buffer concentrations were
mglmL
and 30 mM, re pectively.
Pho phoryla e activity wa as ayed in the direction of glycogen
ynthesi by mea uring the relea e of P, from G 1P. The reaetion
rnixture for the pho phoryla e b eontained 40 mM tri -a etate
buffer, pH 7.0.2 mM EOT A, .2 mg/mL glycogen, 1.6 mM AMP,
00 mM G-I-P, and enzyme preparation in a total volume of I mL.
The reaetion mixture for pho phoryla e a laeked EOTA and AMP.
and the G 1P eoneentration wa 50 mM. After incubation al 30C
for 45 mino the reaction was topped by the addition of 0.1 mL of
20% (w/v) SOSo After centrifuging, P, in the upernatant was
determined by the method of Saheki et al. (1985).
Dala Analysis
ERRANO
ET
L.
REGULATIO
OF MYTILU
GLY
EN PHOSPHORYLA
161
lO,
,.1
.-.. ..
',t
.'
,..
,.1
t
00
10
20
'09 (GIP)""
"
'0
e
1
lo
().I-----..----+__
r).o
o. -
==~-L~)~~~
--l-
n'~
(l.")
(b~0~)~~~~--~(~I~I!.=4==~=~=)=0
1 . ()
r\
Figure 1. Double recprocal plot of initial velocity of pho phorylase a
as funclion of G lP concentration
in the pre ence of several gluco e
concentratons: ( ) O rnM, (e) 10 mM, (6) 20 111M.
1~' l rn
40
"
o.
>-o~
')0
40
t:
t
-)
"
-n
00
zc
tO
lO
Ioq{CIPJmlot
0+
_~-n
.:n..
~, ~
II
1 -
)~-
00
o.r
,1 F
O. '
1.0
r'IM
o. 7
'/[()
::! 08
1 .
IP]
11
1<,2
162
SAN
TABLE
AN SERRANO
1.
ET AL.
phosphorylase
nH
Vm (1 /mL)
whereas
direction
Taking
Km (111M)
(glucose).
Km and Ki respectively,
the value of Km
is 95-fold
are within
in
regulated regardless 01' the mobilization of glyco01' glucose--phosphate phosphaiase activity in the rnantle tissue of Mytilus, which involves an irnportant
difficulty for the gluco e to be the final expression 01' the glycogenolitic effector system (Cres po 1989). and the high values 01' Ki
(70-130
mM).
b FORM
[glycogen]: 3.2 I11g/I11L
IAMP): 1.6 111M
IG6Pl
0.0 mM
0.5 mM
1.5 111M
0.475 3 x 10--'
182.38 2 x 10-2
296.59 6 x 10-'
1699.0 6 x 10-'
0.415 7 x 10
0.491 5 x 10--'
0.8
1.5
1.4
191.98 6 x 10-'
217.546x
10-'
0.445 3 x 10
0.395 5 x 10
1.3
1.9
[glucose]
10.0 111M
20.0 mM
a FORM
1: 3.2 rng/ml,
obtained
[glycogen
Furthermore,
[G6PI
0.0 mM
0.5 mM
1.5 111M
1.1
1.49 5 x 10
0.89 1 x 10-'
0.81 4 x 10--'
0.1433x
0.6
0.6
10-'
1.127xI0--'
1.34 1 x 10-'
el al. 1988).
main interaction
0.9
0.8
of G6P, it
effect
0.112 4 x 10--'
inhibitory
[glucose]
10.0 mM
20.0 mM
(Crespo
tissue
activity
01' glucose-6-
a regu-
Results are expressed as mM (Km - SIl~) and IU/ml SEM (standard error
lating effect on this pathway to this rnolecule, in this lis ue, which
is more important than that of gluco e. as also eern lO occur in
the Iar-body 01' in ects (Applebaum
1973) and in yeasts (Sagardia
means).
el al. 1971).
1983, Carabaza et al. 1992). In ihe case 01' the Mytilus enzyme, we
observed
0.139
1 x 10-'
on
heterotropic
effects
by the inhibitor
as G6P
frorn the
mximum
velocity
decrease four-fold
and mximum
velocity
of the non-pho
1). Moreover,
is particularly
on the affinity
by the
phorylaied
effect of b th
ni
development,
glycogen
degradation
enzymatic
energy
form is described
dernand,
of a greater interaction
with the active
enzyrne
of form a, this
The inhibitory
only,
effect
can be explained
irnportance
in reaction
rever. e direction
of Mytilus glycogen
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