Comp. Biochem. Physiol. Vol. 106][I, No. 4, pp.

925-932, 1993

0305-0491/93 $6.00 + 0.00

1993 Pergamon Press Ltd

Printed in Great Britain

A KINETIC STUDY OF GLYCOGEN PHOSPHORYLASE b

FROM THE MANTLE TISSUE OF MYTILUS
GALLOPRO VINCIALIS, Lmk
FUENCISLA SAN JUAN-SERRANO, MARGARITA FERN.~NDEZ-GONZ~LEZ, JOSl~ LUIS S~NCHEZ-I.~PEZ

and L. OscAR GARciA-MARTiN

Departamento de Bioquimica y Biologia Molecular, Facultad de Farmacia, Universidad de Santiago de
Compostela, Avda de ias Ciencias s/n, 15706 Santiago de Compostela, Spain
(Received 14 April 1993; accepted 2 June 1993)
Abstract--1. In order to assign a meaningful role to the phosphorolytic pathway in Mytilus glycogen
metabolism the kinetic mechanism of phosphorylase b, and its allosteric control, were studied.
2. The kinetic parameters of phosphorylase b from the mussel Mytilus galloprovincialis were determined.
Michaelis constants (Kin or S0.5)were in the range of 0.32-2.49 mg/ml for glycogen, 7-16 mM for Pl and
114-423/~M for AMP. In the direction of glycogen synthesis, the Km value for glucose-l-P was
approximately 180 mM.
3. The enzyme displayed homotropic co-operativity towards the binding of co-suhstrate and AMP (Hill
coefficients of 2 and 1.4, respectively) and heterotropic co-operativity between substrates and AMP.
4. The concentration of glycogen in the Mytilus mantle is between 38- and 125-fold higher than the
apparent Km of phosphorylase b; the concentration of AMP varies throughout the year from 10 to
175 gM, up to a value close to the apparent Km for the effector.
5. The apparent Km for Pi is close to the concentration found in the mantle. This ligand showed more
important regulatory effects than the effector AMP.

ditions and plays a central role in the regulation

of glycogen metabolism. The enzyme can be
The main energetic fuel, supporter of gametogenic
regulated by covalent modification (phosphoryldevelopment, in bivalve molluscs, is glycogen (Gabation-dephosphorylation) and by allosteric modubott, 1975; Bayne, 1976; Pieters et al., 1980; Bayne
lation exerted at the molecular level by various
et aL, 1982). There exist two metabolic pathways for
physiological factors acting through conformational
glycogen mobilization: direct phosphorolysis mediated by the enzyme glycogen phosphorylase (EC changes of the enzyme. Four separate ligand binding
2.4.1.1) and hydrolysis plus phosphorylation medi- sites are localized in glycogen phosphorylase of vertebrates: the active site, which binds the substrates,
ated by the enzymes amyloglucosidase (EC 3.2.1.3)
Pi,
glycogen and glucose-l-P; the nucleotide site or
and hexokinase (EC 2.7.1.1). Despite the presence of
AMP
allosteric site, consisting of three different
the enzyme of both pathways in Mytilus mantle tissue
subsites,
namely the phosphoryl group, the ribosyl
(Alemany and Rosell-P6rez, 1973; Zaba, 1981; San
Juan et aL, 1991), the partial contribution to the residue and the purine base-binding loci; the nuglobal process of glycogen degradation, is still un- cleoside (or inhibitor) site; and the glycogen storage
site (Dombr~idi, 1981; Newgard et al., 1989; Johnson
known.
et
al., 1989).
In mammals, phosphorylase and glycogen synthase
Numerous
kinetic studies have been performed on
(EC 2.4.I.I I) control glycogen metabolism via interthe
glycogen
phosphorylase of vertebrate muscle
conversion of active and inactiveforms mediated by
hormones and the blood-glucose level. Despite the (Cori et al., 1943; Appleman et al., 1966; Engers et aL,
abundant occurrence of glycogen in mantle tissueof 1969; Bonamusa and Baanante, 1990). In general
bivalve molluscs and the seasonal variationobserved terms, it is difficult to subject the phosphorylase
in glycogen content in relationto gametogenic devel- reaction to kinetic analysis because glycogen is simulopment, the phosphorolytic pathway in glycogen taneously the substrate and product and the substrate
metabolism has been less extensivelyinvestigatedin acts, at the same time, as an effector molecule.
these organisms (V~zquez-Baanante and Rosell- Another source of difficulty lies in the allosteric
P6rez, 1979; Ebberink and Salimans, 1982; Hata properties of phosphorylase (Madsen and Shechosky,
et al., 1987).
1967; Lorek et al., 1984) which further complicate the
Glycogen phosphorylase (1,4-~-D-giucan: or- kinetic analysis.
thophosphate ~-giucosyltransferase) catalyses the
The present paper describes the kinetic characterizbreakdown of glycogen under physiological con- ation and allosteric control of the unphosphorylated
INTRODUCTION

cnPB~06/~,--x

925

926

FUENCISLASAN JUAN-SERRANOet al.

form (phosphorylase b) of glycogen phosphorylase

from the mantle tissue of Mytilus galloprovincialis.
MATERIALS AND METHODS

Animals and reagents

Mussels measuring between 7 and 10 cm in length
were collected from Villagarcia de Arosa (Galicia,
NW Spain). The mantle tissue was stored at - 3 0 C
until utilized.
Substrates, enzymes and coenzymes were obtained
from Sigma Chemical Co. (St Louis, MO) and
Boehringer (Mannheim, Germany). All the other
chemicals were obtained from Merck (Darmstadt,
Germany).
Preparation of extracts
Tissues were homogenated in 2 vol (w/v) of ice-cold
40mM Tris-acetate buffer, pH7.0, containing
EDTA, imidazole and 2-mercaptoethanol in a concentration of 5 mM. The homogenate was centrifuged
at 15.400g,v for 20 min and the pellet was discarded.
Aliquots of this crude extract and the purified enzyme
described previously (San Juan et al., 1991), with
specific activities of 0.08-0.10 and. 6.17-6.33 ;t kat/mg
of protein, respectively, were utilized for kinetic
assays.
Phosphorylase assays
Phosphorylase activity was examined in the direction of glycogen breakdown with a coupled enzyme
system, essentially the same described in Childress
and Sacktor (1970). The reaction mixture contained
40 mM Tris-acetate buffer, pH 7.0, 5 mM imidazole,
2 mM EDTA, 1.4 mM 2-mercaptoethanol, 5 mM
acetate-Mg, 5 g M glucose-l-6-diphosphate, 0.6 mM
NADP, 80 mM phosphate buffer, pH 7.0, 1.6 mM
AMP, 16 mg/ml glycogen (Gly), 2.50 nkat of glucose6-phosphate dehydrogenase (EC 1.1.1.49), 1.67 nkat
of phosphoglucomutase (EC 5.4.2.2) and the enzyme
preparation in a total volume of 1.0 ml. The unit of
activity (kat) was defined as the amount of enzyme
catalysing the formation of 1 mol glucose-l-phosphate/sec at 20C.
Phosphorylase activity was assayed in the direction
of glycogen synthesis by measuring the release of Pi
from glucose-l-phosphate. The reaction mixture contained 40mM Tris-acetate buffer, pH 7.0, 2 m M
EDTA, 3.2 mg/ml glycogen, 1.6 mM AMP, 800 mM
glucose-l-phosphate and enzyme preparation in a
total volume of 1 ml. After incubation at 30C for
45 min, the reaction was stopped by the addition of
0.1 ml of 20% (w/v) SDS. After centrifugation, Pi in
the supernatant was determined by the method of
Saheki et al. (1985).
Calculations
The apparent Km and Vn~ values, presented in the
results, were estimated using Lineweaver-Burk
double-reciprocal plots. When the double-reciprocal

plots were non-linear (i.e. enzyme activity did not

follow Michaelis-Menten kinetics), the apparent Km
(S0.5) and Vmaxvalues were evaluated from plots of 1/v
vs (l/S) 2. Such plots have been used previously in the
analysis of kinetic parameters for deoxythymidine
kinase (EC 2.7.4.9) (Okazaki and Kornberg, 1964),
rabbit phosphorylase b (Madsen, 1964) and lobster
muscle phosphorylase (Assaf and Graves, 1969). S0.5
values were evaluated from Hill plots. It will be
shown that the S0.5 values from the Hill plots agree
with the values obtained from the negative intercepts
on the abscissa of 1/v vs (l/S) 2.
RESULTS

Phosphorylase from the mantle tissue of Mytilus is

primarily present as the AMP-dependent form, phosphorylase b (San Juan et al., 1991). The kinetic
parameters of this enzymic form were determined
using aliquots of the purified enzyme and using crude
tissue extracts, before purification, giving identical
results.
Substrate, co-substrate and effector affinities were
determined. By decreasing the co-substrate and effector concentrations to non-saturating values, the binding mode and the interaction between the different
molecular species were observed.

Glycogen
The affinity constant with respect to glycogen was
estimated to be 0.32mg/ml. In saturating concentrations of AMP (1.6 mM), a 9-fold increase in the Km
is observed when the Pi concentration is decreased.
However,
in
lower
AMP
concentrations
(0.8-0.4 mM), this loss of affinity for glycogen is
significantly smaller (Fig. 1) (Table 1). When the
AMP concentration is reduced, a similar effect is
observed. In this case, in a saturating concentration
of Pi (80 raM), the Km for the glycogen ranged from
0.32mg/ml at 1.6mM AMP to 1.6-1.8mg/ml at
400 #M AMP. In non-saturating Pi concentrations,
the decrease in affinity for glycogen is smaller and in
very low Pi concentrations (15 mM), a slight increase
can be seen (Table 1).
In the absence of AMP, both the Vr~ and Km are
strongly affected, reaching values of 0.28 nkat/ml and
5.6 mg/ml, respectively. When the Pi concentration
increases, a slight activation is observed.
In all the cases studied, the straight lines obtained
employing double-reciprocal plots to estimate the
apparent K m for glycogen, showed a lack of cooperativity between substrate binding sites.
Inorganic phosphate
The apparent S0.5 of the Mytilus mantle phosphorylase b for Pt was 7 mM. In high glycogen and
AMP concentrations (24 mg/ml and 1.6-0.8 mM, respectively), the Vm~ is reached at 30 mM Pi and at
40 mM Pi when the AMP concentration is decreased
to 0.4 mM. However, in low glycogen concentrations

Mytilus glycogen

phosphorylase b

927

5.4
A

~3.6
A

"6
C

~- 1.8

I
0.6

t
1.2

I
f
I
0.6
1.2
1.8
1/[Glycogen] m g / m l

f
1.8

f
0.6

I
1.2

I
1.8

Fig. 1. Double-reciprocal plots of initial velocity of phosphorylase b as a function of substrate

concentration at fixed levels of AMP: (A) 1.6mM, (B) 0.8 raM, (C) 0.4 raM, and at several Pi
concentrations: (O) 80 raM, (O) 40 raM, (A) 15 mM
(3.2 mg/ml) and in the same AMP concentrations, the
Vm~ is reached at 60 and 80 mM Pi, respectively.
From the last concentration of Pi, the enzymic activity is largely inhibited in all glycogen and AMP
concentrations. It should also be pointed out that at
zero AMP, phosphorylase b is not inhibited at any Pi
concentration.
The most notable kinetic characteristic of phosphorylase b as regards the co-substrate Pi is the
sigmoidicity of the saturation curve and the non-linearity of the double-reciprocal plots (Fig. 2), similar
to those found in phosphorylases from other sources
(Engers et al., 1969; Childress and Sacktor, 1970;
Sabeki et al., 1985; Okazaki and Kornberg, 1964;
Madsen, 1964; Assaf and Graves, 1969; Leaver and
Burt, 1981; Vaandrager et al., 1987). The non-linear
character was more pronounced at low levels of
substrate and/or AMP. This pronounced curvature in
Lineweaver-Burk plots is usually taken as an expression of allosteric behaviour, i.e. homotropic cooperativity between substrate binding sites.
Table 1. Kinetic parameters of glycogen phosphoryl.
ase b from mantle tissue with respect to glycogen at
several levels of Pi and AMP
[Pi]: 80mM
[AMP]
1.6 mM
0.8 mM
0.4 mM
0.0 mM

/ ~ (mg/ml)
0.36
0.82
1.68
2.01

Vm, (nkat/ml)
1.82
1.63
1.52
0.70

[Pi]: 40 mM
[AMP]
1.6 m M
0.8 m M
0.4 mM
0.0 mM

1.19
1.13
2.41
3.79

1.97
1.40
1.50
0.47

[Pil: 15raM
[AMP]
1.6 m M
0.8 m M
0.4 m M
0.0 mM

2.97
1.66
2.49
5.59

1.57
1.38
1.12
0.28

When the glycogen concentration was decreased,

the affinity for Pi decreased to a half and the reaction
rate by 1.5-fold. The Hill coefficient (nil), with values
always around 2, increased slightly from 1.8 to 2,
mainly at low effectOr concentrations (Table 2). On
the other hand, a decrease in AMP concentration
induced very small variations in both S0.5 and the Hill
coefficient for Pi. The S0.5 values ranged from 7 mM
at 1 . t m M AMP to 9raM at 400#M AMP when
glycogen was 24 mg/ml and from 13 to 16 mM in low
glycogen concentrations (3.2mg/ml). Under such
conditions, the Vw~ decreased slightly and the Hill
coefficients increased (Fig. 3) (Table 2).
In the absence of AMP, the mussel phosphorylase
b follows Michaelis--Menten kinetics. The doublereciprocal plots become linear. Both affinity and Vm~
decrease notably and the Hill coefficient decreases to
a value of one (Fig. 4) (Table 2).
AMP

The requirement for the AMP effector by glycogen

phosphorylase b of Mytilus mantle has been shown
previously (San Juan et al., 1991). Despite the low
level of activity in the absence of this effector
(14-20% of the activity in the presence of AMP), the
requirement can be considered as absolute because,
under these conditions, the kinetic constants and
behaviour are strongly altered, as indicated above.
These are also different to that found for phosphorylase a (San Juan et al., 1993).
The S0.5 of Mytilus phosphorylase b for the AMP
was I14#M. An inhibition of catalytic activity by
high AMP concentrations (above 2 mM) and a progressive loss of linearity in the double-reciprocal
plots, when substrate concentrations decrease, are
also observed (Fig. 5).
When the Pi concentration ranged from 80 to
15 raM, the affinity for AMP decreased 2- or 3-fold
depending on the glycogen concentrations. The S0.5

928

FUENCISLA SAN JUAN-SERRANO et al.

4.5
A

AS.O

E
v

'~ 1.5

0.05

0.10

0.15

0.20
0.05
1/[Pi] mM

0.10

0.15

0.20

Fig. 2. Double-reciprocal plots of initial velocity of phosphorylase b as a function of P~concentration at

two levels of glycogen: (A) 24 mg/ml, (B) 3.2 mg/ml, and at several AMP levels: (O) 1.6 raM, (O) 0.8 raM,
(A) 0.4 raM.
increased from I 1 4 # M to a value of 271#M at
24 mg/ml glycogen and from 137 to 423/~M when the
glycogen concentration was 3.2 mg/ml. The Hill coefficient increased from 0.6 at 80 mM P~ to 1.7 at
15 mM Pi when the glycogen concentration was saturating, and from 1.3 to 1.5 when the glycogen concentration was low (3.2mg/ml). The V~x remained
constant when the P~ concentration decreased, except
at low glycogen concentrations, where the Vmaxdecreased at high Pi values (Fig. 6) (Table 3). At all Pi
concentrations, a decrease in glycogen concentration
resulted in an increase in S05 and the Hill coefficient
for AMP (Table 3).
Glucose- l-phosphate
In order to obtain more data on the glycogen
phosphorylase b reaction in the Mytilus mantle,
which might help to clarify the kinetic model, experiments on the reverse (synthetic) direction of the
reaction were carded out. We tried to demonstrate a
possible control of the phosphorylase activity by the
product of the reaction glucose-l-phosphate.
The apparent Km for this substrate was around
150-180 mM. This value is much higher than the ones
described in the literature for other phosphorylases
(Bonamusa and Baanante, 1990; Assaf and Graves,
Table 2. Kinetic parameters of glycogen phosphorylase b from
mantle tissue with respect to Pi at several levels of glycogen and AMP
[Gly]: 24 mg/ml
[AMP]
1.6 mM
0.8 mM
0.4 mM
0.0 mM

S0.5 (mM)
7.09
8.19
8.87
19.68*

Vm.~ (nkat/ml)
1.88
1.70
1.38
0.40

nn
1.8
1.9
1.8
1.1

[Gly]: 3.2 mg/ml

[AMP]
1.6 mM
0.8 mM
0.4 mM
0.0 mM

12.72
12.59
16.05
29.85*

1.32
1.17
1.03
0.23

1.8
1.9
2.0
1.0

*K. value.

1969; Hergenhahn, 1983) but, on the other hand, it

is comparable to the Km for rat liver phosphorylase
(Tan, 1975). At the same time, the Hill coefficient was
always 1, showing that the glucose-l-phosphate interacts with only one binding site on the enzyme.

DISCUSSION

Glycogen phosphorylase b has been purified and

kinetically characterized from a wide variety of
sources, showing special features in certain cases.
These special characteristics were mainly attributed
to adaptations of the organism to the environment
(Cherian and Philip, 1984), or to organisms far away
from each other on the evolutionary scale (Thomas
and Philip, 1983; Vaandrager et al., 1987; Newgard
et aL, 1989).
The Mytilus phosphorylase b, under certain conditions can catalyse the glycogen phosphorolysis at
the same rate as phosphorylase a (active form) (San
Juan et al., 1993). Thus, the b form must be controlled by the concentration of various natural
metabolites, i.e. substrates, activators and inhibitors.
From our results, it seems that the kinetic characteristics of the enzyme are similar to those of other
organisms reported above. However, several aspects
of the kinetic properties that will be discussed below,
appear to be different.
The value of the apparent K~ for the substrate
glycogen is larger than in vertebrates. This result
explains why the phosphorylase-glycogen complex
formation is not a suitable method for the purification of Mytilus phosphorylase (San Juan et al.,
1991) and is different from rabbit muscle, Squalus
sucklii (Cohen et aL, 1971; Cohen et al., 1973) and
scallop muscle enzymes (Hata et al., 1987).
Glycogen kinetics did not exhibit homotropic cooperativity in any AMP and Pi studied concentrations,
showing
evident
Michaelis-Menten

B///

Mytilus glycogen phosphorylase b

4.5
"'

~. 3.0

-,%

,,..

,:

929

,.,

"~- 1.5
I' U

-%.,
i

1
4
1 / [ P i ] 2 mM x 102

,{,, t.,a,,~,

..3

Fig. 3. Reciprocal plots of initial velocity of phosphorylase b as a function of reciprocals of Pi

concentrations squared at fixed levels of glycogen: (A) 24 mg/ml, (B) 3.2 mg/ml, and in several AMP
concentrations: (O) 1.6 mM, (0) 0.8 raM, (A) 0.4 raM. Inset: Hill plots for determining values of S0.5
and nH.

behaviour, different from other organisms (Engers

et al., 1969; Childress and Sacktor, 1970). If we
assume the existence of two binding sites for glycogen, as in vertebrate phosphorylase, this behaviour
could be explained by the high affinity of the glycogen
storage site, which masks the binding of substrate to
the active centre. However, a positive heterotropic
co-operativity by Pi and AMP was observed. These
molecules modify the intrinsic catalytic activity at the
same time as the affinity for glycogen as substrate. We
can consider them as positive allosteric effectors of
mixed-type with respect to glycogen (Segel, 1975).
The effect of AMP as an activator must be due to
its binding to the nucleotide site. However, at low
co-substrate concentrations (15 raM), the behaviour
of AMP is the opposite of what is expected: when the
AMP concentration decreases, a slight increase of the
affinity for the glycogen is observed (Table 1). This

13.5

<4 . 5
0.05

0.10

0.15

0.20

1 / [ P i ] mM

Fig. 4. Double-reciprocal plots of initial velocity of phos-

phorylase b as a function of Pi concentration in the absence

of AMP and at two levels of glycogen: (O) 24 mg/ml, (0)
3.2 mg/ml.

seems to reveal a positive heterotropic effect of Pi on

the affinity for AMP. The positive co-operativity of
Pi could be due to its secondary interaction with the
specific subsite f o r the phosphoryl group in the
nucleotide site. This locus can work as an activator
or an inhibitor depending on which subsites are
bound to fixed ligands (Morange et al., 1976; Eguchi
et al., 1977). In this way, our results showing a greater
increase in the affinity for glycogen when Pi concentration is increased, seem to indicate that Pi is a better
activator than AMP, although without eliminating
the requirement for the latter effector (Table 1).
The values of Km for glycogen, examined in different concentrations of Pi and AMP, were always lower
than the levels of glycogen in cells of Mytilus mantle
(12-40 mg/ml) (Crespo, 1989). This suggests that in
mussel mantle, glycogen phosphorylase is constantly
saturated by substrate and its regulation is not subject
to variations in glycogen content.
The kinetic behaviour of the enzyme with respect
to P~ is the most complex because this molecule has
a double role, as substrate and effector. The different
saturating levels of P~, as a function of the glycogen
and AMP concentrations, can be explained through
a higher affinity of the active centre than the binding
subsite in the nucleotide locus for the co-substrate.
The inhibition of enzymic activity in high P~ concentrations is probably due to a desensitization of the
enzyme for this substrate (Leaver and Burt, 1981).
The kinetic studies for the co-substrate P~, carried
out at different concentrations of glycogen and AMP,
led to the observation of homotropic and heterotropic co-operativity. As a function of the variation
in glycogen levels, the data obtained showed a clear
positive heterotropic co-operativity of the substrate
on P~ binding: glycogen binding to the specific storage
site promotes P~ binding to the active centre (Kasvinsky et ai., 1978). On the other hand, P~ binding to the
active centre should originate a positive homotropic

930

FUENCISLA SAN JUAN-SERRANOet al.

3.0
A
2.4-

1.8

1.2.

0.6.

6
1/[AMP] mM

Fig. 5. Double-reciprocal plots of initial velocity of phosphorylase b as a function of AMP concentration

two levels of glycogen: (A) 24 mg]ml, (B) 3.2 mg/ml, and at several Pi levels: (O) 80 raM, (0) 40 raM,
(Z~) 15 mM.

at

effect, inducing its binding to specific subsites in the

nucleotide locus, too. This justifies Hill coefficient
values near 2. At low glycogen concentrations, the
described effect is smaller and the Pi binding to the
active centre is less efficient. However, a slight increase of the Hill coefficient, when AMP concentrations are also low, is observed. This could be due
to the co-substrate binding to the site of the allosteric
effector (Lorek et al., 1984) (Table 2). The very small
modification in the S0.s, Vm~ and Hill coefficient for
Pi observed, as a function of the variation in AMP
levels, could be due, indirectly, to its effects on
glycogen binding. However, in the absence of AMP,
the strong alteration of the kinetic constants shows a
loss of the allosteric behaviour.
All the Km or S0.s values found for P~ are within the
values of physiological concentrations determined in
the mantle tissue of the mussel (10/amol Pi/g). Similarly, apparent Km values of glycogen phosphorylase

b for the effector AMP are within the physiological

range of this nucleotide in the mantle tissue of
Mytilus (10-175/aM) (Ibarguren, 1990). Therefore,
small changes in Pi and AMP levels could have an
important regulatory effect on glycogen phosphorylase b.
At AMP concentrations higher than 2 mM, the
phosphorylase b activity is inhibited because of the
secondary binding of the effector to the nucleoside
site. The sigmoidal AMP saturation curve and the
non-linearity of the double-reciprocal plots suggest
that, in Mytilus phosphorylase, the AMP is an effector that acts by an allosteric mechanism and that
shows co-operative effects, similar to Pi and similar to
the ones described in other species (Assaf and Graves,
1969; Engers et al., 1969; Childress and Sacktor,
1970; Leaver and Burt, 1981).
A clear positive heterotropic co-operativity of P~
and glycogen on the kinetic properties, with respect

3.0

B
2.4

1"

E 1.8J--

> 1.2.

0.61

_,,-'
I

t4

21

28

35
7
1 / l A M P ] 2 mM

I,

-~I~,~.l~

14-

21

28

35

Fig. 6. Reciprocal plots of initial velocity of phosphorylase b as a function of reciprocals of AMP

concentrations squared at fixed levels of glycogen: (A) 24 mg/ml, (13) 3.2 mg/ml, and in several Pi
concentrations: (O) 80raM, (0) 40mM, (A) 15raM. Inset: Hill plots for determining values of S0.5
and nH.

931

Mytilus glycogen phosphorylase b

Table 3. Kinetic parameters of glycogen phosphorylase b from

mantletissuewithrespectto AMP at severallevelsof glycogenand Pi
[Gly]: 24 mg/ml
[Pi]
80 mM

40 mM
15 mM
[Gly]: 3.2 mg/ml
[i'd
80 mM
40 mM
15mM

S0.5 ~ M )
114

Vm,, (nkat/ml)
1.82

na
0.6

188
271

1.55
1.58

1.5

137

1.08

1.3

295
423

1.48
1.37

1.6
1.5

1.7

to AMP, was observed. Pi binding to the enzyme

results in a conformation where A M P binding to its
specific activator site (nucleotide site) is promoted.
When the Pi concentration decreases, this positive
beterotropic effect decreases, too, and the A M P can
now bind to the nucleoside site (inhibitor site). This
is indicated by the increase in Hill coefficient to values
around 2. This is also shown by the results obtained
with respect to glycogen: in low Pi concentrations, the
affinity for the substrate increases when the A M P
concentration decreases. This is explained because, in
such conditions, the effector is mainly bound to the
nucleoside site, leading to its inhibitory effect in
decreasing the affinity of the active centre for the
substrates. Therefore, when the A M P concentration
decreases, the inhibition decreases, too, and a decrease of glycogen Km is found (Table 1).
If the values of the Hill coefficient and initial
velocities at different substrate concentrations are
analysed together, we observe the interdependent
effects of the three studied ligands. We can also
predict how the inhibitory effect is exercised by the
A M P binding to the nucleoside site. As in the phosphorylase from vertebrate liver, this locus must have
a physiological regulatory role, working as a negative
heterotropic effector site. In this way, A M P binding
to the nucieoside site prevents glycogen and Pi
binding to the active centre, probably because it
stabilizes an inactive conformation (Dombrfidi, 1981;
Kasvinsky et al., 1981).
At saturating levels of Pi and low glycogen concentrations, the S0.5 for A M P seems to be in disagreement with the low initial velocity value found.
However, this can be explained because, under these
conditions, the binding of Pi to the active centre is not
favoured. Thus, the co-operative effect of Pi on the
A M P binding decreases. Moreover, the excess of Pi
that is not bound to the active centre would be
competing for the nucleotide site with AMP. Therefore~ this should be bound mainly to the inhibitor site.
When the Pi concentration decreases, the competition
for the nucleotide site allows more A M P binding to
the activator site, and the reaction velocity and Hill
coefficient increase (Table 3).
The kinetic data, as regards glucose-l-phosphate,
leads us to conclude that there is no regulatory role
for this metabolite on mussel phosphorylase activity,
since its Km values are very high.

The kinetic properties of glycogen phosphorylase b

from the Mytilus mantle reveal analogous binding
sites for the ligands as described in the molecular
structure of the vertebrate enzyme. These studies also
suggest that there are co-operative interactions between these specific sites for substrates and activators,
which supports the idea that the Mytilus phosphorylase b is controlled by allosteric mechanisms. Contrary
to the vertebrate phosphorylase b, and bearing in
mind the glycogen, Pi and A M P concentrations found
in mantle tissue, t h e s e mechanisms appear to be
functional in vivo, at basal levels of phosphorylase b
activity. Also, certain characteristics discussed, such
as the Michaelis kinetics relative to glycogen, more
important regulatory effects of the Pi compared to the
AMP, the inhibition of activity in high Pi concentrations, the different kinetic behaviour of phosphorylase b in the absence of A M P and the
insignificant regulatory role of glucose-l-phosphate,
suggest a divergence in the metabolic control of
mussel phosphorylase compared to the vertebrate
model. Although w e do not yet understand their
implications, these particular features could be related to evolution, and/or adaptation to the environment, of marine mussels.
Acknowledgements--This work was supported by a grant
(XUGA 20305 B90; Consellerla de Educaci6n) from the
Autonomic Galician Government (Spain).

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