Inr. J. B&hem.

1357-2725(95)OW9-3

Cell Bid. Vol. 27, No. 9, pp. 911-922, 1995

CoavriPht 0 1995 Elsevier Science Ltd
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Printed tn Grea~Britain.
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1357-2725/95 $9.50 + 0.00

Modikation
of Kinetic Parameters of Glycogen
Phosphorylase from Mantle Tissue of Mytilus
galloprovincialis by a Phosphorylation Mechanism
FUENCI$LA SAN JUAN SERRAN?,*
MARGAkmA FERNiiNoEZ GONZALEZ,
JO& LUIS Ss&NCHEZ LOPEZ, L. OSCAR GARCIA MARTbl
Departamento de Bioquimica y Biologia Molecular, Facultad de Farmacia, Universidad de Santiago
de Compostela, Avda de las Ciencias s/n, 15706 Santiago de Compostela, Spain
Initial rate and aflhity studies on mantle Myths phospborylasea were carried out in Order to
find possible differences in its kinetic properties with respect to phosphylase b. PhespBaryhse
a was nut stimulated for any AMP concentrations. Michaelis constants (K,) are O.OSmg/ml
glycogen, 1.15mM inorganic phosphate and 1.5OmM glucose-l-phosphate. The K-s for the
substrates,in the direction of glycogenbreakdown, are enhancedby wa+aturathg amcentratioas
of cost&t&rate, without reducing the apparent maximum velocity. First order and hyperbolic
kinetics and valuesof the allosteric constant smaller than 2 were observed.These results suggest
a catalytic mechanism different to that shown for mantle Mytirus pkosphorylase b.
Keywords: Mytih

Phosphorylase Kinetic parameters

Int. J. Biochem. Cell Biol. (1995) 27,917-922

Whitton, 1980; Madsen, 1986; Madsen et al.,

1983).
Each one of these processes promotes a
different conformational
state of the enzyme,
an active state R or an inactive state T
(Newgard et al., 1989). The integration of both
these mechanisms allows for the regulation of
phosphorylase activity by external stimuli, as
well as by intracellular signals that reflect the
energy balance of the tissue.
The enzyme b form predominates in Mytilus
mantle tissue at rest before changing to the
a form, in response to the activating process
of phosphorylation,
thus increasing glycogen
breakdown (San Juan et al., 1995).
In previous papers (San Juan et al., 1991,
1993) we also described the isolation and
allosteric regulation of phosphorylase b from
Mytilus mantle. This paper describes the modifications of kinetic properties of phosphorylase
a relative to the b form, as a result of the
conformational change induced by phosphorylation.

INTRODUCTION
Mytilus mantle tissue is an organ analogous in
many functional aspects to mammalian liver.
It serves as an important storage organ for
accumulation of energy reserves. Mobilization
of glycogen supphes from mantle tissue of
Mytilus, mainly as a fuel for gametogenic development (Gabbott, 1975, 1976; Bayne, 1976;
Pieters et al., 1979), is dependent on the activity
of various enzymatic systems required for
degradation of these reserves.
Enzymatic release of glucose- 1-phosphate
from glycogen has been extensively studied.
Glycogen phosphorylase activity is regulated in
a complex manner by various small molecules
and ions, and by the reversible covalent transition (phosphorylation) of the enzymatic molecule from b form to a form (inactive and active
in absence of AMP, respectively) (Hems and
*To whom all correspondence should be addressed.
Received 13 July 1994; accepted 1 May 1995.
917

918

Fuencisla San Juan Serrano

MATERIALS

AND

METHODS

Animals and reagents

Mussels measuring between 7 and 10 cm in

length were collected from Villagarcia de Arosa
(Galicia, NW Spain). Their mantle tissue was
stored at -30C until utilized.
Substrates, enzymes and coenzymes were
obtained from Sigma Chemical Company and
Boehringer. All other chemicals were obtained
from Merck.
Phosphorylation

of phosphorylase b

The interconversion of phosphorylase b in

its a form was performed by endogenous
phosphorylase kinase in crude homogenates of
Mytilus mantle as indicated by San Juan et al.
(1995). The mantle tissues were homogenized in
40 mM Tris-acetate buffer pH 7.0, imidazol and
/I-mercaptoethanol in a concentration of 5 mM
and 0.12 M KC1 1: 2 (w/v) ratio, with the aid of
a motor driven teflon-glass Potter homogenizer
at 4C. The homogenate was filtered through
glass wool and incubated with ATP, MgCl,
and CaCl, in a concentration of 2 mM. Endogenous phosphatase activity was inhibited by
the addition of NaF 10mM. Incubations were
carried out at 20C.
For kinetic studies, crude homogenate was
centrifuged at 15,4OOg,, for 20 min. The supernatant, containing the phosphorylated form,
was dialysed against 30 vol of extraction buffer
without KCl, plus 5 mM EDTA and 10mM
NaF.
Determination

of phosphorylase activity

Glycogen
phosphorylase
activity
was
measured spectrophotometrically
in the direction of glycogen breakdown, as described by
Childress and Sacktor (1970). The reaction
mixture for phosphorylase a contained 40 mM
Tris-acetate buffer pH 7.0, 5 mM imidazol,
1.4 mM j?-mercaptoethanol, 5 mM Mg-acetate,
5 PM glucose 1,6-diphosphate, 0.6 mM NADP,
8 mg/ml glycogen, 30 mM phosphate buffer
pH 7.0,0.5 IU of glucosed-phosphate dehydrogenase, 0.1 IU of phosphaglucomutase
and
enzyme preparation in a total volume of 1.O ml.
One international unit (IU) is defined as the
amount of enzyme catalysing the formation of
1 pmol glucose-1-phosphate/min at 20C.
Phosphorylase activity was assayed, in the
direction of glycogen synthesis, by measuring
the release of Pi from glucose- 1-phosphate. The
reaction mixture contained 40 mM Tris-acetate

et a/.

buffer pH 7.0, 3.2 mg/ml glycogen, 50 mM

glucose- 1-phosphate and enzyme preparation
in a total volume of 1 ml. The reaction was
stopped, after incubation at 30C for 45 min, by
adding 0.1 ml of 20% sodium dodecyl sulphate.
The Pi in the supernatant resulting from
centrifugation was determined by the method of
Saheki et al. (1985).
Calculations

Apparent K, and V,, values, presented in the

results, were estimated using Lineweaver-Burk
double reciprocal plots. When the double reciprocal plots were nonlinear (enzyme activity
did not follow Michaelis-Menten kinetics), the
apparent K,,, (,!&), V,,,,, and interaction coefficients (nH ) values were evaluated from Hill
plots.
Apparent V,,, values obtained from these
plots were used to determine the allosteric
constant (L) and dissociation constants for Pi
and AMP. Data was analysed according to
the equation derived by But (1967) from the
exclusive binding model shown below:

where V = initial velocity, V, = velocity observed at a given concentration of phosphate,

and at saturation of AMP, which is = V,,,,,
L,, = T/R in absence of both phosphate
and AMP, the concentration of any other
ligand remaining constant, P = phosphate
concentration, A = AMP concentration, Kp =
dissociation constant of enzyme-bound phosphate and KA = dissociation
constant of
enzyme-bound AMP.
In this derivation the enzyme is assumed to
belong to a K system of allosteric enzymes,
where an increase in substrate concentration [in
this case inorganic phosphate (Pi)] should
reduce the enzyme requirement for the allosteric
effector (in this case AMP).
As suggested by But 1967 data were plotted
versus AMP at
in the form of ,/#?
various constant levels of Pi concentrations.
All phosphate lines were linear and intersected
at a common point on the abscissa equal to
the dissociation constant for AMP. Each of the
ordinate intercepts of phosphate lines is equal to
mO(l
f P/K,). These values were plotted
versus phosphate concentration. The abscissa
intercept is the dissociation constant, and the
allosteric constant can be calculated from ordinate intercept J17L.

Kinetic modifications of Myfilus phosphorylase by phosphorylation

RESULTS

AND DISCUSSION

Phosphorylase
from mantle Mytz7a.r is
primarily present as the AMP dependent form,
phosphorylase b (100%). This form can be
transformed by a phosphorylation process into
the a form (100%) mediated by an endogenous
glycogen phosphorylase kinase (San Juan et al.,
1995).
Both forms, phosphorylated and unphosphorylated, showed the same V,,,. Stimulation
of phosphorylase activity by AMP, measured
under standard conditions, is shown in Fig. 1.
The phosphorylase b activity was stimulated
7-fold by 1.6 mM AMP and this forms K,
value for AMP was 114pM (San Juan et al.,
1991). However, after phosphorylation,
the
requirement for this effector to reach I,,,,, disappeared and phosphorylase a activity was not
stimulated by any AMP concentration, unlike
other phosphorylases a studied in vertebrates
and invertebrates (Childress and Sacktor, 1970;
Cohen et al., 1971; Dombradi et al., 1986;
Vaandrager et al., 1987; Van Marrewijk et al.,
1988; Morishima and Ueno, 1990). In other
literature, only lobster phosphorylase a activity
was not increased by AMP. In this case, however, AMP could be bound by the enzyme,
but the level of enzymatic activity remained
the same (Cowgill and Cori, 1955). Though
AMP binding to Mytilus phosphorylase a
has not been verified, this could explain why
AMP decreases the inhibition for nucleosides.
This inhibition by 5 mM caffeine, analogous
nucleosides, is 12% of total phosphorylase
activity in the presence of 1.6 mM AMP, but

10

25

-3

-2

-1

log [AMP]

mM

Fig. 1. E&t of AMP concentration on both dependent (0)

and independent (0) phosphorylase activities of Myrilus
galloprovincialis mantle.

0.007
1

tI
0.5

0.014

0.02 I

0.028

1
1
1.5

I
2.0

1.0
l/[GlP]

mM

2. Determination of apparent K, values for gfucose-lphosphate. Reciprocals of initial velocity of phosphorylase

b (0) and phosphorylase a (0) as a function of reciprocals
of glucose-l-phosphate concentration. Phosphorylase b
activity was measured in the presence of 1.6mM AMP
and 3.2mg/ml glycogen (as primer). Phosphorylase
a
activity was measured in the absence of AMP. Obtained
values of apparent K,,, for glucose-l-phosphate were 1.5
and 15&180 mM for the a and b phosphorylase forms,
respectively.
Fig.

increases to an inhibition of 67% in the absence

of AMP (data not shown).
The affinity of both forms of Mytih
phosphorylase for glucose-l-phosphate in the direction
of glycogen synthesis is very different
(Fig. 2). Apparent K,,, of the a form has a value
similar to that described in literature for other
organisms (Cori et al., 1943; Assaf and Graves,
1969; Applebaum
and Schlesinger, 1973;
Santiago et al., 1974; Hergenhahn, 1983). However, apparent K,,, of the b form increases
to relatively high values, comparable only to
values obtained from rat liver (Tan, 1975).
In the a form, contrary to what is expected, V,,
is smaller than in the b form. The Hill coefficient
value in both a and b forms is 1, which indicates
that glucose- 1-phosphate interaction takes place
with only one part of the enzyme, i.e. the
active centre by necessity. The large modification of phosphorylase a glucose- 1-phosphate
affinity with respect to that of phosphorylase b,
although not very significant from a physiological point of view, may be due to important
alterations in the enzyme structure caused by
the phosphorylation process.
Substrate affinities and phosphorylase a
activity rates were determined in the direction

920

Fuencisla San Juan Serrano et al.

Table 1. Kinetic parameters of both Myli/us phosphorylase forms
Phosphorylase

Phosphorylase

Pi
(mM)
30
5

K, WYC)
(w/ml)
0.05
2.98

(I$%)
0.108
0.110

Glyc
(mg/mU
8.0
1.6

4, 09
MM)
1.16
2.98

(I$%)
0.110
0.110

1.0
1.0

Pi
bM)
80
15

Km,(JYc)
h&l)
-__
0.36
2.91

rl
0.9
0.8

Glyc
(m&4
24.0
3.2

Km (Pi)
W4
7.09
12.72

&%)
0.113
0.079

n
1.8
1.8

AMP
NM)
1.6
0.4

Km,VYC)
(m&4
0.36
1.68

&Al)
0.109
0.091

n
1.0
1.0

of glycogen degradation, for saturating concentrations of cosubstrate and the absence of

AMP. Michaelis constant (K,,,) and interaction
coefficient, calculated from Hills equation (n,,),
for glycogen and Pi are given in Table 1, in
comparison with the same constants (II;, or S,,,)
for phosphorylase b from Mytilus mantle (San
Juan et al., 1993).
Both Michaelis constants (K,,,) for glycogen
and Pi are 74imes smaller than in phosphorylase b. The lowering of Pi concentration to
non-saturating values increased apparent K,,, for
glycogen markedly to the same value as the
one in phosphorylase b, showing a positive
heterotropic cooperativity of Pi on the glycogcn
binding. A slight increase of Km for Pi is
also observed when glycogen concentration
decreases to non-saturating values.
Myths phosphorylase a would appear to be
a K system, similar to that of the phosphorylase a prototype enzyme from rabbit skeletal
muscle and phosphorylases a from other sources
(Engers et al., 1970; Vaandrager et al., 1987). In
this way, the ligands have a minimal effect on
V max9while causing a consistent decrease on
dissociation constants for the substrates. This is
different from the case of phosphorylase b,
where both Pi and AMP are allosteric effecters
of a mixed K and V system (Table 1).
In comparing phosphorylase a with b, it is
clear that phosphorylase a is a much more
eficient enzyme, not only because it is active in
the absence of AMP, but also because it binds
substrates much more tightly.
It is difficult to interpret the role of tissue
glycogen since it is always at very high levels in
Myti~us mantle (12-40 mg/ml) and is concentrated in granula (Crespo, 1989). This may
allow a much higher local concentration and a
lower accessibility, which may be a regulatory

(I$&
O.iF--0.094

?zH
1.0
1.0

factor of activity for both phosphorylase forms.

Mantle Pi content (10 pmol Pi/g) exceeds by
lo-fold the K,,, for Pi of phosphorylase a, but
equals the K,,, for Pi of phosphorylase b. This
suggests that Pi is only an effective regulator of
phosphorylase b activity.
The phosphorylase a from other studied
organisms, in the absence of AMP, exhibits
a pronounced curvature in Lineweaver-Burk
plots, similar to phosphorylase b. This is usually
taken as an expression of allosteric behaviour
(i.e. homotropic cooperativity between substrate binding sites). In contrast, the curvature
of the double reciprocal plots decrease, in the
presence of increased levels of AMP, indicating
a less homotropic interaction (Childress and
Sacktor, 1970; Vaandrager et al., 1987; Morishima and Ueno, 1990; Metzger et al., 1968;
Engers et al., 1970; Leaver and Burt, 1981).
This catalytic mechanism of phosphorylase a is
essentially the same as the one observed with
phosphorylase b, except that the affinity for
both substrates, glycogen and Pi is much greater
than that of phosphorylase b.
Phosphorylase a of Mytzlus mantle, in contrast to that described above, did not appear to
be stimulated by AMP; neither catalytic rate
nor substrate affinities were modified by any
AMP concentration (1 PM-1 .6 mM). Apparent
K,,, values of this form for substrates in the
absence of AMP was lower than that of phosphorylase a from other organisms, and similar
to that in saturating AMP concentrations
(Childress and Sacktor, 1970; Vaandrager et al.,
1987; Engers et al., 1970). On the other
hand, first order and hyperbolic kinetics were
observed at all substrate levels (Fig. 3). These
unusual kinetic properties of Myribs phosphorylase a, and the fact that this form has a
covalently bound phosphate residue, suggest

921

Kinetic modifications of Mytifus phosphorylase by phosphorylation

8

6
60

15

r(b)

30

45
60 _
I
2
3
80 mM Pi 0 24.0 mglml Glyc
I5 mM Pi 0 3.2 mg/ml Glyc

0
l

0.5

1.0

1.5

[AMP] m&l
60
8.0
30
0
0.5
l/[Glyc]

1 .o

I.5

mg/mI

2.0

0.5

1.o
l/[Pi]

1.5

2.0

mM

Fig. 3. Determination of phosphorylase behaviour and

apparent K,,, values with respect to substrates. Reciprocal of
initial velocity of both phosphorylase activities as function
of reciprocals of glycogen and phosphate concentrations, at
two fixed levels of cosubstrate. Determinations of the a form
were performed in the absence of AMP (a) and the AMP
concentration in the b form determinations was 1.6 mM (b).

that the change in the quaternary and tertiary

conformation,
promoted by phosphorylation
(Barford et al., 1991), induced notable modifications in the allosteric site (nucleotide site), and
therefore, in its catalytic behaviour.
This idea is in accordance with the values of
the allosteric constant, L (constant equilibrium
for molar ratio between inactive and active
conformers: T/R) for the two enzymatic forms
from Myths. The L constant for phosphorylase
b has been estimated to be 50. The same constant for phosphorylase a is more than 25
times smaller (Fig. 4). Both constants are significantly smaller than those of other studied
organisms (Assaf and Graves, 1969; Madsen,
1986), which suggests a very low energy barrier
for this conformation transition. However, the
phosphorylated form is present exclusively in
its active conformation R, thus escaping from
allosteric control. Moreover, this form is
stable at moderate changes in effector ligand
concentrations, to which phosphorylase b is
subject.
From our results, we may consider the phosphorylation of the Mytilus phosphorylase to
be similar to an allosteric activation that is
temporarily stable, and different to the metabolic control in the vertebrate model. Their
importance and contribution
in periods of
high energetic demand and biosynthetic activity,

10

1.5

20

[Pi] mM
Fig. 4. Allosteric and dissociation constants at several fixed
levels of Pi, for glycogen phosphorylase from Mytiim
mantle. The Pi and AMP dissociation constants obtained
agree with the association constants for these effecters at
saturating glycogen levels. In absolutely AMP-independent,
phosphorylase a, &(I
+ P/K,) was calculated as
JV/V, - V (lower plot). (0) 60 mM Pi, (0) 40 mM Pi,
(A) 20 mM Pi, (A) 15 mM Pi.

such as gametogenic development, are presently

being studied at our laboratory.
Acknowledgements-This work was supported by a grant
(XUGA 20305 B90; Conselleria de Educacibn) from the
Autonomous Government of Galicia (Spain).
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