Comp. Biochem. PhysioL Vol.98B, No. 1, pp.

33-39, 1991

0305-0491/91 $3.00+0.00
1991PergamonPress plc

Printed in Great Britain

PURIFICATION A N D MOLECULAR PROPERTIES OF

GLYCOGEN PHOSPHORYLASE b FROM MANTLE TISSUE
OF MUSSEL, M Y T I L U S G A L L O P R O V I N C I A L I S
F. SAN JUAN SERRANO,M. FERNANDEZGONZALEZ,J. L. SANCHEZLOPEZ
and L. O. GARClAMARTIN
Departamento de Bioquimica y Biologia Molecular, Facultad de Farmacia, Universidad de Santiago de
Compostela, 15706 Santiago de Compostela, Spain

(Received 24 May 1990)

Abstraet--l. Glycogen phosphorylase b from mantle of Mytilus galloprovincialis has been purified to
homogenity as judged by native- and SDS-gel electrophoresis.
2. The enzyme was purified 70-fold with a final spec. act. of 0.4 U/rag prot and an overall yield of 34%.
3. The mol. wt of native and purified phosphorylase was estimated to be 340,000 from gel filtration
studies. On SDS-gel electrophoresis, the subunit mol. wt was 86,000. The enzyme was shown to be present
as a tetramer.
4. Maximum activity of the enzyme was obtained at pH 7.2 and 30C.

INTRODUCTION

MATERIALS AND METHODS

Glycogen phosphorylase (u-l,4 glycogen, orthophosphate glucosyltransferase EC 2.4.1.1) catalyzes

the degradation of glycogen to glucose-l-phosphate. The enzyme has been purified and crystallized from various animal species and its structure and properties have been extensively reviewed
(Graves and Wang, 1972; Fischer et al., 1971;
Busby and Radda, 1976; Dombrfidi, 1981; Steele,
1982).
In general the enzyme can exist in two
forms, designated phosphorylase a and phosphorylase b, being active and inactive in the absence
of AMP, respectively. The two forms are interconvertible by phosphorylation-dephosphorylation
reactions.
Despite it being known that glycogen is one
of the major energetic fuels in the mantle of
bivalve molluscs (Zandee et al., 1980) and that
different work has made evident the presumable
relation between its degradation and gametogenic
development (Gabbott, 1975; Bayne et al., 1982),
there are few studies concerning the alternative
metabolic pathways of glycogen metabolism in
these organisms (Alemany and Rosell-P6rez, 1973;
Zaba, 1981), as well as involved enzymes (Zammit
and Newsholme, 1976; Vfizquez-Baanante and
Rosell-P6rez, 1979; Ebberink and Salimans, 1982;
Hata et al., 1987). Because of this, the molecular mechanism used by the bivalve molluscs for
the mobilization of the stored glycogen is still
unknown.
As a part of our investigation on the relevance of
different types of glycogenolitic pathways probably
operating in the mussel Mytilus galloprovincialis, this
paper describes the purification and the molecular
properties of glycogen phosphorylase from the mantle
tissue of this mollusc.
cBPa 9s/t--c

Animals
Sea mussels were supplied by a purification plant in
Villagarcia de Arosa (Galilcia, NW Spain) in September
1988. Animals were collected after a stay of 24-48 hr in the
purification plant. Mussels with shell lengths between 7 and
10 cm were selected for the experiments. They were transported to the laboratory within 1 hr of collectionand excision
of their mantle tissues was commenced immediately. The
tissues were stored at -30C until use.

Reagents
The substrates, enzymes and coenzymes were obtained
from Sigma Chemical Company (St Louis, MO). DEAESephacel, Sephacryl S-300, 5' AMP-Sepharose and protein
calibration set for mol. wt determination, were purchased
from Pharmacia Fine Chemicals (Uppsala, Sweden). Magnesium acetate, Tris-hydroxymethyl aminomethane and
imidazole were purchased from Merck (Darmstadt, FRG).
All other chemicals and salts were of the best purity
commercially available.

Determination of enzyme activity

Glycogen phosphorylase activity was assayed in the
direction of glycogendegradation by coupling the production
of glucose-1-phosphateto the reduction of NADP + by phosphoglucomutase and glucose-6-phosphate dehydrogenase.
The assay medium was the same used by Childress and
Sacktor (1970). The final vol of the reaction mixture was I ml.
One unit of activity was defined as the amount of enzyme
catalyzing the formation of 1/zmol glucose-l-phosphate/rain
at 20C. Specific activity is expressed as U/mg prot. Protein
concentration was measured by the procedure of Smith
(1985), using a solution of 2 mg/ml of bovine serum albumin
as protein standard.

Purification procedure
All purification steps were carried out at 0-4C. Unless
otherwise indicated the bufferscontained EDTA, imidazole,
and 2-mercaptoethanol in a concentration of 5 x 10-3 M.
This purification procedure joins different steps previously
33

F. SAN JUAN SERRANO et al.

34

for 4 hr against 40 mM Tris-acetate buffer pH 7.0. The

dialysis buffer was replaced twice.

used by several authors (Childress and Sacktor, 1970; Miller

et al., 1975). As reference methods have been utilized, in
principle, those employed in the purification of glycogen
phosphorylase from invertebrate organisms (Hergenhahn,
1983; Van Marrewijk et al., 1988).

AMP-Sepharose 4B column chromatography

The dialyzed enzyme solution was applied to an AMPSepharose 4B column (3.5 2 cm) equilibrated with extraction buffer without KC1. The flow rate was 10ml/hr. The
column was washed out with the same buffer (I00 ml) and
the enzyme was eluted by passing a solution of 10 mM AMP
added to the equilibration buffer through the column. The
eluate was collected in 1.8 ml fractions and assayed for
enzymatic activity. The active fractions were pooled and
concentrated by ultrafiltration (PM-10 filter).

Extraction of enzyme and ammonium sulphate fractionation

Mussel mantles (50 g) were homogenized in 2.0 vol (w/v)
of ice-cold 40 mM Tris-acetate buffer, pH 7.0, containing
0.12 M KCI using a Potter homogenizer. The homogenate
was centrifuged at 15,400gay for 20 min and the pellet was
discarded. The particle-free supernatant was filtered through
glass-wool to remove any fat. This suspension was dialyzed
against 30 vols of extraction buffer without KCI, the dialysis
buffer being replaced twice. After dialysis 100 ml of crude
homogenate was obtained.
The crude extract ammonium sulphate fractionation
between the limits of 30-60% was carried out as described
in Wood (1976). The required vol of a 3.8 M solution of
ammonium sulphate to reach 30% saturation (0.452 ml
of 3.8 M solution/ml of enzymatic solution) was added.
The mixture was then stirred for 30 min and allowed to
precipitate for at least 2 h r at 4C. The ammonium
sulphate suspension was centrifuged at 15,400gay for
20 min and the supernatant was decanted. This supernatant,
containing phosphorylase activity, was 60% saturated
by the addition of 0.86 ml of 3.8 M ammonium sulphate
solution/ml supernatant.
After 12 hr (overnight) the suspension was centrifuged as
previously described and the supernatant was discarded. The
pellet obtained was resuspended in a minimum vol of 40 mM
Tris-acetate buffer pH 7.0. The solution was dialyzed against
30 vols of the same buffer, this buffer being replaced twice.
The final vol obtained, after dialysis and posterior concentration using an Amicon ultrafiltration cell (PM-10 filter),
was 16ml.

Sephacryl S-300 gel filtration

The concentrated enzyme solution was applied to a
Sephacryl S-300 column (60 x 2.5 cm) equilibrated with the
extraction buffer and eluted with the same buffer. The flow
rate was 30 ml/hr and 3 ml fractions were collected. Fractions
with enzymatic activity were pooled and concentrated in the
way previously indicated.

Molecular weight estimations

The mol. wt of native and purified enzyme was determined
by gel filtration on Sephacryl S-300 (Andrews, 1964). The
column used for this study was the same utilized in the final
step of the purification procedure. For this purpose the
column was calibrated with chymotrypsinogen A (mol. wt.
25,000), ovalbumin (43,000), aldolase (158,000), catalase
(232,000), ferritin (440,000) and thyroglobulin (669,000).

Electrophoresis

DEAE-Sephacel column chromatography

The dialyzed and concentrated enzyme solution was then
applied to a DEAE-Sephacel column (41 x 2.5 cm) which
had been equilibrated with the extraction buffer, without KC1.
The enzyme was eluted overnight using a linear salt gradient
formed by two 250 ml vessels, containing 40 mM Tris-acetate
buffer pH 7.0 as initial solution and this buffer plus 0.6 M
KC1 as the final solution. The flow rate was 40 ml/hr and
2.8 ml fractions were collected. Fractions containing enzyme
activity were pooled and concentrated by ultrafiltration
(PM-10
filter). This concentrated solution was then dialyzed

Polyacrylamide gel electrophoresis was performed

according to the method of Davis (1964) on 5% acrylamide
gel containing 0.005% glycogen at pH 8,3. Electrophoresis
was carried out at 2 mA/tube for 150 min at 4C. The
phosphorylase activity was detected by staining the gel with
iodine after incubation in 40 mM Tris-acetate (pH 7.0) containing 2 mM EDTA, 20 mM glycogen, 1.6 mM AMP and
50mM glucose-l-phosphate for 90min at 30C. Sodium
dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis
was performed as described in Weber and Osborn (1969).
The protein solution was treated with 1% SDS in the presence
of 1% 2-mercaptoethanol for 2 hr at 37C and subjected to
electrophoresis on 10% gel containing 0.1% SDS. Myosin
(212,000), ct2-macroglolbulin (170,000), fl-galactosidase
(116,000), transferrin (76,000) and glutamic dehydrogenase
(53,000) were used as standards. Protein was stained in
Coomassie Blue.
15

0.6_ 10

e-

g
e~

.O

<

I
50

100

150
Fraction

200

"

250

"

300

n -o

Fig. 1. Elution profile of phosphorylase b from mantle of Mytilus galloprovincialis on DEAE-sephacel,

(
) Absorbance at 280 nm; ( - - - ) enzyme activity (see text).

Mytilus glycogen phosphorylase b

35

0.2

E
-

I
I
l

~0.1
.Q

3~

<

0.0

<

I
0

0
50

25

Fraction

100

75
n _

Fig. 2. Gel filtration of phosphorylase b from mantle of Mytilus galloprovincialis on Sephacryl S-300.
(
) Absorbance at 280 nm; ( - - - ) enzyme activity (see text).
RESULTS

Purification and purity of the enzyme

The elution profiles from DEAE-Sephacel and
Sephacryl S-300 columns are shown in Figs 1 and 2,
respectively. The elution profile from AMP-Sepharose
4B column chromatography is not shown because
of the interference in the values of the absorbance at
280 nm caused by the high A M P concentrations in
the eluate of the column. However, no particularities
are present in this elution profile.
As is shown in Fig. 1, the enzymatic activity is
eluted in a peak, corresponding to the maximum of
activity to 0.26 M KC1. The final vol of the enzyme
solution in this purification step was 6.6 ml, with a
spec. act. of 58 10 -3 U/mg prot.
The elution of the enzymatic activity from the
AMP-Sepharose 4B column was in a single peak
and the fraction with higher activity was at 120 ml
from the beginning and after passing 20ml buffer
with A M P through the column. The vol of the active
solution recovered was 6.5 ml with a spec. act. of
209 10 -3 U/mg.
Figure 2 shows the elution profile from gel filtration
on Sephacryl S-300. It can be observed that the
enzymatic activity was eluted in a single peak. However, the graphic representation of the absorbance at
280 nm shows the presence of proteic contamination.
A typical purification scheme for glycogen phosphorylase is presented in Table 1. The final spec. act.
of the enzyme was approximately 0.4/zmol glucose-lphosphate formed min- ~mg p r o t - ~, which represents
a 70-fold purification of the protein relative to the

enzyme in the homogenate prepared from mantle of

mussels. The purity of the enzyme preparation was
examined by polyacrylamide gel electrophoresis in
native conditions and denaturing conditions in the
presence of 0.1% SDS (Fig. 3). In both cases the
sample exhibited a single protein band on the gel.
Furthermore, Fig. 3 shows that phosphorylase activity
was located on the gel at the same position as the
protein band. The purified enzyme is more unstable
and sometimes became visible as two or four bands,
all associated with glycogen phosphorylase activity.

212 K
170 K
116 K
76 K
53 K
m

Fig. 3. Polyacrytamide gel electrophoresis of purified phosphorylase b. (a) Activity stain; (b) protein stain; (c) SDSacrylamide gel purified phosphorylase; (d) SDS protein
standards.

Table 1. Summaryof the purificationprocedureof the glycogenphosphorylaseb from mantle tissueof Mytilus

galloprovincialis

Steps
Crude extract
30-60% (NH4)2 SO4 precipitate
DEAE-Sephacel
5'-AMP-Sepharose 4B
Sephacryl S-300

Volume
(ml)
100.800
16.367
6.656
6.525
9.299

Specific
Protein Activity activity Purification Yield
(rag)
(U)
(U x 10~/mg) factor
(%)
1139.0
6.026
5.287
1.000
100.00
379.3
59.4
12.1
5.5

4.544
3.452
2.546
2.044

11.959
58.045
209.696
371.449

2.262
10.979
39,663
70,257

75,40
57.28
42.26
33.92

loo

~ 50

I-I

l:\"

o
0

12

18

24

30

Days

Fig. 4. Effect of different storage conditions on purified phosphorylase b activity from Mytilus mantle.
(C)
C)) Control (4C); (&
&) I mg/ml BSA (4C); (A
A) 100 mM glycogen (4C); (O
O)
1.6 mM AMP (4C); (I-3
r-q) 50% glycerol (4C); (ll
I1) frozen to - 80C; (O
O) lyophilized.
100
Tyroglobulin ( ~
Ferritin c ~
~

Glycogen phosphorylase

o
X

Ovalbumin ~NN~ u
~J

Chymotripsinogen A 0

I
100

I
I
200
300
400
Vol. (ml)
Fig. 5. Molecular weight determination of mussel glycogen phosphorylase b by gel filtration chromatography on Sephacryl S-300.
100

Myosin
c~2- Mac roglobu li n ~ . . . ~

10
0~

~..~cog
en phosphorylase
13-Galactosidase
Transferrin
Glutarnic dehydrogenase ~ 0

_e
0

O.

0.2

0.4

0.6

Mobility

Fig. 6. Subunit mol. wt determination of glycogen phosphorylase b from Mytilus mantle by SDS-gel
electrophoresis. The mobility was calculated as,
M = distance of protein migration x length before staining
length after destaining
distance of dye migration"
36

Mytilus glycogen phosphorylase b

37

jO~O

100

100

o
o

O
>

.m

~s0

~50

>

._>

I11
e~

re

10

Temperature

pH

Fig, 7. Effect of pH on phosphorylase b activity from

Mytilus mantle. Temperature assay was carried out at 20C.
Aliquots of this purified protein solution were
utilized for study of the best conditions of storage,
Fig. 4. The enzyme was stable for several months at
- 8 0 C or lyophilized.

100

~s0
.>_
e,,,

/
/

I0

20

20
30
qO
50
Temperature (_oc)
Fig. 8. Effect of temperature on phosphorylase b activity
from Mytilus mantle. The enzyme activity was assayed for
20 rain at pH 7.0.

30

qo

50

(o_c)

Fig. 9. Effect o f temperature on stability o f phosphorylase

b from Mytilus mantle after incubation for 1 hr at indicated

temperatures.

Molecular weight
From gel filtration results, the tool. wt of glycogen
phosphorylase from mussel mantle was estimated
to be 340,000 + 5000 (Fig. 5), The same value was
obtained when the experiment was carried out with
an aliquot either from the crude extract or from the
purified protein. These results seem to suggest that
the purification procedure has no effect on the native
structure of the enzyme. On SDS-polyacrylamide gel
electrophoresis, the subunit mol. wt of purified phosphorylase was estimated to be 86,000 (Fig. 6). Thus,
the phosphorylase of mussel mantle was shown to be
present as a tetramer.

Effect of pH on activity
The effect of pH on this enzyme activity was
examined at 20C utilizing Tris-maleate and phosphate
buffers. As shown in Fig. 7, the optimum pH was
around 7.2.
Effect of temperature
Enzyme activity was assayed at various temperatures in the standard buffer at pH 7.0 after 20 rain
incubation (Fig. 8). Maximum activity was reached at
30C. The enzyme solution was incubated for 1 hr
between 15 and 50C. The residual activity of this enzyme decreased with increasing temperature (Fig. 9).
When the enzyme was incubated for 1 hr at 50C, the
residual activity was 0%.
Activation by A M P
The crude mantle extract showed residual phosphorylase activity without A M P (30% activity in the
presence of AMP). However, this residual activity

F. SAN JUAN SERRANOet al.

38
100

75

"'~ 50

U
,<

jo f

al

,,
25

0
-~

-2

-I

Iog

r A M P ] mM

Fig. 10. Effect of AMP concentration on phosphorylase b

activity from mantle tissue of Mytilus galloprovincialis.

decreased during the purification process to 14%.
The effect of AMP, in various concentrations, on the
activity of the purified enzyme is shown in Fig. 10.
The phosphorylase activity was stimulated 7-fold by
1.6 mM AMP. The K, value of this enzyme for A M P
was l I 4 # M .
DISCUSSION

Phosphorylase b was purified from the mantle

tissue of mussel to apparent homogenity as judged by
native- and SDS-polyacrylamide gel electrophoresis.
The enzyme was purified by successive chromatography on DEAE-Sephacel, AMP-Sepharose 4B
and gel filtration on Sephacryl S-300. At the end of
the process, an enzyme solution with a spec. act. of
371 x 10 -3 U/mg and a yield of 34% was obtained.
The purification factor was 70-fold. This purification
procedure joins different steps previously used by
several works in the purification of the glycogen phosphorylases from various animal sources, as indicated
in Material and Methods. However, in the course of
previous studies carried out to test the effectiveness of
the different steps in the purification procedure, the
inefficacy of mussel enzyme in the process normally
included in the purification schemes for the glycogen
phosphorylases from diverse organisms, such as
rough decrease in the pH value of the dissolution has
been demonstrated (Fischer and Krebs, 1958) or pH
increase and posterior incubation at 41C (Childress
and Sacktor, 1970). Both manipulations cause total
loss of activity in the Mytilus enzyme.
The glycogen phosphorylase present in the crude
extract, as well as the purified molecule, were both

AMP-dependent activity forms. Those results show

that the enzyme from mantle tissue of Mytilus is
primarily present as phosphorylase b.
The molecular weight of the enzyme obtained by
gel filtration studies was found to be 340,000 and
86,000 by SDS-electrophoresis, which suggests that
this enzyme is in a tetrameric form and that this could
be the normal structure of the molecule in Mytilus.
These results agree with the range of values mentioned in the references for the same enzyme from
different sources (De Vincenzi and Hedrick, 1967;
Cohen et al., 1971), but not with the theoretical
quaternary structure of phosphorylase b in vitro
(Dombrddi, 1981).
Different storage forms of the purified enzyme were
examined as shown in Fig. 4. The freezing and
lyophilizing maintain total phosphorylase activity for
several months.
Despite that the maximum rate for the enzyme was
obtained at 30C, the assay was carried out at 20C,
as at this temperature, the enzyme shows the highest
affinity for its principal substrates (data not shown).
In the crude extract, the phosphorylase showed
activity without addition of AMP, but the AMPindependent activity decreased during the purification
procedure and the purified phosphorylase preparation
showed very weak activity without addition of AMP.
The ratio of the AMP-independent activity to the
total activity of the enzyme decreased 1/7 at the final
step of purification. These results show that the phosphorylase of mussel mantle is primarily present as the
AMP-dependent form, phosphorylase b. The decrease
of A M P independent activity can be attributed to
either the removal of A M P or its inactivation by
dephosphorylation during the purification procedure
(unpublished results).
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