From the
Skeletal muscle glycogen phosphorylase b binds to
sarcoplasmic reticulum (SR) membranes with a dissociation constant of
1.7 ± 0.6 mg of phosphorylase/ml at 25 °C at physiological
pH and ionic strength. Raising the temperature to 37 °C produced a
2-3-fold decrease in the dissociation constant. The SR membranes
could bind up to 1.1 ± 0.1 mg of glycogen phosphorylase
b/mg of SR protein, whereas liposomes prepared with endogenous
SR lipids and reconstituted Ca
Glycogen phosphorylase (phosphorylase, EC 2.4.1.1) plays an
important role in controlling glycogen metabolism and in the cell is
found associated with glycogenolytic particles (Meyer et al.,
1970), which are associated with the sarcoplasmic reticulum
(SR)
Glycogen mediates the
association of phosphorylase with the SR-glycogenolytic particle
complex because digestion with
Because of the importance that these findings may
have in regulating muscle bioenergetics, we have studied the
association of phosphorylase with SR membranes under experimental
conditions approaching normal physiological conditions (concentrations
of phosphorylase and of SR membranes, pH, temperature, ionic strength,
and the allosteric activators of phosphorylase b, AMP and
IMP). In addition, we have investigated the kinetic properties of
phosphorylase b present in SR membranes prepared from skeletal
muscle and the effect of phosphorylase b on the Ca
The Ca
The AMP deaminase
and phosphoglucomutase activities of SR membrane preparations were
determined from measurements of the AMP concentrations at different
times as described (Cuenda et al., 1994). On average, the AMP
deaminase and phosphoglucomutase activities of our SR membrane
preparations were found to be (1.12 ± 0.55)
The correction of the effect of the
AMP deaminase activity on the activity of phosphorylase b in
the presence of SR membranes was done as follows. The decrease in AMP
concentration and the increase in IMP concentration at every SR
concentration at the given time of incubation were measured. By making
use of the saturation curves for the phosphorylase activity induced by
AMP and IMP, determined under the same experimental conditions, the
loss of activity due to partial desaturation of AMP or IMP (1 -
Y) was corrected using the following relationship:
activity
The preparations of SR
membranes contain glycogen (see ``Materials and
''Methods`` and Wanson and Drochmans(1972)). Since treatment
with
Fig. 3A shows
that for purified phosphorylase b, the
K
Scatchard plots of the
results obtained in a typical experimental series are presented in
Fig. 5
. The scatter of the data at low phosphorylase b concentrations is likely due to the variation in the content of
phosphorylase in the SR membrane preparations used in these experiments
(from 0.03 to 0.1 mg of phosphorylase/mg of total SR protein). It can
be readily observed that the data demonstrate that the binding of
phosphorylase b to SR membranes shows, at most, weak positive
cooperativity. Fig. 5illustrates that an SR membrane
concentration of 2 mg of protein/ml can bind up to 2.0-2.5 mg of
phosphorylase/ml and that the endogenous content of phosphorylase in
our SR preparations accounts for only 3-10% of the maximum
binding capacity of SR membranes. From the results shown in
Fig. 5
, a K
The conversion of
phosphorylase b to a produces dissociation of fully
active phosphorylase from SR membranes since phosphorylase a binds
The results reported in this paper show that binding of
phosphorylase b to SR membranes produces inhibition of the
activity of this enzyme and a severalfold increase in the
K
Inhibition of phosphorylase b was produced by SR membranes,
but not by other membrane preparations, such as erythrocyte membranes,
or by liposomes prepared from lipids extracted from these membranes
(Cuenda et al., 1991; data not shown). Therefore, the
inhibition of phosphorylase b by SR membranes must be caused
by its interaction with a non-lipid component of SR membranes. In spite
of the high content of Ca
1) The inhibition of the activity of
purified phosphorylase b by SR membranes is strongly
potentiated by incubation of SR membranes in a medium containing all
the components required for polysaccharide synthesis by the
phosphorylase associated with these membranes. In addition, the
spectral characteristics of the I
2) The V
3) Endogenous
phosphorylase b associated with SR membranes has a
K
The simplest hypothesis of
how phosphorylase b binds to the SR membranes in vitro is that it initially associates with glycogen particles on the
membrane, which act as anchoring points until new binding sites are
formed by the elongation of the polysaccharide. Light scattering
studies show that the SR membranes preferentially associate with low
branched polysaccharides,
Values were obtained from titrations with
glycogen, P
We gratefully acknowledge Dr. Dario Alessi for
critical reading of the manuscript and suggestions.
-ATPase were unable to
bind glycogen phosphorylase. Binding of glycogen phosphorylase b to SR membranes is accompanied by inhibition of its activity in
the presence of AMP. The V
for glycogen
phosphorylase b associated with SR membranes is 40 ± 5%
of that for purified glycogen phosphorylase and shows a decreased
affinity for its allosteric activators, AMP and IMP. These kinetic
effects are also observed with purified glycogen phosphorylase b when starch or
-amylose is used as substrate instead of
glycogen. Treatment of SR membranes with
-amylase produced
dissociation of glycogen phosphorylase b from the SR
membranes. Thus, linear polysaccharide fragments of glycogen bound to
the SR membranes are likely mediating the binding of glycogen
phosphorylase b to these membranes.
(
)
membrane (Wanson and Drochmans, 1972;
Busby and Radda, 1976; Entman et al., 1976, 1980). This
association increases the rate at which glycogen phosphorylase b kinase phosphorylates phosphorylase (Entman et al.,
1980). It also has been shown that phosphorylase is present in SR
membranes prepared using different procedures (Pickart and Jencks,
1982; Gutiérrez-Merino, 1983; Ross and McIntosh, 1987; Cuenda
et al., 1994). We have shown that binding of purified
phosphorylase to the SR membrane leads to partial loss of its catalytic
activity and that the phosphorylated form of phosphorylase
(phosphorylase a) binds with much lower affinity to SR
membranes than the dephosphorylated form (phosphorylase b)
(Cuenda et al., 1991). In addition, phosphorylase b can be dissociated from SR membranes by simple dilution and upon
dissociation is fully active, thus showing that this is a reversible
process (Cuenda et al., 1994).
-amylase largely decreases the
amount of enzyme bound to this complex (Meyer et al., 1970;
Wanson and Drochmans, 1972). Interestingly, it has been found that
glycogen associated with SR membranes is less branched compared with
glycogen particles (Wanson and Drochmans, 1968), but the functional and
physiological role of this observation is not known. Recent results
obtained in our laboratory support the view that the association of
glycogen phosphorolysis with SR membranes in skeletal muscle cells can
produce low amounts of ATP in the vicinity of these membranes, which
support Ca
transport into the SR (Cuenda et
al., 1993).
pump of SR membranes.
Preparation of Phosphorylase b, SR Membranes, and
Ca
Phosphorylase b and SR membranes were prepared from rabbit (New Zealand White)
back and hind leg skeletal muscle as described (Gutiérrez-Merino
et al., 1980, 1989). SR membranes were fractionated into heavy
and light fragments following the method of Meissner(1984, 1986). Based
on the SDS gel electrophoretic pattern (Laemmli, 1970), phosphorylase
b was found to be >98% pure and did not show a detectable
AMP deaminase activity (measured with 100 µg of protein/ml).
Ca-ATPase
-ATPase (EC 3.6.1.38) was purified from SR
membranes as described (Gutiérrez-Merino, 1987) and
reconstituted with egg lecithin or with lipids extracted from SR
membranes as described by Mata and Gutiérrez-Merino(1985) using
1 mg of lipid/mg of protein. The protein concentration was determined
using the method of Lowry et al.(1951) with bovine serum
albumin as a standard and, in the case of phosphorylase b,
also spectrophotometrically using a molar absorption coefficient at 280
nm of
= 1.29
10
M
cm
at pH 6.9
(Buc et al., 1971).
Determination of Glycogen Content of SR
Preparations
The glycogen concentration of SR membranes was
measured using the phenol sulfuric method (Dubois et al.,
1956) with glycogen as a standard and was found to be on average 32
± 10 µg of glycogen/mg of membrane protein (n = 10).
Enzyme Assays
The phosphorylase activity was
measured in the direction of both glycogen synthesis and degradation as
described by Cuenda et al.(1991, 1994). In the synthesis mode,
the assay medium contained 10 mM glucose 1-phosphate, 0.45
g/liter glycogen, 0.1 mM AMP, 1 mM EGTA, 150
mM KCl, 10 mM TES (pH 7.4) or 10 mM MES (pH
6.0), and 0.1 mg/ml purified phosphorylase b. The inorganic
phosphate released was determined following the method of Fiske and
SubbaRow(1925) as described by Leloir and Cardini(1957). Unless stated
otherwise, the initial activity in the direction of glycogen synthesis
was measured after a 1-min reaction. The phosphorylase activity in the
direction of glycogen phosphorolysis was measured in the following
assay medium: 12 mM HPO
K, 10
mM magnesium acetate, 0.63 mM NADP
,
50 mM imidazole (pH 6.9), 1 mM AMP, 0.45 g/liter
glycogen, 25 µg of phosphoglucomutase, and 15 µg of
glucose-6-phosphate dehydrogenase plus purified phosphorylase b (
1 µg/ml) or SR membranes (30-50 µg of
protein/ml). Each data point of phosphorylase activity in the figures
is the average of triplicate experiments done with at least three
different preparations of phosphorylase b or of SR membranes,
and the error bars shown are the standard errors (n
9).
When needed, the concentration of glucose 1-phosphate in the medium
used for phosphorylase activity was measured from the increase in
absorbance at 340 nm after the addition of 0.63 mM
NADP
, 25 µg of phosphoglucomutase, and 15 µg
of glucose-6-phosphate dehydrogenase by interpolation in the
calibration line obtained with solutions of known concentrations of
glucose 1-phosphate.
-ATPase activity was
measured at 25 °C using the coupled enzyme system pyruvate
kinase/lactate dehydrogenase (Warren et al., 1974) as
described (Gutiérrez-Merino et al., 1989; Cuenda et
al., 1990), with the following assay medium: 100 mM TES
(pH 7.4), 0.1 M KCl, 0.1 mM CaCl
, 5
mM MgCl
, 35 µg of pyruvate kinase, 30 µg
of lactate dehydrogenase, 0.25 mM NADH, 0.42 mM
phosphoenolpyruvate, and 2.5 mM ATP. On average, the
Ca
-dependent ATPase activity of our preparations
assayed in the presence of calcimycin (0.1 µg/µg of protein)
ranged from 3 to 5 µmol of ATP hydrolyzed per min/mg of protein.
The Ca
-independent ATPase activity was measured in
the presence of 3.5 mM EGTA and was <10% of total uncoupled
ATPase activity. The steady-state level of calcium accumulation was
monitored using arsenazo III as a metallochromic dye as described
(Fernández-Salguero et al., 1990).
10
and (0.86 ± 0.33)
10
µmol of product/min/mg of membrane protein (n = 10), respectively.
= activity
(1/Y).
Determination of Phosphorylase Content in SR
Membranes
Pyridoxal 5`-phosphate (PLP) was resolved from
phosphorylase following the method of Strausbauch et al. (1967) with minor modifications, and PLP was taken as a
measurement of phosphorylase content of SR membranes because no other
proteins in the SR membranes contain PLP (Cuenda et al.,
1994). Briefly, to resolve PLP from phosphorylase, the enzyme was
treated with 0.6 M perchloric acid for 30 min at 30 °C
with mild stirring, and the sample was centrifuged for 30 min at 15,000
g. The concentration of PLP in the supernatant was
determined using absorbance measurements at 410 nm of the
PLP-phenylhydrazone derivative (
= 3
10
M
cm
).
Essentially, 1 volume of sample was mixed with 0.05 volume of 2%
phenylhydrazine in 5 M NH
SO
and then
allowed to stand for 30 min before taking absorbance readings. The
absorbance of samples of reconstituted Ca
-ATPase with
endogenous SR lipids treated as described above was used as the blank
value in measurements of phosphorylase bound to SR membranes and was
found to be <0.005 A at 410 nm (20 nmol of PLP gives an
absorbance reading of 0.110 ± 0.005 A).
Chemicals
Bovine serum albumin, AMP, ATP,
-amylose, PLP, phosphoenolpyruvate, EGTA, phenylmethylsulfonyl
fluoride,
-mercaptoethanol, sodium dodecyl sulfate, Sephadex G-50,
glucose 1-phosphate, glucose 6-phosphate, Trizma (Tris base), and TES
were obtained from Sigma. Glycogen, NADH, NADP
,
calcimycin,
-amylase, pyruvate kinase (200 IU), lactate
dehydrogenase (550 IU), phosphoglucomutase (200 IU), and
glucose-6-phosphate dehydrogenase (350 IU) were purchased from
Boehringer Mannheim. Starch (70-80% amylopectin and 20-30%
-amylose) and all other chemicals used in this study were obtained
from Merck.
RESULTS
Inhibition by SR Membranes of the Activity of Purified
Phosphorylase b Depends on the time of Preincubation of SR Membranes
with the Reaction Mixture
The activity of purified phosphorylase
b is inhibited when the enzyme is incubated with SR membranes
at 25 °C (Cuenda et al., 1991). The inhibition is fully
reversed by dilution of the phosphorylase b/SR membrane
mixture, thus showing that it is a reversible process. This excluded
the possibility that the inhibition could be due to irreversible
processes, such as proteolysis and/or denaturation of phosphorylase
b. Increasing the temperature of the incubation medium from 25
to 37 °C enhances the inhibition of purified phosphorylase b by SR membranes (Fig. 1A). This result indicates
that the inhibition of phosphorylase b activity by SR
membranes is stronger at physiological temperatures than at 25 °C.
In addition, the inhibition appears to be insensitive to physiological
pH changes (6.0 and 7.4) (Fig. 1B). This inhibition is
specific for SR membranes because neither incubation with liposomes
from egg lecithin or from SR lipids nor incubation with plasma membrane
vesicles from erythrocytes produces a significant inhibition of
phosphorylase b activity (Cuenda et al.,
1991).(
)
The
Ca
,Mg
-ATPase is the major protein
component of the SR membrane, accounting for 70-80% of total SR
protein (Andersen, 1989). However, preincubation of SR membranes with
purified phosphorylase b (up to 8 mg/ml) had no effect on the
Ca
-ATPase activity, on Ca
binding
to SR membranes, or on Ca
uptake by these membranes
at pH 7 (data not shown). This and the lack of inhibition of
phosphorylase b by incubation with Ca
-ATPase
reconstituted in egg lecithin (data not shown) suggest that interaction
of phosphorylase b with another component(s) of the SR
membrane should be producing the observed inhibition of its activity.
Figure 1:
Effect of temperature and pH on the
activity of purified phosphorylase b bound to SR membranes.
A, effect of temperature. After a 15-min preincubation of SR
membranes in the assay medium (10 mM TES (pH 7.4), 0.15
M KCl, 10 mM glucose 1-phosphate, 0.45 g/liter
glycogen, 1 mM EGTA) at 25 °C () or 37 °C
(
), the phosphorylase activity was measured at 25 or 37 °C,
respectively, after the addition of 0.1 mM AMP and 0.1 mg/ml
phosphorylase b. The activity in the absence of SR membranes
was taken as 100%. B, effect of pH. After a 15-min
preincubation of SR membranes at 25 °C in the assay medium
described for A at pH 7.4 (
) or in 10 mM MES (pH
6.0) (
), 0.15 M KCl, 10 mM glucose 1-phosphate,
0.45 g/liter glycogen, 1 mM EGTA, the phosphorylase activity
was measured as described for A.
Fig. 2
shows that the extent of inhibition of purified
phosphorylase b by SR membranes is dependent on the time of
incubation of SR membranes with the assay medium not containing AMP,
prior to the addition of purified phosphorylase b. The time
taken to achieve 50% inhibition (t) is linearly dependent on
the concentration of SR membranes in the reaction medium (Fig. 2,
inset). This inhibition is dependent on the presence of
glucose 1-phosphate since if it is omitted from the incubation medium,
no time-dependent inhibition of phosphorylase b activity was
observed in the presence of up to 4 mg of SR protein/ml. Withdrawal of
any other of the components of the incubation medium did not
significantly alter the time course of inhibition shown in
Fig. 2
. The inhibition of phosphorylase b activity by
preincubation of SR membranes with the assay medium cannot be explained
in terms of glucose 1-phosphate depletion because the concentration of
glucose 1-phosphate only decreased from 10 to 9.9 mM after a
120-min incubation with SR membranes (2 mg of protein/ml), a decrease
that would account for <5% inhibition. In addition, this
time-dependent inhibition was also observed in the presence of
saturating concentrations (30 mM) of glucose 1-phosphate. A
small amount of phosphoglucomutase activity is associated with the SR
membranes (Cuenda et al., 1994). Since glucose 6-phosphate is
a well known allosteric inhibitor of phosphorylase b (Graves
and Wang, 1972), we considered the possibility that this time-dependent
inhibition could be due to glucose 6-phosphate production from glucose
1-phosphate. As shown in Fig. 2, production of glucose
6-phosphate cannot account for the time-dependent inhibition observed.
Figure 2:
Effect of preincubation of SR membranes
with the substrates needed for glycogen synthesis on the activity of
purified phosphorylase b. The assay medium contained 10
mM TES (pH 7.4), 0.15 M KCl, 10 mM glucose
1-phosphate, 0.45 g/liter glycogen, 1 mM EGTA, and 1
(circles), 2 (squares), or 4 (triangles) mg
of SR protein/ml. SR vesicles were incubated at 25 °C for different
times in the assay medium, and the reaction was started by the addition
of 0.1 mM AMP and 0.1 mg of phosphorylase b/ml. The
100% value of phosphorylase b activity ranged from 20 to 25 IU
in different experiments. Filled and opensymbols correspond to uncorrected experimental data and data corrected for
the inhibition caused by glucose 6-phosphate accumulated during
preincubation of SR membranes, respectively. This correction was done
as follows. We measured the phosphoglucomutase activity of our SR
membrane preparations as described under ``Materials and
Methods'' and also the glucose 6-phosphate concentration at
different incubation times. With these data and the titration curve of
phosphorylase b activity with glucose 6-phosphate, the
time-dependent inhibition was corrected for the contribution of
inhibition by glucose 6-phosphate. Each data point is the average of
triplicate experiments and has an average standard deviation of
approximately ±5%. Inset, dependence of the t value for the inhibition of the activity of phosphorylase b on the concentration of SR membranes in the incubation
medium.
Linear Polysaccharides Inhibit the Activity of Purified
Phosphorylase b with Glycogen as Substrate
Despite the absence
of AMP in the reaction medium used in the incubation of SR membranes,
there is a low but significant phosphorylase activity in the direction
of glycogen synthesis ((0.40 ± 0.02) 10
µmol/min/mg of SR protein (n = 10)). Taking
into account that the SR membrane preparations used in this study
contain <0.1 mg of phosphorylase b/mg of SR protein (Cuenda
et al., 1994; see below), this activity is close to that
reported elsewhere for purified phosphorylase b in the absence
of AMP, 0.1 IU (Madsen and Shechosky, 1967). The polysaccharide
synthesized by phosphorylase b activity is linear (Graves and
Wang, 1972), and the spectral characteristics of the complex of the
polysaccharide synthesized by SR membranes with I
confirmed
this point and the lack of glycogen branching activity in these
membranes (Cuenda et al., 1994).
-amylase dissociates endogenous phosphorylase b from
SR membranes (Cuenda et al., 1994), we considered the
possibility that inhibition of purified phosphorylase b by SR
membranes could be due to interaction with linear polysaccharide
fragments associated with these membranes. The kinetic properties of
purified phosphorylase b with starch and
-amylose as
substrates support this hypothesis.
values obtained with glycogen, starch,
and
-amylose are approximately the same. However, the
V
values for purified phosphorylase b with starch (16 ± 2 IU) and with
-amylose (7 ±
1 IU) were much lower than the V
that we
obtained with glycogen (28 ± 3 IU). In addition, the
K
values with starch and
-amylose as
substrates obtained from Hill plots of the data of Fig. 3B (170 ± 20 and 220 ± 20 µM,
respectively) are severalfold higher than the K
with glycogen as substrate (30 ± 5 µM)
(Fig. 3B and ).
Figure 3:
Phosphorylase activity with -amylose,
glycogen, and starch as substrates. The phosphorylase activity was
measured with purified phosphorylase b (
1µg/ml) at 30
°C with
-amylose (
), glycogen (
), or starch
(
) as substrate. A, titrations with polysaccharides under
the following experimental conditions: 12 mM
H
PO
K, 1 mM AMP, 10 mM
magnesium acetate, 0.63 mM NADP
, 50
mM imidazole (pH 6.9), 25 µg of phosphoglucomutase, and 15
µg of glucose-6-phosphate dehydrogenase. B, titrations
with AMP using the assay medium described for A and 0.45 mg/ml
polysaccharide.
The SR preparations used
in this study have a phosphorylase activity in the presence of 1
mM AMP between 0.3 and 0.75 µmol of glucose 1-phosphate
released per min/mg of SR membrane protein. At least 95% of this
activity is in the unphosphorylated b form since this activity
depends on the presence of AMP (Cuenda et al., 1994). The
content of phosphorylase in these membranes was measured as described
under ''Materials and Methods`` by assaying the content of
PLP and also by using polyclonal antibodies against phosphorylase. The
values obtained ranged from 0.03 to 0.1 mg of phosphorylase/mg of
membrane protein. Therefore, we obtained a specific V at 30 °C and pH 6.9 for phosphorylase b bound to SR
membranes of 12 ± 6 IU (n = 14). This
V
is clearly lower than that for soluble and
purified enzyme (25-30 IU) and close to the V
obtained using starch as substrate instead of glycogen
(Fig. 3A).
Kinetic Properties of Phosphorylase Bound to SR
Membranes
If the inhibition of phosphorylase b activity
by SR membranes is due to the use of low branched polysaccharide
associated with SR membranes instead of glycogen as substrate, the
dependence of the activity on AMP concentration should be altered (see
above). Fig. 4and show that the activation by AMP
and IMP of the phosphorylase b activity associated with SR
membranes is shifted to higher nucleotide concentrations. The apparent
K was 100 ± 10 µM for
phosphorylase b associated with SR membranes. This is
3-fold higher than that obtained for the purified enzyme (30
± 5 µM) with glycogen as substrate
(Fig. 4A), and it is closer to the
K
with starch as substrate, 170 ± 20
µM (Fig. 3B). The slope of the Hill plot of
the dependence of the activity on AMP concentration was 1.7-1.8
and therefore shows characteristics of positive cooperativity in the
activation process, like purified phosphorylase b (Morange
et al., 1976). Similar results were obtained from the analysis
of the activation by IMP of phosphorylase b bound to SR
membranes (Fig. 4B).
Figure 4:
Dependence on AMP and IMP of the
phosphorylase activity of SR membranes and of purified phosphorylase
b. The phosphorylase activity was measured at 30 °C under
the following experimental conditions: 12 mM
HPO
K, 10 mM magnesium acetate, 0.63
mM NADP
, 50 mM imidazole (pH 6.9),
0.45 g/liter glycogen, 25 µg of phosphoglucomutase, and 15 µg
of glucose-6-phosphate dehydrogenase plus either purified phosphorylase
b (
1 µg/ml) (
) or isolated SR membranes
(30-50 µg of protein/ml) (
) and the AMP and IMP
concentrations indicated on the abscissae of A and
B, respectively.
shows that
association of phosphorylase b with SR membranes had no effect
on the Kvalues for P
and
glycogen and on the K
for glucose
6-phosphate. If AMP causes the dissociation of phosphorylase b from the SR membranes, this can account for the apparent lack of
effect of SR membranes on these kinetic parameters since they were
obtained under saturating concentrations of AMP. Therefore, we measured
the dissociation constant for purified phosphorylase b from SR
membranes in the absence and presence of AMP, and the results obtained
are presented below.
Binding of Purified Phosphorylase b to SR
Membranes
SR membranes (2 mg of protein/ml) were incubated with
different concentrations of purified phosphorylase b (0.5-15 mg/ml) at 25 °C in 10 mM TES/KOH (pH
7.4), 0.1 M KCl. After a 15-min incubation, SR membranes were
pelleted by centrifugation at 35,000 g for 90 min at
25 °C. In control experiments, we demonstrated that SR membranes
were completely pelleted by this procedure because no significant
Ca
-ATPase activity was measured in the supernatant.
Furthermore, purified phosphorylase b under these conditions
remained in the supernatant, as shown by the fact that the
phosphorylase activity in the supernatant was identical before and
after centrifugation in the absence of added SR membranes. The
concentration of phosphorylase b in the supernatant (free
phosphorylase b) was determined by measuring the concentration
of PLP as described under ''Materials and Methods.`` The
concentration of phosphorylase b bound to the membrane was
calculated as the difference between total and free phosphorylase
b. Identical values were obtained by directly measuring the
concentration of PLP in the membrane pellet.
of 1.7 ± 0.6 mg
of phosphorylase b/ml was obtained, a value that is well below
the phosphorylase concentration in skeletal muscle cells (
10
mg/ml) (Fischer et al., 1978). This K
is close to the concentration of SR membranes producing 50% of
the maximum inhibition of phosphorylase activity in titrations of
purified phosphorylase b with SR membranes
(Fig. 1A). It is noteworthy that the inhibition of
phosphorylase b by light, heavy, and unfractionated SR
membranes was found to be the same within experimental error (data not
shown).
Figure 5:
Scatchard plots for the binding of
phosphorylase b to SR membranes. Scatchard plots are shown of
the data obtained at 25 °C after a 15-min incubation of SR
membranes (2 mg of protein/ml) with different phosphorylase b concentrations in 10 mM TES (pH 7.4), 0.1 M KCl
in the absence () and presence (
) of 1 mM AMP. See
``Results'' for further experimental details. Each data point
is the average with the standard error (n
10)
corresponding to an experimental series carried out with at least
triplicate phosphorylase b and SR membrane preparations.
Solid and brokenlines are included to
illustrate (from abscissaintersections) the most
probable range of phosphorylase b binding to SR membranes at
saturation.
Fig. 5
shows that in the presence of saturating AMP
concentration (1 mM), the dissociation constant for
phosphorylase b from SR membranes is 3-fold higher than
the one obtained in its absence. Therefore, if inhibition of
phosphorylase b is a consequence of its binding to SR
membranes, it can be predicted that the K
obtained from titrations of the activity of purified
phosphorylase b with SR membranes should be dependent on the
concentration of AMP. Plotted in Fig. 6are the results obtained
from these titrations in the presence of the following AMP
concentrations: 0.05, 0.1, and 0.5 mM, which produced
endogenous phosphorylase activities of SR membranes of
25, 50, and
95% of the V
at saturation by AMP, respectively
(Fig. 4). An
3-fold increase in the
K
is produced when the AMP concentration
is raised from 0.05 to 0.5 mM, in good agreement with the
conclusions derived from the Scatchard plots in Fig. 5.
Fig. 5
also shows that in the presence of AMP, the SR membranes
can bind at saturation approximately twice the amount of phosphorylase
b than in the absence of AMP. This latter result can be simply
rationalized taking into account that AMP shifts the dimer
tetramer equilibrium toward the tetramer of phosphorylase b (Merino et al., 1976).
Figure 6:
Effect of AMP on the activity of
phosphorylase in the presence of different concentrations of SR
membranes. The phosphorylase activity was measured at 25 °C with
0.1 mg of phosphorylase/ml in the following assay medium: 10
mM glucose 1-phosphate, 0.15 M KCl, 1 mM
EGTA, 0.45 g/liter glycogen, and 10 mM TES (pH 7.4) plus the
concentrations of SR membranes indicated on the abscissa and
0.05 mM (), 0.1 mM (
), or 0.5 mM
(
) AMP. The SR membranes were preincubated for 15 min with the
assay medium (minus AMP), and the reaction was started by the addition
of 0.1 mg/ml phosphorylase b and the corresponding
concentration of AMP. The effect of the AMP deaminase activity on
phosphorylase activity was corrected for as described under
``Materials and Methods.''
DISCUSSION
The association of phosphorylase b with SR membranes
is reversible, and upon dissociation, phosphorylase is fully active
(Cuenda et al., 1994), suggesting that SR membranes can act as
a phosphorylase b reservoir in skeletal muscle cells. The
results presented in this paper support this hypothesis since the
dissociation constant (K) for
phosphorylase b from the SR membranes determined in this study
at 25 °C at physiological pH and ionic strength is 1.7 ± 0.6
mg of phosphorylase b/ml. A 2-3-fold decrease in the
K
is produced by raising the temperature
from 25 to 37 °C. Owing to the high content of phosphorylase in
skeletal muscle cells (
10 mg/ml) (Fischer et al., 1978;
Varsanyi and Heilmeyer, 1981) and to the large membrane network of SR
in these cells (>1.5 mg of protein/ml) (MacLennan, 1970), it can be
estimated that a large fraction of phosphorylase b must be
bound to SR membranes in vivo.
10-fold more weakly than phosphorylase b (Cuenda et al., 1991). In addition, we show in this study
that the association constant for phosphorylase b with SR
membranes is 3-fold lower in the presence of AMP, the allosteric
activator of phosphorylase b, which produces a conformational
change in this enzyme close to that observed upon phosphorylation
(Barford et al., 1991). On the basis of in vivo concentrations of relevant metabolites, it has been estimated that
in normal skeletal muscle during contraction,
10-14% of
total phosphorylase activity can be attributed to the allosteric
activation of this enzyme by AMP (Busby and Radda, 1976). It is
noteworthy, however, that the effect of compartmentation of
phosphorylase was not taken into account when doing these calculations.
of activation by AMP and IMP. As a result, the
allosteric activation of phosphorylase bin vivo by
nucleotides (AMP and IMP) must be lower than previously assumed, at
least by a factor of 2. Thus, the association of phosphorylase with SR
membranes will result in a more efficient cutoff of glycogenolysis
after muscle contraction ceases. The association of AMP deaminase with
SR membranes (Cuenda et al., 1994) should also contribute to
this end since this will result in even further decreases in the local
AMP concentration near phosphorylase b bound to SR membranes.
-ATPase in SR membranes,
purified Ca
-ATPase reconstituted with egg lecithin or
endogenous SR lipids failed to produce inhibition of phosphorylase
(data not shown). SR membrane preparations contain 32 ± 10
µg of glycogen/mg of protein, enriched in linear polysaccharide
fragments with respect to glycogen purified from skeletal muscle (see
''Materials and ``Methods'' and Wanson and
Drochmans(1968)). It is likely that this glycogen is tightly bound to
the glycogen-targeting subunit of protein phosphatase-1,
PP1
, a protein component of the SR membranes (Hubbard and
Cohen, 1993). Because treatment of SR membranes with
-amylase
promotes dissociation of phosphorylase b from the SR membrane
(Wanson and Drochmans, 1972; this paper), we considered the possibility
that the inhibition of phosphorylase is a consequence of binding to
linear polysaccharide fragments tightly associated with SR membranes.
This hypothesis is supported by the following experimental results
reported in this paper.
-polysaccharide complex
showed that the polysaccharide synthesized is an
-amylose type.
for purified phosphorylase b with
-amylose as substrate (7 ± 1 IU) is only 25% of
the V
with glycogen (28 ± 3 IU). With
starch, the V
(16 ± 2 IU) is about half
the V
with glycogen, and it is close to the
activity obtained for phosphorylase b associated with SR
membrane preparations (12 ± 6 IU).
severalfold higher than that for purified
phosphorylase b with glycogen as substrate. This is also seen
with purified phosphorylase b when
-amylose or starch is
used as substrate instead of glycogen.
and this is consistent with the
fact that glycogen associated with the SR membranes has long end chains
with a mean length of 16-18 glucose units (Wanson and Drochmans,
1968). Since the V
for phosphorylase b is much lower when
-amylose is used as substrate instead of
glycogen, binding to the SR membranes results in a high level of
inhibition of phosphorylase activity measured in the presence of
glycogen. From the results obtained with purified phosphorylase b and
-amylose as substrate, a maximum inhibition of
75-80% should be expected after a long preincubation of the SR
membranes with the assay medium, which allows for a large elongation
(through
-1,4-glycosidic bonds) of the end chains of the glycogen
associated with the SR membranes. The maximum inhibition experimentally
measured (80-90%) is in good agreement with this prediction.
Neither the Ca
dependence nor the V
for the Ca
pumping activity of SR membranes
mediated by the Ca
-ATPase is altered by this
association.
Table: Kinetic constants for substrates and ligands
of phosphorylase
, AMP, or IMP at 30 °C in 10 mM magnesium acetate, 0.63 mM NADP
, 50
mM imidazole (pH 6.9), 25 µg of phosphoglucomutase, and 15
µg of glucose-6-phosphate dehydrogenase plus phosphorylase b (1 µg/ml) or SR membranes (30-50 µg of protein/ml).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.