©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interaction between Glycogen Phosphorylase and Sarcoplasmic Reticulum Membranes and Its Functional Implications (*)

Ana Cuenda (§) , Manuel Nogues (§) , Fernando Henao , Carlos Gutiérrez-Merino (¶)

From the (1) Departamento de Bioqumica y Biologa Molecular, Facultad de Ciencias, Universidad de Extremadura, 06080 Badajoz, Spain

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-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.


INTRODUCTION

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)() 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).

Glycogen mediates the association of phosphorylase with the SR-glycogenolytic particle complex because digestion with -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).

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 pump of SR membranes.


MATERIALS AND METHODS

Preparation of Phosphorylase b, SR Membranes, and Ca-ATPase

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 (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 10M 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 HPOK, 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.

The Ca-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).

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) 10 and (0.86 ± 0.33) 10 µmol of product/min/mg of membrane protein (n = 10), respectively.

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 = 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 10M cm). Essentially, 1 volume of sample was mixed with 0.05 volume of 2% phenylhydrazine in 5 M NHSO 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).

The preparations of SR membranes contain glycogen (see ``Materials and ''Methods`` and Wanson and Drochmans(1972)). Since treatment with -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.

Fig. 3A shows that for purified phosphorylase b, the Kvalues 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 HPOK, 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 HPOK, 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 Kfor 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.

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 Kof 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 Kis 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 Kobtained 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 Kis 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 Kis 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.

The conversion of phosphorylase b to a produces dissociation of fully active phosphorylase from SR membranes since phosphorylase a binds 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.

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 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.

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-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.

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-polysaccharide complex showed that the polysaccharide synthesized is an -amylose type.

2) The V 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).

3) Endogenous phosphorylase b associated with SR membranes has a K 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.

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, 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

Values were obtained from titrations with glycogen, P, 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).



FOOTNOTES

*
This work was supported in part by Projects PB87-1020 and PB91-0311 from the Spanish Dirección General de Investigación Cientfica y Técnica. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a predoctoral fellowship from the Junta de Extremadura.

To whom correspondence should be addressed. Tel.: 34-24-289422; Fax: 34-24-271304.

The abbreviations used are: SR, sarcoplasmic reticulum; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; MES, 4-morpholineethanesulfonic acid; PLP, pyridoxal 5`-phosphate.

A. Cuenda, M. Nogues, F. Henao, and C. Gutiérrez-Merino, unpublished results.


ACKNOWLEDGEMENTS

We gratefully acknowledge Dr. Dario Alessi for critical reading of the manuscript and suggestions.


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