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 degrees C at physiological pH and ionic strength. Raising the temperature to 37 degrees 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(2+)-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 Vmax 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 alpha-amylose is used as substrate instead of glycogen. Treatment of SR membranes with alpha-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.
Highlights
From the Departamento de Bioquimica y Biologia Molecular, Facultad de Ciencias, Uniuersidad de Extremadura, 06080 Badajoz, Spain
This result indicates that the inhibition of phosphorylase b activity by sarcoplasmic reticulum (SR) membranes is stronger at physiological temperatures than at 25 °C
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)
Summary
From the Departamento de Bioquimica y Biologia Molecular, Facultad de Ciencias, Uniuersidad de Extremadura, 06080 Badajoz, Spain. The Vmax 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. 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). 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 Ca2 + transport into the SR (Cuenda et al, 1993)
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