Phosphorylase removes glucosyl units from the terminal branches of glycogen through phosphorolysis, forming glucose-1-P. It is present in two interconvertible forms, phosphorylase α and b. The α form is the active form and is rate limiting in glycogen degradation. The activities of phosphorylase α and of total phosphorylase as conventionally measured exceed the activities of glycogen synthase R (active form) and of total synthase by ~10-and 20-fold. Thus, unless phosphorylase α is inhibited or compartmentalized or its substrates are exceedingly low in vivo, net glycogen synthesis could not occur. In addition, following an administered dose of glucose, phosphorylase α activity changes little when glycogen is being synthesized, is stable, or is being degraded, suggesting an important role for allosteric effectors in regulation. Therefore, we have determined the effect of potential modifiers of enzyme activity at estimated intracellular concentrations. Purified liver phosphorylase α was used. Activity was measured in the direction of glycogenolysis, at 37°C, pH 7.0, and under initial rate conditions. Both a K(m) and a near-saturating concentration of inorganic phosphate (substrate) were used in the assays. A physiological concentration of AMP was saturating. It decreased the K(m) for P(i) by ~50% and stimulated activity. ADP, ATP, and glucose inhibited activity. Fructose-1-P inhibited activity only at a high and nonphysiological concentration. Glucose-6-P and UDP-glucose were not significant inhibitors. Inhibition of activity by ADP was little affected by the addition of AMP. However, AMP partially abolished the inhibitory effect of ATP and completely abolished the inhibitory effect of glucose. When AMP, ADP, ATP, glucose-6-P, UDP-glucose, glucose, and fructose-1-P were added together, the net effect was no change in phosphorylase α activity compared to the activity without any effectors. In addition, changes in glucose concentration did not affect activity. K glutamine modestly stimulated activity. Numerous other metabolites were tested and were without effect. The present data indicate that the known endogenous allosteric effectors cannot explain the smaller than expected in vivo phosphorylase α activity or the regulation of phosphorylase α activity.
Bibliographical noteFunding Information:
Supported by Merit Review Research Funds from the Department of Veterans Affairs and by Grant DK43018 from the National Institutes of Health. The authors thank Claudia Durand for her expert secretarial assistance.
- physiological regulation