ALLOSTERIC REGULATION OF LIVER PHOSPHORYLASE A - REVISITED UNDER APPROXIMATED PHYSIOLOGICAL CONDITIONS

Phosphorylase removes glucosyl units from the terminal branches of gly cogen through phosphorolysis, forming glucose-1-P. It is present in tw o interconvertible forms, phosphorylase a and b. The a form is the act ive form and is rate limiting in glycogen degradation. The activities of phosphorylase a and of total phosphorylase as conventionally measur ed exceed the activities of glycogen synthase R (active form) and of t otal synthase by similar to 10- and 20-fold. Thus, unless phosphorylas e a is inhibited or compartmentalized or its substrates are exceedingl y low in vivo, net glycogen synthesis could not occur. In addition, fo llowing an administered dose of glucose, phosphorylase a activity chan ges little when glycogen is being synthesized, is stable, or is being degraded, suggesting an important role for allosteric effecters in reg ulation. Therefore, we have determined the effect of potential modifie rs of enzyme activity at estimated intracellular concentrations. Purif ied liver phosphorylase a was used. Activity was measured in the direc tion of glycogenolysis, at 37 degrees C, pH 7.0, and under initial rat e conditions. Both a K-m and a near-saturating concentration of inorga nic phosphate (substrate) were used in the assays. A physiological con centration of AMP was saturating. It decreased the K-m for P-i by simi lar to 50% and stimulated activity. ADP, ATP, and glucose inhibited ac tivity. Fructose-1-P inhibited activity only at a high and nonphysiolo gical concentration. Glucose-6-P and UDP-glucose were not significant inhibitors. Inhibition of activity by ADP was little affected by the a ddition of AMP. However, AMP partially abolished the inhibitory effect of ATP and completely abolished the inhibitory effect of glucose. Whe n AMP, ADP, ATP, glucose-6-P, UDP-glucose, glucose, and fructose-1-P w ere added together, the net effect was no change in phosphorylase a ac tivity compared to the activity without any effecters. In addition, ch anges in glucose concentration did not affect activity. K glutamine mo destly stimulated activity. Numerous other metabolites were tested and were without effect. The present data indicate that the known endogen ous allosteric effecters cannot explain the smaller than expected in v ivo phosphorylase a activity or the regulation of phosphorylase a acti vity. (C) 1996 Academic Press, Inc.