Effects of Adenylic Acid on the Kinetics of Muscle Phosphorylase A.

It has long been known that muscle phosphorylase b has a strict requirement for adenylic acid (5’-AMP) (1). Phosphorylase a, on the other hand, is active without 5’-AMP (2) and although 5’-AMP may increase velocity somewhat, the increase according to most reports is not more than 50%. In connection with the use of phosphorylase and glycogen to measure low levels of inorganic orthophosphate, it was noticed that rates at 38” were very slow unless 5’-AMP was present. In exploring this further, it has been found that 5’-AMP can increase phosphorylase a activity 40 times or more when levels of glycogen, Pi, or glucose l-phosphate are low. At 25” the 5’-AMP concentration necessary to produce half-maximal stimulation is very small, less than 1% of that required for phosphorylase b and only 10% of the concentration found by Cori, Cori, and Green (2) to produce halfmaximal increments in phosphorylase a activity at high substrate levels. An increase in temperature to 38” markedly increases the requirement for 5’-AMP and has a profound effect on the kinetic parameters of the enzyme even in the presence of 5’-AMP. With both glycogen and Pi at low levels, the temperature coefficient actually becomes negative above 25” with or without 5’-AMP present. The studies with low substrate levels have also shown an unusual degree of interdependence between the Michaelis constant for one substrate and the concentration of the other. The fact that the rather striking effects of 5’-AMP on muscle phosphorylase a were not observed earlier is ascribed (a) to the fact that most measurements have been made at room temperature and (b) to the use of high substrate concentrations in most studies. By the use of highly sensitive fluorometric procedures, it was possible to make valid kinetic measurements at very low substrate levels.