A study on the autoactivation of rabbit muscle phosphorylase kinase.
Under conditions favoring its autocatalytic reaction, phosphorylase kinase may be activated and phosphorylated in 2-(N-morpholino)ethanesulfonate (Mes) buffer to a much higher level than in beta-glycerophosphate buffer. The fact that the reaction is autocatalytic is supported by several observations: (a) the progress curve of the reaction exhibits a pronounced lag phase, (b) the reaction is strongly inhibited by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetate, which inhibits phosphorylase kinase, (c) the pH profile of the reaction resembles that of the phosphorylase b to a reaction as catalyzed by nonactivated phosphorylase kinase, and (d) the reaction is not significantly affected by adenosine 3':5'-monophosphate (cAMP) nor by the heat-stable protein inhibitor of cAMP-dependent protein kinases. When fully autoactivated, phosphorylase kinase possesses an activity that is 100% higher than that of the protein kinase-activated form. The results suggest that autophosphorylation of phosphorylase kinase may be an important regulatory mechanism. The autocatalytic reaction involves phosphorylation of the two larger subunits of phosphorylase kinase, i.e. subunits A and B, with a combined total of 7 to 9 phosphates incorporated per mol of enzyme. Although the cAMP-dependent protein kinase also catalyzes the phosphorylation of subunits A and B, the two mechanisms of phosphorylation appear to involve different sites. Prior phosphorylation of phosphorylase kinase by the protein kinase has little effect on the level of autophosphorylation. Thus activation of phosphorylase kinase may be brought about by phosphorylation of the enzyme at different sites.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002021:Buffers; D002118:Calcium / Q000494:pharmacology; D004789:Enzyme Activation; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009132:Muscles / Q000201:enzymology*; D010764:Phosphorylase Kinase / Q000378:metabolism*; D011817:Rabbits
D002021:Buffers; D000255:Adenosine Triphosphate; D010764:Phosphorylase Kinase; D008274:Magnesium; D002118:Calcium