Effect of Mg2+ concentration on the cAMP-dependent protein kinase-catalyzed activation of rabbit skeletal muscle phosphorylase kinase.
Phosphorylase kinase was found to be activated and phosphorylated at 10mM Mg2+ by the cAMP-dependent protein kinase-catalyzed reaction ot much higher levels than observed previously when reactions were carried out in 1 to 2 mM Mg2+ (Cohen, P. (1973) Eur. J. Biochem. 34, 1; Hayakawa, T., Perkin, J.P., and Krebs, E.G. (1973) Biochemistry 12, 574). That the reaction at 10 mM Mg2+ is protein kinase-catalyzed is supported by several observations: (a) the reaction is facilitated by the addition of protein kinase; (b) the reaction depends on cAMP when protein kinase holoenzyme is uded; (c) the reaction is not inhibited by 1 mM ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetate which is known to inhibit autoactivation and autophosphorylation of phosphorylase kinase; and (d) the protein inhibitor of protein kinase inhibits this reaction. The phosphorylation and activation of phosphorylase kinase seem to occur in two phases. At low Mg2+ only the first phase is manifested and involves the incorporation of 2 mol of phosphate, 1 mol into each of Subunits A and B. At high Mg2+ additional sites are phosphorylated almost exclusively on Subunit A, with phosphate incorporation approaching the final level of 7 to 9 mol. Enzyme activity at high Mg2+ is 2 to 3 times higher than that observed when activation is studied at low Mg2+. The observation that both casein and type II histone are phosphorylated to the same extent at 1 mM and 10 mM Mg2+ suggested that high Mg2+ may be altering the conformation of phosphorylase kinase thus rendering more phosphorylation sites accessible to protein kinase. Since the phosphorylation of phosphorylase kinase by either the protein kinase-catalyzed or autocatalytic reaction can result in the incorporation of 7 to 9 mol of phosphate, the finding that only about seven sites become phosphorylated by both mechanisms acting together suggest that activation by these two mechanisms may involve common phosphorylation sites.
D000818:Animals; D002364:Caseins; D000242:Cyclic AMP / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D009132:Muscles / Q000201:enzymology*; D010764:Phosphorylase Kinase / Q000378:metabolism*; D006656:Protamine Kinase / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits
D002364:Caseins; D000242:Cyclic AMP; D011494:Protein Kinases; D010764:Phosphorylase Kinase; D006656:Protamine Kinase; D008274:Magnesium