Dephosphorylation of rabbit skeletal muscle phosphorylase kinase. Evidence against the operation of the "second-site phosphorylation" mechanism of regulation.
The dephosphorylation of rabbit skeletal muscle phosphorylase kinase was studied using two purified rabbit skeletal muscle protein phosphatases. The first enzyme (Mr = 32,000) corresponds to the form we have previously termed protein phosphatase C. Phosphorylase kinase was found to be rapidly dephosphorylated by this enzyme. The site of dephosphorylation was examined, and it was shown that this enzyme was relatively specific for the dephosphorylation of the beta-subunit phosphate, as compared to the alpha-subunit phosphate, of phosphorylase kinase. Phosphate release from the beta-subunit was approximately 100-fold faster than from the alpha-subunit. More importantly, dephosphorylation of the beta-subunit phosphate was not significantly affected by phosphorylation of the alpha-subunit. The dephosphorylation of phosphorylase kinase by a second low molecular weight protein phosphatase, Mr = 33,500, was also studied. The specific activity of this enzyme toward phosphorylase kinase was only a fraction of that exhibited by the Mr = 32,000 phosphatase. This enzyme removed phosphate from both the alpha- and beta-subunits but more rapidly (about 4-fold) from the alpha-subunit. With neither of these enzyme preparations was there any evidence for the regulation of beta-subunit dephosphorylation by phosphorylation of the alpha-subunit as proposed by Cohen and Antoniw ((1973) FEBS Lett. 34, 43-47).
D000818:Animals; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification / Q000378:metabolism*; D010764:Phosphorylase Kinase / Q000378:metabolism*; D010766:Phosphorylation; D011817:Rabbits; D013379:Substrate Specificity
D007527:Isoenzymes; D046911:Macromolecular Substances; D010764:Phosphorylase Kinase; D010749:Phosphoprotein Phosphatases