Purification and properties of the cardiac isoenzyme of phosphorylase kinase.
This study reports the purification of bovine cardiac phosphorylase kinase to near homogeneity (approximately 1240-fold), as based upon SDS gel electrophoresis and sucrose density gradient ultracentrifugation (co-migration of enzyme activity and protein with constant specific activity throughout the peak). The molecular weight of the enzyme following purification is identical with that in the soluble extract from beef hearts, and similar to that of the rabbit skeletal muscle isoenzyme (i.e. approximately 1.3 X 10(6)) based on co-migration in sucrose density gradient ultracentrifugation. The molecular weights of the subunits determined by SDS gel electrophoresis are: alpha', 134,000; beta, 125,000; gamma, 48,000. The subunit stoichiometry is determined to be alpha 1 beta 1.01 gamma 1.35 with a faint band co-migrating with purified bovine brain calmodulin. The enzyme displays little, if any, activity below pH 6.0, but activity increases markedly in the pH range of 6.8 to 8.2. After phosphorylation with pure cAMP-dependent protein kinase catalytic subunit and [gamma-32P]ATP (0.14 mM ATP, 4 mM magnesium acetate) in which the beta subunit was maximally phosphorylated (stoichiometry, 0.25 mol/mol; ratio of 32P in the alpha' and beta subunits, 1.90:1), enzyme activity was increased approximately 2-fold at pH 6.8, while at higher pH values, the effect of phosphorylation was less marked (20 to 50% increase at pH 8.2). Ca2+ is required for enzyme activity; ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (0.5 mM) completely inhibited the activity of th nonphosphorylated and phosphorylated enzyme, and activity was restored by the addition of Ca2+ (Ka for Ca2+, 1.94 and 1.35 microM for the nonphosphorylated and phosphorylated enzyme, respectively.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D004533:Egtazic Acid / Q000494:pharmacology; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D009206:Myocardium / Q000201:enzymology*; D010764:Phosphorylase Kinase / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D013379:Substrate Specificity
D007527:Isoenzymes; D046911:Macromolecular Substances; D008345:Manganese; D004533:Egtazic Acid; D011494:Protein Kinases; D010764:Phosphorylase Kinase; D008274:Magnesium; D002118:Calcium