Isolation and partial characterization of two forms of rat heart glycogen phosphorylase.
Rat heart glycogen phosphorylase a has been purified to apparent homogeneity by a procedure involving precipitation with ammonium sulfate, DEAE-Sephacel chromatography, and AMP-Sepharose affinity chromatography. In contrast to the skeletal muscle enzyme which appeared as a single peak upon ion-exchange chromatography, heart phosphorylase was separated into two distinct peaks (I and II), both only active in the presence of AMP. The isoelectric points of skeletal muscle phosphorylase b (dephosphorylated form) and heart phosphorylase Ib were both at 6.2, that of heart phosphorylase IIb was 5.2. The Km of phosphorylase IIb for AMP was threefold lower (7 microM) than that of Ib (22 microM). The dissociation constant K8 of phosphorylase Ia (phosphorylated form) was 0.37 mM for glycogen and 5.6 mM for Pi. Increasing the levels of glycogen decreased the apparent Km for Pi and vice versa. No such interaction between substrate binding was observed with phosphorylase IIa, since the Ks values for glycogen (0.27 mM) and Pi (3.8 mM) were not significantly influenced by increasing concentrations of the other substrate. The specific activity of both isoenzymes was 46 units/mg of protein at pH 6.8 and 30 degrees C. The subunit Mr of both forms was 92,500. By incubation with purified phosphorylase kinase and [gamma-32P]ATP both forms were converted to their corresponding forms by 32P incorporation of 1 mol/mol of subunit. This suggests the existence of two native forms of phosphorylase b in rat heart.
D000818:Animals; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D009132:Muscles / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology*; D010710:Phosphates / Q000378:metabolism; D010762:Phosphorylase a / Q000302:isolation & purification / Q000378:metabolism*; D010763:Phosphorylase b / Q000302:isolation & purification / Q000378:metabolism*; D006005:Phosphorylases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D007527:Isoenzymes; D010710:Phosphates; D010762:Phosphorylase a; D010763:Phosphorylase b; D006005:Phosphorylases