Liver Glycogen Phosphorylase Kinase

Liver glycogen phosphorylase kinase was assayed by measuring the radioactivity of [y-32P]ATP incorporated into rabbit skeletal muscle phosphorylase b. Using this assay two forms of phosphorylase kinase, Kinase I and Kinase II, were pirified about 70and 130-fold, respectively, from rabbit liver cytosol by DEAE-Sephadex A50 column chromatography, ammonium sulfate fractionation, gel filtration on a Sepharose CL-6B column, followed by phosphocellulose column or Cibacron blueSephadex affinity chromatography. The phosphorylation reaction was accompanied by the concomitant activation of phosphorylase b. Both Kinases I and II were able to phosphorylate and activate liver inactive phosphorylase. The molecular weight was about 1,300,OOO for Kinase I and 110,000 for Kinase II. Kinetic properties of Kinases I and II were similar: the pH optimum was 7; the K,,, value for ATP was 3.0 x 10m5 M; and the K,,, value for muscle phosphorylase b was 300 gg/ml, and that for liver inactive phosphorylase was 80 gg/ml. The liver phosphorylase kinase was inhibited partly, at most 60 to 70%, by 0.05 mM ethylene glycol bis(P-aminoethyl ether)N,N,iV,W-tetraacetate, and reactivated by Ca’+. The K, value for free Ca2+ was about 3 x lo-’ M at pH 6.8. Kinase II appeared to be a proteolytic artifact that was produced from Kinase I probably due to a lysosomal protease, cathepsin B. Kinase I was also converted to a smaller form that was indistinguishable from Kinase II by limited proteolysis with Ca2+-dependent protease or trypsin. The catalytic activity was enhanced about 60% during this proteolysis. No evidence has been obtained indicating the existence of active and inactive forms of liver phosphorylase kinase.