Ca2+-dependent activation of phosphorylase by phosphorylase kinase in adipose tissue.
Phosphorylase kinase (EC 2.7.1.38) activity in crude cytosol preparations of chicken adipose tissue was assayed using as substrate either the endogenous phosphorylase b in the preparation or added purified rabbit skeletal muscle phosphorylase b. The results obtained with the two substrates were similar. The phosphorylase kinase reaction was markedly inhibited by ethyleneglycol-bis(beta-aminoethylether)-N,N',-tetraacetic acid (EGTA), maximum inhibition (about 90%) occurring at approx. 0.2 mM. This inhibition was readily reversed by addition of Ca2+. Full reversal was achieved with 0.3 mM Ca2+ in the presence of 0.5 mM EGTA; the estimated free Ca2+ concentration required was 4 muM. The activation of phosphorylase b was blocked immediately and completely by EGTA added during the course of the assay; reversal was achieved without a time lag by the addition of Ca2+. The Ca2+ requirement was also demonstrated directly by preparing an enzyme fraction from which Ca2+ had been removed and by using Ca2+-free reagents. Under these conditions the Ca2+ concentration needed for half maximum activation was 10 muM and maximum activation was obtained at about 100 muM. The possibility that the effects of EGTA and Ca2+ might be related to changes in phosphorylase phosphatase activity rather than phosphorylase kinase was considered unlikely since the phosphorylase phosphatase activity was inhibited during the phosphorylase kinase assay step by the inclusion of fluoride and beta-glycerophosphate. Phosphorylase kinase activity in rat adipocytes, using endogenous phosphorylase as substrate, was also inhibited EGTA but, whereas the activity in chicken adipose tissue was inhibited by 90%, the activity in rat adipose tissue was inhibited only 60%. These data indicate that adipose tissue phosphorylase kinase has a Ca2+ requirement for optimal activity and is thus qualitatively similar to the enzyme in contractile tissues.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000273:Adipose Tissue / Q000201:enzymology*; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002645:Chickens; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010764:Phosphorylase Kinase / Q000378:metabolism*; D006005:Phosphorylases / Q000378:metabolism*
D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D006005:Phosphorylases; D010764:Phosphorylase Kinase; D008274:Magnesium; D002118:Calcium