Properties of a calmodulin-activated Ca 2+-dependent protein kinase from whrat germ

Abstract

A soluble protein kinase that is largely dependent upon Ca 2+ for activity was partially purified from wheat germ. The protein kinase ( M r 90 000) catalyzes the phosphorylation of casein, histones and of endogenous proteins. Calmodulin activates the protein kinase with histone as substrate, half-maximal activation being obtained with 1.4 μM sheep brain calmodulin. The rate of casein phosphorylation is half-maximal at 0.3 μM free Ca 2+ and maximal at 2.0 μM free Ca 2+. Higher Ca 2+ is required for histone phosphorylation, namely 80 μM and 500 μM free Ca 2+, respectively, for half-maximal and maximal phosphorylation rates. In addition to Ca 2+, millimolar Mg 2+ is required for maximal activity of the enzyme; millimolar Mn 2+ can substitute for the (Ca 2+ + Mg 2+) requirement. The K m for ATP is 31 μM; other nucleoside 5′-triphosphates and ADP inhibit phosphoryl transfer from ATP to protein. Serine and threonine residues of casein or histones are phosphorylated by the enzyme. The protein kinase is inhibited by relatively high concentrations of chlorpromazine and fluphenazine. The low free Ca 2+ required for activation of the enzyme suggests that this type of Ca 2+-dependent protein kinase may be involved in Ca 2+-mediated stimulus-response coupling in plants.