Partial purification and characterization of a second Calmodulin-activated Ca 2+-dependent protein kinase from wheat germ

Abstract

A soluble Ca 2+-dependent protein kinase was partially purified wheat germ by a procedure involving adsorption to DEAE-cellulose, Ca 2+-dependent binding to phenyl-Sepharose, (NH 4) 2SO 4 precipitation and gel filtration. The protein kinase ( M r 86 000) catalyzes the phosphorylation of histones, casein and phosvitin. Calmodulin activates the phosphorylation of histones catalyzed by the protein kinase. The protein is largely dependent upon Ca 2+ for activity. The rate of casein phosphorylation is half-maximal at 0.3 μM free Ca 2+ and maximal at 3 μM free Ca 2+; much higher free Ca 2+ concentrations are required for half-maximal (60 μM) and maximal (500 μM) rates of histone phosphorylation. Millimolar Mg 2+ is required in addition to Ca 2+ for maximal activity of the enzyme and millimolar Mn 2+ can substitute for the (Ca 2+ + Mg 2+) requirement. The protein kinase is inhibited by the calmodulin antagonists, chlorpromaxine and fluphenazine. The K m for ATP is 16 μM and the enzyme phosphorylates serine and threonine residues of casein. The enzyme is very similar to a Ca 2+- and calmodulin-activated protein kinase isolated from wheat-germ chromatin, but differs from this enzyme in various properties, notably apparent molecular size and insensitivity to nucleotides that inhibit the chromatin-derived enzyme.