Regulation of protein kinase through de nove enzyme synthesis in germinating embryos of wheat: enzyme purification and its autophosphorylation

A significant increase (3.4 fold) in a cyclic nuclotide-independent protein kinase activity was witnessed during first 12 h of wheat embryo germination. Three activity peaks of protein kinase were physically separated by ion-exchange (DE-52) chromatography. The major peak on DE-52 was purified to electrophoretic homogeneity by Casein-Sepharose affinity chromatography. The molecular weight of the enzyme under non-denaturing conditions is l00000. The 32P-labelled reaction product of protein kinase was chemically characterized as phosphoserine and phosphothreonine residues. Conclusive proof for de novo synthesis of protein kinase was obtained by the in vivo labelling of the enzyme with [35S]sulphate. The 35S-label was present in the electrophoretically homogeneous preparation of purified kinase. On acid hydrolysis, the 35S-label was recovered predominantly in the methionine residue of the purified enzyme. The purified kinase undergoes autophosphorylation upon incubation with [-32P]ATP and Mg2+ ions in the absence of exogenously added substrate. Chemical characterization of the autophosphorylated reaction product yielded 32P-label in phosphoserine, phosphothreonine and phosphotyrosine residues of the purified protein kinase. Kinetics of autophosphorylation reaction revealed that the reaction was rapid with a pH optimum of 8.0. The Km for ATO for autophosphorylation of protein kinase was l.8μM. The in vitro autophosphorylation of protein kinase failed to modulate the catalytic activity of the enzyme. 31 refs. (author)