Ca2+‐dependent in vitro phosphorylation of soluble proteins from germinating wheat (Triticum turgidum) endosperm

A 40000 g supernatant fraction from extracts of germinating wheat (Triticum turgidum Desf. cv. Edmore) endosperm contains protein kinase activity that phosphorylates several endogenous proteins. In vitro incorporation of radiolabel from [32P]‐ATP into phosphoproteins was maximal in the presence of 1 mM CaCl2 and 5 mM MgCl2Ca2+ at micromolar concentrations greatly stimulated the phosphorylation of 49 and 47 kDa polypeptides and also inhibited the phosphorylation of a few specific polypeptides. The phosphorylation of the 49 and 47 kDa polypeptides was present at 2 days after seed germination and was maximal at 8 days. Quantitative protein changes were also detected during the seed germination, but differences could not be correlated with changes in protein phosphorylation. Phosphoamino acid analysis by two dimensional thin‐layer electrophoresis showed that the Ca2+‐dependent protein kinase phosphorylates a serine residue of the 47 kDa polypeptide. Ca2+‐dependent protein kinase phosphorylates a serine residue of the 47 KDa polypeptide. Ca2+ dependent protein phosphorylktion was inhibited by phenothiazine‐derived drugs. Addition of S‐adenosylmethionine to the in vitro phosphorylation reaction specifically inhibited the Ca2+‐dependent protein phosphorylation.