Protein phosphorylation and its regulation by calcium and calmodulin in membrane fractions from zucchini hypocotyls.

Protein-kinase activity has been found to be associated with a membrane fraction obtained from dark-grown zucchini (Cucurbita pepo L., cv. Senator) hypocotyl hooks. Proteins of this membrane fraction were used as protein substrates. The effects of Mg(2+), Na(+) and K(+) on phosphorylation, measured as (32)P incorporation, was investigated. The kinetics of phosphorylation of the individual protein peptides indicate the presence of specific phosphatase activity. Phosphorylation activity is strongly influenced by Ca(2+). One peptide (relative molecular weight: 180,000) exhibits strong inhibition of (32)P incorporation at physiological Ca(2+) concentrations between 0.1 and 1 μM. Phosphorylation of about 10 other proteins was enhanced by Ca(2+), being maximal in most cases at a concentration of about 3 μM free Ca(2+). Five out of these 10 peptides show increased phosphorylation in the presence of 1 μM calmodulin. This calmodulin-dependent enhancement of phosphorylation could be completely inhibited by the calmodulin antagonist fluphenazine. Cyclic AMP was found to have no stimulating effect on protein phosphorylation.