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A widespread occurrence of Ca2+-dependent protein kinase was shown in various tissues and phyla of the animal kingdom. Phosphatidylserine appeared to be more effective than calmodulin in supporting the Ca2+-dependent phosphotransferase activity. The phospholipid-sensitive Ca2+-dependent protein kinase activity, distributed in both the cytosolic and particulate fractions, was not inhibited by trifluoperazine, a specific inhibitor of calmodulin-sensitive, Ca2+-dependent reactions or processes. The enzyme activity levels, compared to those of cyclic AMP-dependent and cyclic GMP-dependent protein kinases, were exceedingly high in certain tissues (such as brain and spleen) and exhibited a much greater disparity among tissues. The Ka for Ca2+ was about 100 microM in the presence of phosphatidylserine; the value was as low as 2 microM in the presence of phosphatidylserine and diolein. It is suggested that phospholipid-sensitive Ca2+-dependent protein kinase may mediate certain actions of Ca2+ in tissues, acting independently or in a complementary manner with other protein phosphorylation systems stimulated by calmodulin-Ca2+, cyclic AMP, or cyclic GMP.

Soluble and membrane fractions of bovine adrenal medulla contain several substrates for the Ca2+/phospholipid-dependent and cyclic AMP-dependent protein kinases. The phosphorylation of soluble proteins (36 and 17.7 kilodaltons) and a membrane protein (22.5 kilodaltons) showed an absolute requirement for the presence of both Ca2+ and phosphatidylserine; other substrates showed less stringent phosphorylation requirements and many of these proteins were specific for each of the protein kinases. The Ca2+/phospholipid-dependent phosphorylation was rapid, with effects seen as early as at 30 s of incubation. Measurement of enzyme activities with histone H1 as an exogenous substrate demonstrated that the Ca2+/phospholipid-dependent protein kinase was equally distributed between the soluble and membrane fractions whereas the cyclic AMP-dependent enzyme was predominantly membrane-bound in adrenal medulla and chromaffin cells. The activity of the soluble Ca2+/phospholipid-dependent protein kinase of adrenal medulla was found to be about 50% of the enzyme level present in rat brain, a tissue previously shown to contain a very high enzyme activity. These results suggest a prominent role for the Ca2+/phospholipid-dependent protein kinase in chromaffin cell function.

The Ca2+-dependent phosphorylation of a number of proteins in the cytosol of the rat or guinea pig cerebral cortex was profoundly stimulated by phosphatidylserine; calmodulin, on the other hand, had only a minimal effect. The Ca2+-dependent phosphorylation of different proteins from the total particulate fraction of the same tissue, in comparison, was specifically stimulated by either phosphatidylserine or calmodulin. The present findings, in line with the phospholipid-sensitive Ca2+-dependent protein kinase recently recognized, suggest an involvement of phospholipid in regulating Ca2+-dependent phosphorylation of endogenous substrate proteins. This new system presumably functions independent or in a complementary manner with the calmodulin-sensitive Ca2+-dependent protein phosphorylation system previously reported by others.

Effects of melittin, an amphipathic polypeptide, on various species of protein kinases were investigated. It was found that melittin inhibited the newly identified phospholipid-sensitive Ca2+-dependent protein kinase (from heart, brain, spleen and neutrophils) and the cardiac myosin light-chain kinase, a calmodulin-sensitive Ca2+-dependent enzyme. In contrast, melittin had little or no effect on either the holoenzymes of the cardiac cyclic AMP-dependent and cyclic GMP-dependent protein kinases or the catalytic subunit of the former. Kinetic analysis indicated that melittin inhibited phospholipid-sensitive Ca2+-dependent protein kinase non-competitively with respect to ATP (Ki = 1.3 microM); although exhibiting complex kinetics, its inhibition of the enzyme was overcome by phosphatidylserine (a phospholipid cofactor), but not by protein substrate (histone H1) or Ca2+. On the other hand, melittin inhibited myosin light-chain kinase non-competitively with respect to ATP (Ki = 1.4 microM) or Ca2+ (Ki = 1.9 microM), and competitively with respect to calmodulin (Ki = 0.08 microM); although exhibiting complex kinetics, its inhibition of the enzyme was reversed by myosin light chains (substrate protein). The present findings indicate the presence of functionally important hydrophobic or hydrophilic loci on the Ca2+-dependent protein kinases, but not on the cyclic nucleotide-dependent class of protein kinase, with which melittin can interact. Moreover, the kinetic data suggest that melittin inhibited myosin light-chain kinase by interacting with a site on the enzyme the same as, or proximal to, the calmodulin-binding site, thus interfering with the formation of active enzyme-calmodulin-Ca2+ complex.