Cyclic AMP- and Ca²⁺-dependent protein kinases in Plasmodium falciparum

The cyclic AMP- and Ca²⁺-dependent protein kinase activities of Plasmodium falciparum were partially characterized after purification of parasites from host erythrocytes by N₂ cavitation and Percoll gradient centrifugation. Proteins of molecular weights 80, 54, 51, and 31.5 kDa were phosphorylated in a cAMP-dependent manner in cytosolic extracts of isolated P. falciparum. Cytosolic extracts also contained cAMP-dependent histone II-A kinase activity with an average V_max of 131.1 pmol/³²P/min/mg protein and a K_m for cAMP of 85 nM. Upon photoaffinity labeling with [³²P]-8-N₃3-cAMP, a 53-kDa protein was specifically labeled in parasite cytosol. A metabolically labeled protein of the same molecular weight was identified by cAMP-agarose affinity chromatography. The 53-kDa protein cochromatographed with cAMP-dependent histone II-A kinase activity on DEAE-cellulose, suggesting that it is the regulatory subunit of the kinase. Ca²⁺-dependent phosphorylation of proteins of molecular weights 195, 158, 51, 47.5, and 15 kDa was demonstrated in a membrane fraction from parasites free of the erythrocyte membrane. This activity was not stimulated by either calmodulin or phospholipid plus diacylglycerol and was absent from the membranes of uninfected erythrocytes. Of several exogenous substrates tested, none were found to be a substrate for this Ca²⁺-dependent kinase. Both cAMP- and Ca²⁺-dependent kinases phosphorylated serine and threonine residues.