PURIFICATION AND CHARACTERIZATION OF A CA2+-DEPENDENT PROTEIN-KINASE FROM THE HALOTOLERANT GREEN-ALGA DUNALIELLA-TERTIOLECTA

A Ca2+-dependent protein kinase (CDPK) that has been partially purified and characterized previously [Yuasa and Mute (1992) Arch. Biochem. Biophys. 296: 175] was further purified to about 20,000-fold from the soluble fraction of Dunaliella tertiolecta. The enzyme preparation contained 60- and 52-kDa polypeptides both of which phosphorylated casein as a substrate. Both polypeptides showed a Ca2+-dependent increase in mobility during SDS-PAGE and Ca-45(2+)-binding activity after SDS-PAGE and electroblotting onto a nitrocellulose membrane, suggesting that both the 60- and 52-kDa CDPKs directly bind Ca2+. The protein kinase inhibitors, K-252a and staurosporine, inhibited the CDPK competitively with respect to ATP. An antibody raised against the 60-kDa CDPK crossreacted with both the 60- and 52-kDa polypeptides. Both molecular species were autophosphorylated in the presence of Ca2+, and a highly phosphorylated 80-kDa band appeared in addition to these phosphorylated bands at 60 and 52 kDa in SDS-PAGE. However, the specific activity of CDPK was not changed by prior autophosphorylation when the autophosphorylated enzyme was assayed as a mixture of these phosphorylated molecular species. Only the 60-kDa polypeptide was immunodetected in subcellular fractions of Dunaliella cells. The 52-kDa polypeptide increased during storage of the enzyme. These results suggest that the 52-kDa polypeptide is a proteolytic artifact produced during purification. Immunoreactive bands of 60-kDa were detected in extracts of several green algae but not in extracts of higher plants or a brown alga.