PURIFICATION AND CHARACTERIZATION OF THE ZETA-ISOFORM OF PROTEIN-KINASE-C FROM BOVINE KIDNEY

The zeta-isoform of protein kinase C (PKC-zeta) was purified to near homogeneity from the cytosolic fraction of bovine kidney by successive chromatography on DEAE-Sephacel, heparin-Sepharose, phenyl-5PW, hydroxyapatite, and Mono Q. The purified enzyme had a molecular mass of 78 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein was recognized by an antibody raised against a synthetic oligopeptide corresponding to the deduced amino acid sequence of rat PKC-zeta. The enzymatic properties of PKC-zeta were examined and compared with conventional protein kinase C purified from rat brain. The activity of PKC-zeta was stimulated by phospholipid but was unaffected by phorbol ester, diacylglycerol, or Ca2+. PKC-zeta did not bind phorbol ester, and autophosphorylation was not affected by phorbol ester. Unsaturated fatty acid activated PKC-zeta, but this activation was neither additive nor synergistic with phospholipid. These results indicate that regulation of PKC-zeta is distinct from that of other isoforms and suggest that hormone-stimulated increases in diacylglycerol and Ca2+ do not activate this isoform in cells. It is possible that PKC-zeta belongs to another enzyme family, in which regulation is by a different mechanism from that for other isoforms of protein kinase C.