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.
Nakanishi
H
H
Howard Hughes Medical Institute, Nashville, Tennessee.
Exton
J H
JH
D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology; D001921:Brain / Q000201:enzymology; D002417:Cattle; D002845:Chromatography; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D005230:Fatty Acids, Nonesterified / Q000494:pharmacology; D006882:Hydroxyapatites; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D010743:Phospholipids / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000302:isolation & purification* / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity
D005230:Fatty Acids, Nonesterified; D006882:Hydroxyapatites; D007527:Isoenzymes; D010743:Phospholipids; D016718:Arachidonic Acid; D015240:Phorbol 12,13-Dibutyrate; D017886:Durapatite; D011493:Protein Kinase C