Purification of PKC-I, an endogenous protein kinase C inhibitor, and types II and III protein kinase C isoenzymes from human neutrophils.
Human neutrophil protein kinase C (PKC) activity is inhibited by an endogenous protein found primarily in the pellet fraction from homogenized specific granules, which was both heat- and proteinase-sensitive [Balazovich, Smolen & Boxer (1986) J. Immunol. 137, 1665-1673]. We now report that two PKC isoenzymes and the endogenous PKC inhibitor, which we named PKC-I, were purified from human neutrophils. A neutrophil soluble fraction that was subjected to DEAE-Sephacel chromatography yielded highly enriched PKC because, by definition, enzymic activity was strictly dependent on Ca2+ and phosphatidylserine. Hydroxyapatite chromatography resolved two peaks of PKC activity. Type II and Type III PKC isoenzymes were each identified on Western blots by using isoenzyme-specific monoclonal antibodies. Unlike rat brain, from which PKC isoenzymes were also purified, Type I PKC was not detected in human neutrophils. Western blots indicated that both Type II and Type III PKC isoenzymes had molecular masses near 80 kDa. In agreement with other reports, PKC was autophosphorylated in vitro. PKC-I, an endogenous neutrophil inhibitor of PKC, was purified to apparent homogeneity by DEAE-Sephacel and S-400 Sephacel chromatography. PKC-I had a molecular mass of 41 kDa. PKC-I inhibited purified PKC activity stimulated by 1,2-diacylglycerols in a concentration-dependent manner, and inhibited PKC-dependent phosphorylation of proteins present in neutrophil cytosol.
Balazovich
K J
KJ
Department of Pediatrics, University of Michigan Medical School, Ann Arbor 48109.
McEwen
E L
EL
Lutzke
M L
ML
Boxer
L A
LA
White
T
T
D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007120:Immunochemistry; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D009504:Neutrophils / Q000201:enzymology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D011493:Protein Kinase C