Endogenous inhibitor of protein kinase C: association with human peripheral blood neutrophils but not with specific granule-deficient neutrophils or cytoplasts.
A Ca2+-activated and phospholipid-dependent protein kinase (PKC) has been described in several cell systems, including the human neutrophil. We found that less than 30 pmol 32P/10 min/10(6) cell equivalents of phorbol ester- or 1-oleoyl-2-acetylglycerol-stimulated PKC activity was obtained when neutrophil homogenates were used as an enzyme source. However, detergent-soluble and detergent-insoluble fractions prepared from the same homogenates, respectively, catalyzed 578 +/- 50 and 136 +/- 40 pmol 32P/10 min/10(6) cell equivalents. Recombining detergent-soluble and detergent-insoluble fractions resulted in the complete loss of activity. We therefore explored the possibility of an endogenous inhibitor of PKC in neutrophils. Homogenates from neutrophil cytoplasts, which lack the nuclei and intracellular granules of whole neutrophils, yielded 813 +/- 28 pmol 32P/10 min/10(6) cell equivalents. In addition, total neutrophil homogenates from a patient with a specific granule deficiency yielded high activities, namely 424 +/- 48 pmol 32P/10 min/10(6) cell equivalents. Specific granule-deficient neutrophils possessed translocated and activated PKC, and phosphoprotein patterns from these cells resembled those from activated normal neutrophils. Our results suggested the existence of an inhibitor of previously active PKC. That this inhibitor is primarily associated with neutrophil-specific granule membranes was suggested by our finding of high PKC activity associated with cell preparations or combinations of cell fractions that were free of specific granules, but not necessarily of other cellular organelles. Preliminary characterization of the endogenous inhibitor indicated that it was protease- and heat-sensitive, and did not exhibit either protease or phosphatase activity. We speculate that the inhibitor may play a physiologic role in regulating the activity of its target enzyme.
Balazovich
K J
KJ
Smolen
J E
JE
Boxer
L A
LA
D003594:Cytoplasmic Granules / Q000032:analysis; D004075:Diglycerides / Q000494:pharmacology; D006801:Humans; D007425:Intracellular Membranes / Q000032:analysis; D009504:Neutrophils / Q000032:analysis* / Q000187:drug effects; D015240:Phorbol 12,13-Dibutyrate; D010703:Phorbol Esters / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D004075:Diglycerides; D010703:Phorbol Esters; D015240:Phorbol 12,13-Dibutyrate; C038237:1-oleoyl-2-acetylglycerol; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate