Activation of human neutrophil NADPH-oxidase in vitro by the catalytic fragment of protein kinase-C.
Phorbol ester treatment of intact neutrophils both stimulates protein kinase C (PK-C) and causes the rapid proteolytic conversion to a cytosolic, co-factor independent fragment, protein kinase M (PK-M). In intact neutrophils, phorbol ester treatment activates the NADPH-oxidase, the enzyme responsible for the oxidative burst. Addition of purified PK-M to resting neutrophil light density membranes activated the NADPH-oxidase in the presence of PS, ATP and Mg2+. A 3.5-fold greater stimulation of oxidase (ca. 25 nmoles O2-/min/mg membrane protein) was obtained with comparable PK-M concentrations to that observed with the reconstituted PK-C system, and approximately 1/3 that obtained with arachidonic acid (AA) or SDS. In contrast to the reconstituted system using PK-C, PMA and Ca++ were neither required nor affected activity. The effect of PS was unexpected, since PK-M does not require phospholipids for enzymatic activity, and likely represents the action of PS on the oxidase itself or on another component in the plasma membrane fraction. Our studies demonstrate for the first time that purified PK-M permits reconstitution of a physiologic phorbol ester response.
Tauber
A I
AI
Boston University School of Medicine, MA 02118.
Cox
J A
JA
Curnutte
J T
JT
Carrol
P M
PM
Nakakuma
H
H
Warren
B
B
Gilbert
H
H
Blumberg
P M
PM
D000255:Adenosine Triphosphate / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP / Q000494:pharmacology; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D010718:Phosphatidylserines / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D010718:Phosphatidylserines; D009249:NADP; D000255:Adenosine Triphosphate; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D011493:Protein Kinase C; D008274:Magnesium; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium