A specific requirement for protein kinase C in activation of the respiratory burst oxidase of fertilization.
Partially reduced oxygen species are toxic, yet activated sea urchin eggs produce H2O2, suggesting that the control of oxidant stress might be critical for early embryonic development. We show that the Ca2(+)-stimulated NADPH oxidase that generates H2O2 in the "respiratory burst" of fertilization is activated by a protein kinase, apparently to regulate the synthesis of this potentially lethal oxidant. The NADPH oxidase was separated into membrane and soluble fractions that were both required for H2O2 synthesis. The soluble fraction was further purified by anion exchange chromatography. The factor in the soluble fraction that activated the membrane-associated oxidase was demonstrated to be protein kinase C (PKC) by several criteria, including its Ca2+/phophatidylserine/diacyl-glycerol-stimulated histone kinase activity, its response to phorbol ester, its inhibition by a PKC pseudosubstrate peptide, and its replacement by purified mammalian PKC. Neither calmodulin-dependent kinase II, the catalytic subunit of cyclic AMP-dependent protein kinase, casein kinase II, nor myosin light chain kinase activated the oxidase. Although the PKC family has been ubiquitously implicated in cellular regulation, enzymes that require PKC for activation have not been identified; the respiratory burst oxidase is one such enzyme.
Heinecke
J W
JW
Department of Medicine, University of Washington, Seattle 98195.
Meier
K E
KE
Lorenzen
J A
JA
Shapiro
B M
BM
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D004789:Enzyme Activation; D005260:Female; D005306:Fertilization / Q000502:physiology*; D006861:Hydrogen Peroxide / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D019255:NADPH Oxidases; D010063:Ovum / Q000201:enzymology*; D010446:Peptide Fragments / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D012617:Sea Urchins; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium