Characterization of multiple active forms of the NADPH dehydrogenase component of the oxidase complex from rabbit peritoneal neutrophils by photolabeling with an arylazido derivative of NADP+.
A NADPH cytochrome c oxidoreductase purified from membranes of rabbit peritoneal neutrophil was shown to behave as the NADPH dehydrogenase component of the O2- generating oxidase complex. A photoactivable derivative of NADP+, azido nitrophenyl-gamma-aminobutyryl NADP+ (NAP4-NADP+), was synthesized in its labeled [3H] form and used to photolabel the NADPH cytochrome c reductase at different stages of the purification procedure. Control assays performed in dim light indicated that the reduced form of NADP4-NADP+ generated by reduction with glucose-6-phosphate and glucose-6-phosphate dehydrogenase was oxidized at virtually the same rate as NADPH. Upon photoirradiation of the purified reductase in the presence of [3H]NAP4-NADP+ and subsequent separation of the photolabeled species by sodium dodecyl sulfate polyacrylamide gel electrophoresis, radioactivity was found to be present predominantly in a protein band with a molecular mass of 77-kDa and accessorily in bands of 67-kDa and 57-kDa. Evidence is provided that the 67-kDa and 57-kDa proteins arose from the 77-kDa protein by proteolysis. Despite removal of part of the sequence, the proteolyzed proteins were still active in catalyzing electron transport from NADPH to cytochrome c and in binding the photoactivable derivative of NADP+.
Laporte
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F
LBIO/Biochimie, Département de Recherche Fondamentale, Centre d'Etudes Nucléaires, Grenoble, France.
Doussiere
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J
Vignais
P V
PV
D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D046911:Macromolecular Substances; D008566:Membranes / Q000378:metabolism; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology*; D010529:Peritoneal Cavity / Q000166:cytology; D010777:Photochemistry; D011817:Rabbits
D000345:Affinity Labels; D046911:Macromolecular Substances; D009249:NADP; D009251:NADPH-Ferrihemoprotein Reductase