Identification of the NADPH-binding protein of the neutrophil superoxide-generating oxidase of guinea pigs.
A cell-free system prepared from polymorphonuclear neutrophils is capable of NADPH-dependent generation of superoxide anion, but requires the simultaneous presence of plasma membranes, cytosol, arachidonate and guanosine 5'-[gamma-thio]triphosphate (GTP[S]). The isolated membranes from such a preparation are able to catalyse NADPH-dependent superoxide formation independently of added cytosol and activators. Such activated membranes, activated in the cell-free system, must consequently contain all of the essential components required by the oxidase for superoxide formation, including the NADPH-binding component. Arylazido-beta-alanyl-[32P]NADPH (3'-O-(3-[N-(4-azido-2-nitrophenyl)-amino] propionyl)-[32P]NADPH), an NADPH analogue and photoaffinity probe, is shown to act in the dark as a substrate for the oxidase activity in the activated membranes and an irreversible photodependent inhibitor following photoirradiation. The photoaffinity probe has been used to identify the specific NADPH-binding component of the oxidase in the activated membranes. In contrast with the sensitivity of the activated membranes, photoirradiation of arylazido-beta-alanyl-[32P]-NADPH under identical conditions, but with non-activated membranes, did not prevent subsequent activation of the treated membranes by cytosol, arachidonate and GTP[S]. However, photoirradiation of the cytosolic fraction in the presence of arylazido-beta-alanyl-[32P]NADPH resulted in an inhibition of the cytosol's ability to activate superoxide generation upon subsequent incubation with plasma membranes in the presence of arachidonate and GTP[S]. These observations are taken as a strong indication that the NADPH-binding protein of the oxidase is a cytosolic factor which associates with the plasma membrane upon activation. The superoxide-generating activity of the activated membranes was inhibited irreversibly in a concentration- and photo-dependent manner by arylazido-beta-alanyl-NADPH. The arylazido-beta-alanyl-NADPH-photodependent inhibition of superoxide generation in the activated membranes correlated with the photodependent labelling of a protein of 55.6 kDa by the arylazido-beta-alanyl-NADPH. Specificity of labelling was indicated by a lack of labelling of the 55.6 kDa region in the non-activated membranes and protection of the photodependent inhibition and labelling of the 55.6 kDa protein by NADPH. It is proposed that the arylazido-beta-alanyl-NADPH-labelled 55.6 kDa protein present on the activated membranes is the NADPH-binding protein of the neutrophil superoxide-generating oxidase.
Ge
F
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Department of Biochemistry and Biophysics, John A. Burns School of Medicine, University of Hawaii, Honolulu 96822.
Guillory
R J
RJ
D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D006168:Guinea Pigs; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D019255:NADPH Oxidases; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology; D016897:Respiratory Burst; D013481:Superoxides / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D002352:Carrier Proteins; D011480:Protease Inhibitors; D013481:Superoxides; D009249:NADP; C023549:arylazido-beta-alanyl-NADP; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C022709:superoxide-forming enzyme