NADPH binding component of neutrophil superoxide-generating oxidase.
The 2',3'-dialdehyde derivative of NADPH was used as an affinity labeling reagent of a solubilized NADPH-dependent superoxide-generating oxidase preparation of pig neutrophils. The analogue served as both an electron donor and a competitive inhibitor of the NADPH oxidase against NADPH. The apparent Michaelis constant (Km) for the derivative (31 microM) was essentially the same as that for NADPH (33 microM). The activity of the superoxide formation in the presence of 2',3'-dialdehyde NADPH was about a half of that in the presence of NADPH. Incubation of the enzyme with the derivative inactivated the superoxide-generating activity and the inactivation was prevented by the addition of NADPH. We performed the labeling of the oxidase preparation with 2',3'-dialdehyde NADPH and sodium cyanoboro[3H]hydride, based on the above results. A protein of 66,000 daltons was selectively labeled among more than 20 bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis which were visualized with Coomassie Brilliant Blue. The protein was not labeled when the oxidase preparation was pretreated with p-chloromercuribenzoate or it was labeled in the presence of excess NADPH. The protein is suggested to be the NADPH binding component of the neutrophil superoxide-generating oxidase system.
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002729:Chloromercuribenzoates / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP / Q000031:analogs & derivatives / Q000378:metabolism*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D013481:Superoxides / Q000096:biosynthesis*; D013552:Swine; D020245:p-Chloromercuribenzoic Acid
D000345:Affinity Labels; D002729:Chloromercuribenzoates; D009243:NAD; D013481:Superoxides; D009249:NADP; D020245:p-Chloromercuribenzoic Acid; C038805:2',3'-dialdehyde NADP; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases