Detection and isolation of the NADPH-binding protein of the NADPH:O2 oxidoreductase complex of human neutrophils.
Neutrophils assayed with nitro blue tetrazolium (NBT) exhibit intracellular rather than extracellular superoxide-generating activity when stimulated with phorbol myristate acetate. Enzyme activity is stimulated by anionic detergents, reversibly inhibited by 2',3'-NADPH dialdehyde, and present in equal levels in membrane fractions obtained from phorbol myristate acetate-stimulated and resting cell suspensions. Solubilized membrane shows enzyme activity co-eluting on molecular sieving columns with the cytochrome b redox component of the oxidoreductase complex. Enzyme activity was resolved free of the cytochrome b component following passage of solubilized membrane extracts through QAE-Sephadex anion exchange columns. Enzyme activity measured by the NBT assay appears to be that associated with the NADPH binding protein of the oxidoreductase complex. When exposed to NBT and NADPH this component of the oxidoreductase generates superoxide independent of cytochrome b.
Green
T R
TR
Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland.
Pratt
K L
KL
D002352:Carrier Proteins / Q000097:blood* / Q000302:isolation & purification; D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004227:Dithionite / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D009247:NADH, NADPH Oxidoreductases / Q000097:blood* / Q000302:isolation & purification; D009249:NADP / Q000378:metabolism; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D010084:Oxidation-Reduction; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D013053:Spectrophotometry; D013481:Superoxides / Q000097:blood; D014452:Ubiquitins / Q000235:genetics* / Q000378:metabolism
D002352:Carrier Proteins; D046911:Macromolecular Substances; D011498:Protein Precursors; D014452:Ubiquitins; C045677:ubiquitin precursor; D013481:Superoxides; D004227:Dithionite; D009249:NADP; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C022709:superoxide-forming enzyme