title: Purification of the solubilized NADPH:O2 oxidoreductase of human neutrophils. Isolation of its catalytically inactive cytochrome b and flavoprotein redox centers.
abstract: The membrane-bound NADPH:O2 oxidoreductase of human neutrophils has been solubilized in approximately 70% yield and purified on concanavalin A-Sepharose and gel sieving columns of varying bed volumes and sieving ranges. The half-life of the solubilized oxidoreductase stored at 2-4 degrees C in the presence of 25% glycerol at pH 8.6 is approximately 30 h. The oxidoreductase contains a flavoprotein identifiable by its fluorescence spectrum for FAD which binds weakly to concanavalin A-Sepharose and elutes from gel sieving columns at a molecular weight range of approximately 51,000. This flavoprotein accounts for approximately 70% of the total FAD content found in granular membrane fractions recovered from activated neutrophils. Recovery of oxidoreductase activity from both concanavalin A-Sepharose affinity and gel sieving columns is affected by the resolution of the flavoprotein free of the cytochrome b component of the oxidoreductase. The resolved flavoprotein and cytochrome b appear unable to catalyze either NADH nor NADPH oxidase activities with O2, ferricyanide, or nitroblue tetrazolium salt serving as electron acceptors.
authors: Clinical Pathology Service, Veterans Administration Medical Center, Portland, Oregon.
authors: T R
authors: TR
authors: Green
authors: K L
authors: KL
authors: Pratt
mesh_terms: D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003573:Cytochrome b Group / Q000097:blood*; D005182:Flavin-Adenine Dinucleotide / Q000097:blood*; D006207:Half-Life; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000097:blood*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D010084:Oxidation-Reduction; D012995:Solubility
chemical_list: D003573:Cytochrome b Group; D005182:Flavin-Adenine Dinucleotide; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C022709:superoxide-forming enzyme