Isolation from neutrophil membranes of a complex containing active NADPH oxidase and cytochrome b-245.
NADPH-dependent O2- forming activity was extracted with deoxycholate from subcellular particles of guinea-pig neutrophils following stimulation with phorbol myristate acetate. The solubilized enzyme was purified by chromatography on Ultrogel AcA22, by isopycnic glycerol density gradient centrifugation and by treatment with 0.4 M NaCl. This procedure yielded a high-molecular-weight complex containing phospholipids, cytochrome b-245 and NADPH oxidase activity. Cytochrome b was found to be purified to the same extent as NADPH oxidase activity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the various purification fractions showed a progressive enrichment of a band whose molecular weight is 3.2 X 10(4). The enrichment of this protein band paralleled those of NADPH oxidase activity and of cytochrome b, indicating that it is a component of the oxidase system. The possibility that this band corresponds to either cytochrome b or a flavoprotein/cytochrome b complex is considered.
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D003573:Cytochrome b Group / Q000097:blood*; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D009247:NADH, NADPH Oxidoreductases / Q000097:blood*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D010743:Phospholipids / Q000097:blood; D012995:Solubility; D013347:Subcellular Fractions / Q000201:enzymology
D003573:Cytochrome b Group; D010743:Phospholipids; C035130:cytochrome b245; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases