Purification and some properties of the 45 kDa diphenylene iodonium-binding flavoprotein of neutrophil NADPH oxidase.
The 45 kDa diphenylene iodonium-binding flavoprotein of the human neutrophil superoxide-generating oxidase has been purified by affinity chromatography. The polypeptide was eluted from Blue Memsep or 2',5'-ADP-agarose columns with either NADP or low concentrations of the specific inhibitor diphenylene iodonium. The purified protein was shown to bind FAD at a ratio of 1.09 mol of FAD/mol of protein. The reconstituted flavoprotein had a fluorescence spectrum similar, but not identical, to that of free FAD. It had an isoelectric point of approx. 4.0. The reconstituted flavoprotein displayed no diaphorase activity towards a range of artificial electron acceptors. Polyclonal antibodies raised against the pure protein inhibited superoxide generation by solubilized oxidase in a dose-dependent manner, and inhibited superoxide generation when incubated with either cytosol or membrane fractions in a reconstituted system. These antibodies precipitated the 45 kDa polypeptide together with a haem-containing 23 kDa protein thought to be the small subunit of cytochrome b-245. Antibodies raised against cytochrome P-450 reductase also precipitated these two polypeptides. These results are consistent with the 45 kDa polypeptide being the flavoprotein of the neutrophil superoxide-generating oxidase.
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Department of Biochemistry, School of Medical Sciences, University of Bristol, U.K.
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Jones
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D002474:Cell-Free System; D002850:Chromatography, Gel; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005420:Flavoproteins / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007525:Isoelectric Focusing; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D009861:Onium Compounds / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013481:Superoxides / Q000378:metabolism
D005420:Flavoproteins; D009861:Onium Compounds; D013481:Superoxides; C007517:diphenyleneiodonium; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D008058:Dihydrolipoamide Dehydrogenase