Composition of partially purified NADPH oxidase from pig neutrophils.
The superoxide (O2.-)-forming enzyme NADPH oxidase from pig neutrophils was solubilized and partially purified by gel-filtration chromatography. The purification procedure allowed the separation of NADPH oxidase activity from NADH-dependent cytochrome c reductase and 2,6-dichlorophenol-indophenol reductase activities. O2.-forming activity was co-purified with cytochrome b-245 and was associated with phospholipids. However, active fractions endowed with cytochrome b were devoid of ubiquinone and contained only little FAD. The cytochrome b/FAD ratio was 1.13:1 in the crude solubilized extract and increased to 18.95:1 in the partially purified preparations. Most of FAD was associated with fractions containing NADH-dependent oxidoreductases. These results are consistent with the postulated role of cytochrome b in O2.-formation by neutrophil NADPH oxidase, but raise doubts about the participation of flavoproteins in this enzyme activity.
D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003573:Cytochrome b Group / Q000097:blood; D005182:Flavin-Adenine Dinucleotide / Q000097:blood; D009247:NADH, NADPH Oxidoreductases / Q000097:blood* / Q000302:isolation & purification; D019255:NADPH Oxidases; D009251:NADPH-Ferrihemoprotein Reductase / Q000097:blood; D009504:Neutrophils / Q000201:enzymology*; D011808:Quinone Reductases / Q000097:blood; D013347:Subcellular Fractions / Q000201:enzymology; D013552:Swine; D014451:Ubiquinone / Q000097:blood
D003573:Cytochrome b Group; D014451:Ubiquinone; D005182:Flavin-Adenine Dinucleotide; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase; D019255:NADPH Oxidases; D011808:Quinone Reductases; C033633:dichlorophenolindophenol reductase