The membrane-associated component of the amphiphile-activated, cytosol-dependent superoxide-forming NADPH oxidase of macrophages is identical to cytochrome b559.
The superoxide (O2-) forming NADPH oxidase complex of resting phagocytes can be activated in a cell-free system by certain anionic amphiphiles, such as sodium dodecyl sulfate (SDS). For O2- production to occur, the participation of both membrane-associated and cytosol-derived components is required. The purpose of this investigation was to isolate and characterize the membrane component of NADPH oxidase. For this purpose, guinea pig macrophage membranes were extracted with 1 M NaCl, solubilized by 40 mM octyl glucoside, and subjected to a purification sequence consisting of absorption with DEAE-Sepharose, affinity chromatography on heparin-agarose, and chromatography on hydroxylapatite. At each purification step, fractions were assayed for their ability to support SDS-elicited, cytosol-dependent O2- production, following incorporation in liposomes of phosphatidylcholine. We found that membrane oxidase activity copurified strictly with cytochrome b559. Peak hydroxylapatite fractions exhibited specific O2(-)-forming activity in the range of 81-115 mumol of O2-/mg protein/min and a specific cytochrome b559 content of 7-14 nmol of cytochrome b559/mg protein. SDS-polyacrylamide gel electrophoresis analysis of the peak oxidase activity fractions, derived by hydroxylapatite chromatography, revealed essentially two bands that were identified as the beta (54-60 kDa) and alpha (21/22 kDa) subunits of guinea pig cytochrome b559. The relation of the two polypeptides to cytochrome b559 was established by correlation with a spectral signal characteristic of cytochrome b559, immunoblotting with antibodies against defined human cytochrome b559 beta and alpha chain peptides, cross-linking studies, and deglycosylation experiments. Hydroxylapatite-purified membrane oxidase preparations did not contain FAD and were free of cytochrome c reductase activity. Purified membrane oxidase preparations were also capable of cooperating with purified cytosolic components in SDS-elicited cell-free O2- production. We conclude that the membrane-associated component of the O2- generating NADPH oxidase is identical to cytochrome b559.
Knoller
S
S
Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Israel.
Shpungin
S
S
Pick
E
E
D000818:Animals; D015153:Blotting, Western; D002853:Chromatography, Liquid; D003573:Cytochrome b Group / Q000737:chemistry*; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006168:Guinea Pigs; D008264:Macrophages / Q000201:enzymology*; D009245:NADH Dehydrogenase / Q000032:analysis; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry*; D019255:NADPH Oxidases; D045332:Photosystem II Protein Complex; D013481:Superoxides / Q000737:chemistry*
D003573:Cytochrome b Group; D045332:Photosystem II Protein Complex; D013481:Superoxides; C019313:cytochrome b559; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D009245:NADH Dehydrogenase