Activation of the superoxide-forming NADPH oxidase of macrophages requires two cytosolic components--one of them is also present in certain nonphagocytic cells.
The superoxide-forming NADPH oxidase of resting macrophages can be activated in a cell-free system by certain anionic amphiphiles, most notably SDS. Activation requires the cooperation of membrane-associated and cytosolic components. We now report that at least two cytosolic factors are required for SDS-elicited activation of NADPH oxidase of guinea pig macrophages. Treatment of cytosol with ammonium sulfate at 37% saturation led to the partition of the two factors in the supernatant and precipitate fractions (termed components sigma 1 and sigma 2, respectively). Although each fraction by itself was inactive, recombining them resulted in complete recovery of the original ability of native cytosol to support SDS-elicited superoxide production by octyl-glucoside solubilized macrophage membranes. Both components are proteins, as shown by their susceptibility to trypsin and proteinase K, and were inactivated by heating at 60 degrees C. sigma 2, but not sigma 1, was inactivated by treatment with the covalent sulfhydryl reagent N-ethylmaleimide. On high-performance gel filtration, sigma 1 was found to have a molecular mass of 30 to 52 kDa, whereas sigma 2 eluted with molecules of 150 to 440 kDa. Component sigma 1 was partially purified from the ammonium sulfate supernatant fraction of cytosol by hydrophobic interaction chromatography followed by gel filtration. A material behaving like sigma 1 was also found to be present in the cytosol of guinea pig thymus cells, lymph node lymphocytes and brain and of the mouse myeloma cell line MOPC 315. However, sigma 2 appears to be strictly phagocyte specific. The molecular characteristics of sigma 1 components from nonphagocytic cells were similar to those of macrophage sigma 1, as shown by their presence in the supernatant, after treatment of cytosol with ammonium sulfate at 37% saturation, a molecular mass close to 30 to 52 kDa and a similar behavior on hydrophobic interaction chromatography. These findings raise the possibility that cytosolic component sigma 1 might be the bearer of a cellular function, more general than the one suggested by its role in the activation of NADPH oxidase of phagocytes.
Pick
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Department of Human Microbiology, Sackler School of Medicine, Tel Aviv, Israel.
Kroizman
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Abo
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D000645:Ammonium Sulfate; D000818:Animals; D001921:Brain / Q000201:enzymology; D002460:Cell Line; D002474:Cell-Free System; D003600:Cytosol / Q000201:enzymology*; D004789:Enzyme Activation; D005033:Ethylmaleimide; D005590:Fractional Precipitation; D006168:Guinea Pigs; D008198:Lymph Nodes / Q000201:enzymology; D008214:Lymphocytes / Q000201:enzymology; D008264:Macrophages / Q000201:enzymology*; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases; D010447:Peptide Hydrolases; D013481:Superoxides / Q000378:metabolism*; D013696:Temperature
D013481:Superoxides; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D010447:Peptide Hydrolases; D005033:Ethylmaleimide; D000645:Ammonium Sulfate