Activation of the superoxide-generating NADPH oxidase of macrophages by sodium dodecyl sulfate in a soluble cell-free system: evidence for involvement of a G protein.
The superoxide (O2-)-generating NADPH oxidase of resting macrophages can be activated in a soluble cell-free system by certain anionic amphiphiles, such as sodium dodecyl sulfate (SDS). We demonstrate that cell-free activation is specifically enhanced by nonhydrolyzable guanosine 5'-triphosphate (GTP) analogues. Guanosine 5'-diphosphate (GDP) and guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) prevent cell-free oxidase activation and reverse the activated state when added to preactivated oxidase preparations. Nonhydrolyzable GTP analogues have a protective effect on the SDS-stimulated NADPH oxidase, as shown by the maintenance of more than 90% and close to 60% of enzyme activity 6 and 24 hr, respectively, after the addition of SDS to the cell-free preparation. A novel procedure is described for separating the activated NADPH oxidase from SDS and added nucleotides by gel filtration of the SDS-stimulated solubilized membrane-cytosol mixtures through a Sephadex G-25 column. By utilizing this method, it was found that the presence of micromolar concentrations of nonhydrolyzable GTP analogues during activation by SDS results in a marked increase in the recovery of SDS-independent, NADPH-dependent O2(-)-producing activity in the excluded volume of the column. It is suggested that GTP stabilizes the SDS-induced complex between membrane and cytosolic components of the oxidase. Cell-free activation of NADPH oxidase by SDS was found to be cholera and pertussis toxin insensitive. These results serve as evidence of the participation of a G protein in the activation of the O2(-)-generating NADPH oxidase of macrophages by SDS.
Aharoni
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Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Israel.
Pick
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D000818:Animals; D002474:Cell-Free System; D002772:Cholera Toxin / Q000494:pharmacology; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006153:Guanosine Diphosphate / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006168:Guinea Pigs; D008264:Macrophages / Q000201:enzymology*; D009247:NADH, NADPH Oxidoreductases / Q000032:analysis* / Q000037:antagonists & inhibitors; D019255:NADPH Oxidases; D037342:Pertussis Toxin; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology*; D013481:Superoxides / Q000378:metabolism*; D013873:Thionucleotides / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D013873:Thionucleotides; D010566:Virulence Factors, Bordetella; D013481:Superoxides; D006153:Guanosine Diphosphate; D012967:Sodium Dodecyl Sulfate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D002772:Cholera Toxin; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins