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.