Activation of bovine neutrophil oxidase in a cell free system. GTP-dependent formation of a complex between a cytosolic factor and a membrane protein.
The effect of guanine nucleotides on activation of the O2-. generating oxidase in a cell free system consisting of bovine neutrophils membranes, cytosol and arachidonic acid has been studied. In a complete system, GTP-gamma-S was stimulatory and GDP-beta-S inhibitory. When cytosol was omitted, both nucleotides acted as inhibitors. Activation parameters have been explored in a preincubation step prior to the oxidase assay. Stimulation was found to be maximal at 7 to 100 microM GTP-gamma-S. Whereas the time course of activation was monophasic when activation was performed at room temperature, it became biphasic at 2 degrees C, with a first plateau of activation attained after 1 min, followed by a slow rise lasting for more than 30 min. The following lines of evidence demonstrated that oxidase activation resulted from the formation of a complex between cytosolic factor(s) and a target protein in the plasma membrane. 1/ When activated membranes, in a suspension containing cytosol, arachidonic acid and GTP-gamma-S, were separated from soluble components by centrifugation and washed, their oxidase remained fully active. 2/ The activity of the washed membranes was lost upon addition of GDP-beta-S, urea and deoxycholate, but was preserved by addition of glutaraldehyde, a cross-linking reagent. The results of experiments in which cytosol and membrane fractions were incubated separately with GTP-gamma-S, suggested that GTP-gamma-S first interacts with a factor present in the cytosol, before reacting with a target protein in the plasma membrane.
D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000494:pharmacology; D002417:Cattle; D002474:Cell-Free System; D005976:Glutaral / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000097:blood*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D013873:Thionucleotides / Q000494:pharmacology
D001095:Arachidonic Acids; D008565:Membrane Proteins; D013873:Thionucleotides; D006153:Guanosine Diphosphate; D016718:Arachidonic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C023427:guanosine 5'-O-(2-thiodiphosphate); D006160:Guanosine Triphosphate; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D005976:Glutaral