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 O 2 ⨪ generating oxidase in a cell free system consisting of bovine neutrophils membranes, cytosol and arachidonic acid has been studied. In a complete system, GTP-γ-S was stimulatory and GDP-β-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 μM GTP-γ-S. Whereas the time course of activation was monophasic when activation was performed at room temperature, it became biphasic at 2°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-γ-S, were separed 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-β-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-γ-S, suggested that GTP-γ-S first interacts with a factor present in the cytosol, before reacting with a target protein in the plasma membrane.