Activation of O2·- Generating Oxidase of Bovine Neutrophils in a Cell-Free System. Interaction of a Cytosolic Factor with the Plasma Membrane and Control by G Nucleotides

Erzsebet Ligeti, Marianne Tardif, Pierre V. Vignais

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Abstract

Activation of the O2·--generating oxidase of bovine neutrophils was studied in a cell-free system, consisting of a particulate fraction enriched in plasma membrane, cytosol, arachidonic acid, and the non-hydrolyzable nucleotide GTP-γ-S. Activation of the membrane-bound oxidase was accompanied by the disappearance of the activating factor from the cytosol. Above a cytosol to membrane ratio of 25, the excess of added cytosolic factor remained in active state in the soluble fraction. The process could be partially reversed by serum albumin. Disappearance of the cytosolic factor was promoted by unsaturated long-chain fatty acids, but not by saturated ones, and occurred not only in the presence of GTP-γ-S but also in the presence of GTP-β-S or in the absence of Mg ions, although in the latter cases activation of O2·- production was seriously impaired. This suggests that the disappearance of the activating factor from the cytosol and the triggering effect of GTP-γ-S are related, but distinct, events in the oxidase activation process. The disappearance of the activating factor from cytosol can be explained by translocation of the cytosolic factor to the membrane fraction. Yet under some conditions, including the presence of GTP-β-S or EDTA, inactivation was prevailing and could be an alternative explanation for the results. Specific binding of radiolabeled GTP-γ-S could be demonstrated both in the membrane and in the cytosolic fractions. Although a substantial amount of GTP-γ-S was able to bind to the membrane proteins, its effect on oxidase activation was moderate compared to that of GTP-γ-S bound to cytosolic proteins. Oxidase activation was correlated with the binding of GTP-γ-S to cytosolic proteins in a range of concentrations from approximately 0.3 to 2.5 μM GTP-γ-S, with a GTP-γ-S concentration of ≃1 μM corresponding to the half-maximal oxidase activation. A GTP-binding protein of Mr = 23 000 was detected in cytosol; its function in oxidase activation remains to be assessed.

Original languageEnglish
Pages (from-to)7116-7123
Number of pages8
JournalBiochemistry
Volume28
Issue number17
DOIs
Publication statusPublished - Jan 1 1989

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ASJC Scopus subject areas

  • Biochemistry

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