Cytosolic factors in bovine neutrophil oxidase activation. Partial purification and demonstration of translocation to a membrane fraction

Abstract

The O2.--generating oxidase of bovine neutrophils is activated in a cell-free system consisting of a particulate fraction enriched in plasma membrane and containing the dormant oxidase, a high-speed supernatant from neutrophil homogenate (cytosol), Mg ions, GTP.gamma.S, and arachidonic acid [Ligeti, E., Doussiere, J., and Vignais, P. V. (1988) Biochemistry 27, 193-200]. The cytosolic components participating in the activation of the membrane-bound oxidase have been investigated. These components were resolved into several active peaks by Q Sepharose chromatography. The oxidase-activating potency of these peaks was synergistically enhanced by combining samples from separate peaks, or by supplying them with a threshold amount of crude cytosol. Partial purification of two active fractions containing a limited number of proteins of 65, 56, 53, and 45 kDa was achieved by gel filtration of cytosol on Ultrogel AcA44, followed by chromatography on hydroxylapatite and Mono Q. The specific oxidase-activating potency of these partially purified fractions (activating potency per milligram of soluble protein) was 6-8-fold higher than that of crude cytosol; it was enhanced up to 75-fold by complementation with a minute amount of crude cytosol, which per se had a limited efficiency. These data indicate that oxidase activation requires more than one cytosolic component to be activated. To check whether translocation of cytosolic proteins to the membrane occurred concomitantly with oxidase activation, use was made of radiolabeled cytosolic proteins. Cytosol was treated with phenyl [14C]isothiocyanate ([14C]PITC), such that 60% of its activation potency was still present. Translocation was studied under conditions in which production of O2.- was largely modulated by varying the amount of arachidonic acid added to the cell-free system. Maximal oxidase activation with optimal concentration of arachidonic acid resulted in the selective translocation of labeled cytosolic proteins of 65, 53, 45, and 17 kDa to the membrane.