Immunological properties of O•−2 generating oxidase from bovine neutrophils

AbstractTwo antisera have been prepared against the O•−2 generating oxidase purified from bovine polymorphonuclear neutrophils (PMNs). The first antiserum was directed against the enzymatically active fraction obtained after isoelectric focusing (pI oxidase), which consisted of a major protein of Mr 65 000 [(1985) Biochemistry 24, 7231–7239]. The second antiserum was directed against the 65 kDa band excised from an SDS-polyacrylamide gel after electrophoresis of the pI oxidase preparation. The pI oxidase antiserum inhibited O•−2 generation by PMN cells, PMN membranes and detergent-solubilized membranes. The 65 kDa band antiserum was virtually non-inhibitory against PMN cells; in contrast, it was nearly as potent as the pI oxidase antiserum on PMN membranes and detergent-solubilized membranes. Inhibition of O•−2 generation by the pI oxidase antiserum was correlated with the immunoreactivity of four membrane-bound proteins of 65, 54, 18 and 16 kDa; the 65 kDa band antiserum reacted only with the two proteins of 65 and 54 kDa. It is concluded that the 18 and 16 kDa proteins, present in trace amounts in the pI oxidase preparation, are probably potent catalysts of the respiratory burst

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

International audienceThe 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., & 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.(ABSTRACT TRUNCATED AT 250 WORDS