Activation of the superoxide radical generating oxidase in plasma membrane from bovine polymorphonuclear neutrophils by arachidonic acid, a cytosolic factor of protein nature, and nonhydrolyzable analogs of GTP

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Abstract
A reconstitution system for activation of the O2.--generating oxidase from bovine polymorphonuclear neutrophils (PMN) is described. This system consisted of three components, namely, a particulate fraction enriched in plasma membrane, a supernatant fluid (cytosolic fraction) recovered by high-speed centrifugation from sonicated resting bovine PMN, and arachidonic acid. The pH optimum (7.8) and the KM value for NADPH (45 .mu.M) of the activated oxidase were virtually the same as those found in the purified enzyme. All three components had to be present during the preincubation for elicitation of oxidase activity. A further enhancement of oxidase activity was observed with the addition of nonhydrolyzable GTP analogues, such as guanosine 5''-O-(3-thiotriphosphate) (GTP-.gamma.-S) and guanosine 5''-(.beta.,.gamma.-imidotriphosphate) (GMP-PNP), to the preincubation medium. In contrast, GDP-.beta.-S drastically decreased oxidase activation. In a two-stage experiment, a 9-min preincubation of PMN membranes with arachidonic acid and GTP-.gamma.-S followed by a 1-min contact with the cytosolic fraction led to a more marked activation than did preincubation of the cytosol with arachidonic acid and GTP-.gamma.-S for 9 min followed by a 1-min contact with membranes, suggesting the presence of a G-protein in the membrane fraction. In the absence of added cations, the reconstitution system exhibited a substantial oxidase activity which was totally prevented by ethylenediaminetetraacetic acid (EDTA). Mg2+ added at a concentration of 0.5-1 mM enhanced oxidase activation by about 30%, indicating that endogenous Mg2+ or other activating cations were sufficient to ensure 70% of maximal activation. ATP was not required, and all assays carried out to check whether protein phosphorylation occurred during the activation process were negative. In particular, protein kinase C did not appear to be directly involved. The ratio of arachidonic acid to the amount of membrane material was the critical factor in the activation process. Maximal activation occurred with a ratio of 1 mol of arachidonic acid per 4 mol of membrane phospholipids. Long-chain unsaturated fatty acids of the cis and trans conformation were as effective as arachidonic acid. The protein nature of the factor present in the cytosolic fraction was demonstrated by loss of activity upon trypsin and proteinase K treatment. The cytosolic factor was found in PMN and not in the other tissues, such as brain and heart; in contrast, its species specificities were rather broad, as cytosol from bovine PMN could replace cytosol from rabbit PMN to activate the oxidase from rabbit PMN membranes. The maximal rate of O2.- production dependent on the amount of cytosol protein in contact with the membrane fraction, indicating that activation was not a catalytic process but involved a stoichiometric interaction between the cytosolic factor and a strategic component of the membrane fraction. Oxidase activation was temperature-dependent. The plateau of activation was attained in about 5 min at room temperature (22-25.degree.C) and in more than 30 min at 0.degree.C.

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