Parameters of activation of the membrane-bound O2- generating oxidase from bovine neutrophils in a cell-free system

Parameters governing the extent of activation of the O2- generating oxidase in a cell-free system derived from bovine neutrophils were examined. The reconstituted system consisted of the following: a particulate fraction enriched in plasma membrane and containing the oxidase, a soluble fraction containing cytosolic factor(s) required for oxidase a soluble fraction containing cytosolic factor(s) required for oxidase activation, a non hydrolyzable analog of GTP, and either arachidonic acid or sodium dodecyl sulfate. When the amount of arachidonic acid or sodium dodecyl sulfate was maintained at a fixed value with respect to the amount of membrane used, a sigmoidal response of oxidase activity to increasing amounts of cytosol added was observed. In contrast, when the concentration of arachidonic acid or sodium dodecyl sulfate was properly adjusted with respect to that of membrane and cytosol, the curve relating oxidase activity to cytosol was hyperbolic, pointing to a simple michaelian relationship for the dependence of oxidase activation on the activating factor(s) of cytosol. Another parameter affecting oxidase activation was the ionic strength of the reconstitution medium, the extent of activation being lower at high ionic strength.