Multi-step mechanism for activation of active oxygen-producing system in leukocytes

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Mechanism for activation of production of O_2, a primary product among active oxygen metabolites, in polymirphonuclear leukocytes was investigated. It is known that the activation of the producing system, NADPH oxidase, is achieved by an association of membrane factors, such as cytochrome b_<558>, and cytosolic factos, such as p47-phox, p67-phox, rac, etc., but it has not been fully clarified how these proteins are assembled to form the active complex on plasma membranes from the disassemvled resting state. We first observed that membrane perturbation as manifested by transient deporalization and increase in membrane fluidity served as the priming step for the production. We have long been involved in studies to show that production of O_2, was increased in accordance with phosphorylation by PKC and concurrent translocation to cell membranes of a cytosolic 46 kDa protein (equivalent to p47-phox), abd developed the line in this study. It was feasible that introduction of phosphate was a significant step for the ativation and atabilization of NADPH oxidase complex. In fact, treatment with protein phosphatase inhibitor resulted increase in O_2 production. However, amphiphilic acidic compounds, such as arachidonic acid and SDS,induced similar translocation and activation without phosphorylation. Since these compounds cause membrane perturbation and have negative charge, those factors may act synergistically. In conclusion, O_2 production in leukocytes proceeds as through multi-steps and at least two PKC-dependent and-independent ways.