Production of active oxygen metabolites in polymorphonuclear leukocytes and regulatory mechanism for the production

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This research project was planned to characterized NADPH oxidase as well as to elucidate the mechanism for the activation of this enzyme which is dormant in the absence of stimulation. Guinea pig peritoneal polymorphonuclear leukocytes were used as the experimental material.We previously found that cytosolic 46kDa protein was phosphorylated in parallel with 0^2_ production and protein kinase C (PKC) was responsible for the phosphorylation. In the present study, translocation of this protein was found from the cytosol to the cell membranes. It was assumed that active NADPH oxidase complex was formed by the association of the cytosolic protein (s) and membranous proteins, flavoprotein, cytochrome b, etc., and that the translocation was responsible for the activation. As the significance of the phosphorylation by PKC, we proposed from the similar effect of arachidonate and SDS that the phosphorylation worked through introduction of negative charge to the cytosolic protein. In the later st … More age of the present study, we recognized that the 46kDa protein corresponded probably to oneof the cytosolic activating factors reported for human leukocytes.As another evidence for the active complex formation, we found that performed NADPH oxidase complex was greatly stabilized by the treatment of the leukocytes with glutaraldehyde at very low concentration. On the contrary, the pretreatment with glutaraldehyde canceled the activation of NADPH oxidase.Next, we examined preceding (so-called priming) condition for O^2_ production. As compared with isotonic condition, the stimulation under hypotonic condition caused much increase in the production. Treatment of the leukocytes with cytochalasin had similar effect. On the other hand, change in depolarization pattern was observed in the stimulated leukocytes in accordance with the degree of the change in 0^2_ production. These findings indicate that membrane potential and /or structure is involved in the production through providing the priming ondition in the membranes prior to the active complex formation. Less