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

本研究计划对NADPH氧化酶进行表征，并阐明该酶在缺乏刺激的情况下处于休眠状态的激活机制。以豚鼠腹膜多形核白细胞为实验材料。我们之前发现胞质46kDa蛋白在0^2_产生的同时被磷酸化，蛋白激酶C （PKC）负责磷酸化。在本研究中，发现该蛋白从细胞质到细胞膜的易位。假设活化的NADPH氧化酶复合体是由胞质蛋白(s)与膜质蛋白、黄蛋白、细胞色素b等结合形成的，并认为易位是活化的原因。由于PKC磷酸化的意义，我们从花生四烯酸酯和SDS的相似作用中提出磷酸化是通过向胞质蛋白引入负电荷来实现的。在本研究的后期，我们认识到46kDa蛋白可能与报道的人类白细胞的细胞质激活因子之一相对应。作为活性复合物形成的另一个证据，我们发现在极低浓度的戊二醛处理下，已执行的NADPH氧化酶复合物大大稳定了白细胞。相反，戊二醛预处理能抑制NADPH氧化酶的活化。接下来，我们检查了O^2_产生的前面（所谓的启动）条件。与等渗条件相比，低渗条件下的刺激使产量明显增加。用细胞松弛素治疗白细胞也有类似的效果。另一方面，在受刺激的白细胞中观察到退极化模式的变化与0^2_产生的变化程度一致。这些发现表明，在活性复合物形成之前，膜电位和/或结构通过在膜上提供启动条件参与了生产。少

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