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，并阐明该酶在无刺激条件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，我们先前发现胞浆46 kDa蛋白的磷酸化与0^2_2的产生平行，蛋白激酶C(PKC)负责磷酸化。在本研究中，发现该蛋白从胞浆转位到细胞膜。推测胞浆蛋白(S)与膜蛋白、黄素蛋白、细胞色素b等结合形成具有活性的NADPH氧化酶复合体，易位是激活的原因。鉴于PKC磷酸化的意义，我们从花生四烯酸和十二烷基硫酸钠的相似作用出发，提出PKC的磷酸化是通过在胞浆蛋白中引入负电荷来实现的。在后来的st…随着时间的推移，我们认识到46 kDa蛋白可能对应于已报道的人白细胞胞浆激活因子之一。作为活性复合体形成的另一个证据，我们发现，用极低浓度的戊二醛处理白细胞后，所执行的NADPH氧化酶复合体非常稳定。反之，戊二醛的预处理取消了NADPH氧化物酶的激活。接下来，我们考察了预先(所谓的引发)条件对O^2_产生的影响。与等渗条件相比，低渗条件下的增产效果较好。用细胞松弛素处理白细胞也有类似的效果。另一方面，受刺激的白细胞的去极化模式的改变与0^2_产生的变化程度相一致。这些发现表明，在活性复合体形成之前，膜电位和/或结构通过在膜中提供启动条件而参与了合成。较少

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