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

多形核白细胞活性氧代谢产物的产生及其调控机制

基本信息

批准号：
01480492
负责人：
ISHIBASHI Sadahiko
金额：
$ 3.58万
依托单位：
Hiroshima University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1991
项目状态：
已结题

项目摘要

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

该研究项目计划表征NADPH氧化酶，并阐明这种在无刺激情况下处于休眠状态的酶的激活机制。以豚鼠腹腔多形核白细胞为实验材料，我们发现胞浆46 kDa蛋白磷酸化与0^2_产生平行，蛋白激酶C（PKC）参与了这种磷酸化。在本研究中，这种蛋白质的易位被发现从胞质到细胞膜。推测活性NADPH氧化酶复合物是由胞浆蛋白与膜蛋白、黄素蛋白、细胞色素B等结合形成的，易位是激活的原因由于PKC磷酸化的重要性，我们从花生四烯酸和SDS的类似作用中提出，磷酸化是通过向胞浆蛋白引入负电荷而起作用的。在后来的ST ...更多信息在本研究中，我们认识到46 kDa蛋白可能对应于已报道的人白细胞胞浆活化因子之一，作为活性复合物形成的另一证据，我们发现用极低浓度的戊二醛处理白细胞，可大大稳定还原型NADPH氧化酶复合物。相反，戊二醛预处理取消了NADPH氧化酶的激活。接下来，我们检查了前（所谓的引发）条件下O^2_的产生。与等渗条件相比，低渗条件下的刺激使产量显著增加。用细胞松弛素处理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去极化模式的变化与0^2_产生的变化程度一致。这些结果表明，膜电位和/或结构参与生产，通过提供启动条件，在膜活性复合物形成之前。少