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Studies on the production active oxygens by neutrophils and its regulation.

中性粒细胞产生活性氧及其调控的研究。

基本信息

批准号：
02670112
负责人：
TAKESIGE Koichiro
金额：
$ 1.54万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1992
项目状态：
已结题

项目摘要

(1)Studies in a cell-free system(i) We solubilized the membrane component of the NADPH oxidase and fractionated by chromatography on agarose-linked wheat germ agglutinin(WGA).Although each fraction showed no potency for the O_2^--generating activity in a cell-free activation system,that of the membrane component was reconstituted from the fraction passing through the column and that eluted with N-acetylglucosamine.The former fraction could be replaced by a phospholipid,such as azolectin.(ii) We found that Mg^<2+> seems to modulate the activation of the NADPH oxidase at the level of the G-protein.(iii) Because the alignments of the amino acid sequence of the large subunit of the cytochrome b_<558> with those of previously characterized flavoproteins revealed that the middle and C-terminal portions of the cytochrome are likely to be FAD-and NADPH-binding domains,respectively, cytochrome b_<558> appears to be a flavoprotein with an NADPH-binding site of the NADPH oxidase.(iv) The effect of bilirubin on the NADPH-dependent O_<2"> production was studied in a cell-free system,and the results indicate that bilirubin inhibits the activation process of the NADPH oxidase by decreasing the potency of the cytosolic fraction.(2) Studies with intact or electropermeabilized neutrophils(i) We studied the action of iodinated thyronines of O_2^- production of human neutrophils stimulated by a chemotactic peptide,FMLP,in vitro and found that thyroxine and tri iodothyronine interfere with the binding of FMLP to the receptor,leading to the inhibition of neutrophil functions such as O_2^- production.(ii) We showed by using electropermeabilized human neutrophils that c AMP inhibited the activation of the NADPH oxidase not only the at the site before the protein kinase C but also at another site after the kinase.

(1)在无细胞系统中的研究(i)我们将NADPH氧化酶的膜组分溶解，并在琼脂糖连接的小麦胚芽凝集素（WGA）上进行色谱分离。虽然在无细胞活化系统中，每个组分都没有表现出生成O_2^的活性，但通过柱的组分和n -乙酰氨基葡萄糖洗脱的组分可以重建膜组分的活性。前一部分可以用磷脂代替，如唑粘素。（ii）我们发现Mg^<2+>似乎在g蛋白水平上调节NADPH氧化酶的激活。（iii）由于细胞色素b_<558>的大亚基氨基酸序列与先前鉴定的黄蛋白的氨基酸序列比对表明，细胞色素的中间和c端部分可能分别是fad和NADPH结合域，因此细胞色素b_<558>可能是具有NADPH氧化酶NADPH结合位点的黄蛋白。（iv）在无细胞系统中研究了胆红素对NADPH依赖性O_<2”>产生的影响，结果表明胆红素通过降低胞质部分的效力抑制NADPH氧化酶的激活过程。(1)在体外研究了趋化肽FMLP刺激人中性粒细胞产生O_2^-的碘化甲状腺原氨酸的作用，发现甲状腺素和三碘甲状腺原氨酸干扰FMLP与受体的结合，导致中性粒细胞产生O_2^-等功能受到抑制。（ii）通过电渗透人中性粒细胞，我们发现c AMP不仅在蛋白激酶c之前的位点抑制NADPH氧化酶的激活，而且在激酶c之后的另一个位点也抑制NADPH氧化酶的激活。