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Purification and Molecular Mechanisms of O^-_ generating System in Polymorphonuclear Leukocytes and Thyroid Cells

多形核白细胞和甲状腺细胞O^-_生成系统的纯化及分子机制

基本信息

批准号：
63480505
负责人：
ISHIMURA Yuzuru
金额：
$ 4.29万
依托单位：
Keio 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：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

1. O^-_ generating system in PMNsO^-_ generating enzyme system in polymorphonuclear leukocytes (PMNS) is called NADPH oxidase. We studied the activation mechanisms of the oxidase in a cell-free preparation from porcine PMNS. To activate the oxidase in vitro, membrane fraction (the enzyme), cytosol fraction and fatty acid were required. The activation was reversible. The activated oxidase was deactivated by removal of fatty acid and reactivated by replenishment of both fatty acid and fresh cytosol fraction. Effective component in cytosol fraction (cytosolic factor, CF) seemed to be consumed or translocated to the membrane fraction during the activation. To clarify further mechanisms of the activation we tried to purify and characterize the CF. By anion exchange chromatography it was fractionated into two essential components. One of them was purified to near homogeneity which gave a main band at 60 kD on SDS polyacrylamide gel electrophoresis. Antibody raised against the 60 kD protein inhibited the activation of the oxidase. Function of the CF is under investigation. Purification of another CF and components of NADPH oxidase is now in progress.2. O^-_ generating system in thyroid cellsIt is widely known that hydrogen peroxide (H_2O_2) is required for thyroid hormone biosynthesis. However, little is known about molecular mechanisms of the H_2O_2 forming oxidase system in thyroid cells. Our new method for discrimination between H_2O_2 and O^-_ revealed that the plasma membrane fraction of the cells produced 02- as the primary metabolite of oxygen by using NADPH as an electron donor. On the other hand, intact thyroid cells released only H_2O_2 into extracellular medium. As superoxide dismutase activity in the cells was more than 10 times higher than that in neutrophils, we concluded that O^-_ was produced by the oxidase initially and then efficiently dismutated to H_2O_2 for the reaction with thyroid peroxidase in physiological conditions.

1. 多形核白细胞（PMNS）生成O^ _酶系统中的O^ _生成系统称为NADPH氧化酶。我们研究了猪PMNS无细胞制剂中氧化酶的活化机制。为了在体外激活氧化酶，需要膜组分（酶）、细胞质组分和脂肪酸。激活是可逆的。活化的氧化酶通过去除脂肪酸而失活，通过补充脂肪酸和新鲜细胞质组分而重新激活。胞浆因子（cytosolic factor， CF）在活化过程中被消耗或转移到膜组分中。为了进一步阐明其活化机制，我们对其进行了纯化和表征。通过阴离子交换色谱法将其分离为两种主要成分。其中一株经纯化后均质性较好，在SDS聚丙烯酰胺凝胶电泳上有60 kD的主带。抗60kd蛋白的抗体抑制了氧化酶的激活。CF的功能正在调查中。另一种CF和NADPH氧化酶组分的纯化正在进行中。甲状腺细胞O -_生成系统众所周知，过氧化氢（H_2O_2）是甲状腺激素生物合成所必需的。然而，甲状腺细胞中H_2O_2形成氧化酶系统的分子机制尚不清楚。我们新的区分H_2O_2和O^-_的方法表明，细胞的质膜部分以NADPH作为电子供体产生o2 -作为氧的主要代谢物。另一方面，完整的甲状腺细胞仅向细胞外介质中释放H_2O_2。由于细胞内超氧化物歧化酶的活性比中性粒细胞高10倍以上，因此我们认为，在生理条件下，O^-_首先由氧化酶产生，然后有效地歧化成H_2O_2与甲状腺过氧化物酶反应。