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

1.多形核白细胞（PMNS）中的O^-_产生系统多形核白细胞（PMNS）中的O^-_产生酶系统称为NADPH氧化酶。我们研究了猪PMNS无细胞制剂中氧化酶的激活机制。为了在体外激活氧化酶，需要膜组分（酶）、胞质组分和脂肪酸。激活是可逆的。通过去除脂肪酸使活化的氧化酶失活，并通过补充脂肪酸和新鲜细胞质组分使其重新活化。胞质中的有效成分（胞质因子，CF）似乎在激活过程中被消耗或转移到膜组分中。为了进一步阐明活化机制，我们试图纯化和表征CF。通过阴离子交换色谱法，它被分馏成两个基本组分。其中一株经SDS聚丙烯酰胺凝胶电泳，在60 kD处有一条主带。针对60 kD蛋白质的抗体抑制了氧化酶的活化。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。甲状腺细胞内的氧生成系统众所周知，过氧化氢（H_2O_2）是甲状腺激素生物合成所必需的。然而，对甲状腺细胞中H_2O_2形成氧化酶系统的分子机制知之甚少。我们的新方法区分H_2O_2和O^-_表明细胞质膜部分通过NADPH作为电子供体产生O 2-作为氧的初级代谢产物。而完整甲状腺细胞仅释放H_2O_2进入细胞外液。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，超氧化物歧化酶首先产生O^-，然后有效地歧化为H_2O_2，与甲状腺过氧化物酶反应。

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.