Sulfhydryl-disulfide modulation and the role of disulfide oxidoreductases in regulation of the catalytic activity of nitric oxide synthase in pulmonary artery endothelial cells.
Sulfhydryl-disulfide modulation and the role of disulfide oxidoreductases in regulation of the catalytic activity of nitric oxide synthase in pulmonary artery endothelial cells.
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硫氢基二硫键调节和二硫键氧化还原酶在肺动脉内皮细胞一氧化氮合酶催化活性调节中的作用。
DOI：
10.1165/ajrcmb.13.3.7544597
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Block,ER
中科院分区：
文献类型：
--
作者：
Patel,JM;Block,ER
本文研究了巯基、二硫键和二硫氧化还原酶在调节猪肺动脉内皮细胞（PAEC）一氧化氮（NO）合酶膜结合型异构体催化活性中的作用。用SH烷基化剂N-乙基马来酰亚胺（NEM）（10至50 μ M）或分子内二硫键形成剂二酰胺（20至100 μ M）处理完整的PAEC或从PAEC分离的总膜制备物，导致NO合酶活性以剂量依赖性方式降低。当从PAEC的膜部分中纯化的NO合酶在NEM存在下孵育时，观察到类似的酶活性损失。NEM和二酰胺处理的制剂的膜蛋白SH含量的损失与NO合酶活性的损失有关。相比之下，当完整的PAEC或分离的总膜来自PAEC用增加浓度（1至5 mM）的二硫化物还原剂二硫苏糖醇（DTT），但不氧化DTT，NO合酶活性增加了20至85%。DTT减少天然二硫化物从NEM处理的制剂或二酰胺处理后形成的二硫化物的膜逆转NO合酶活性的抑制。类似地，硫氧还蛋白/硫氧还蛋白还原酶的酶促还原，但不是谷氧还蛋白，逆转了从二酰胺处理的细胞中分离的膜组分和纯化的NO合酶的抑制。这种酶催化的二硫键还原比DTT诱导的还原效率高> 1，000倍。（250字处删节）
The role of sulfhydryl groups (SH) and disulfide bonds as well as disulfide oxidoreductases in regulation of the catalytic activity of the membrane-bound constitutive isoform of nitric oxide (NO) synthase from porcine pulmonary artery endothelial cells (PAEC) was examined. Treatment of intact PAEC or a total membrane preparation isolated from PAEC with the SH alkylating agent N-ethylmaleimide (NEM) (10 to 50 microM) or with the intramolecular disulfide-forming agent diamide (20 to 100 microM) resulted in the reduction of NO synthase activity in a dose-dependent fashion. Similar loss of enzyme activity was observed when purified NO synthase from the membrane fraction of PAEC was incubated in the presence of NEM. The loss of membrane protein SH content from NEM- and diamide-treated preparations was associated with loss of NO synthase activity. In contrast, when intact PAEC or isolated total membranes derived from PAEC were treated with increasing concentrations (1 to 5 mM) of the disulfide-reducing agent dithiothreitol (DTT), but not oxidized DTT, NO synthase activity was increased by 20 to 85%. DTT reduction of native disulfides from NEM-treated preparations or of disulfides formed after diamide treatment of membranes reversed the inhibition of NO synthase activity. Similarly, enzymatic reduction by thioredoxin/thioredoxin reductase, but not by glutaredoxin, reversed the inhibition of membrane fraction and purified NO synthase isolated from diamide-treated cells. This enzyme-catalyzed disulfide reduction was > 1,000-fold more efficient than the DTT-induced reduction.(ABSTRACT TRUNCATED AT 250 WORDS)