The functional role of thiol groups in protease-solubilized NADPH-cytochrome c reductase from pork-liver microsomes.
The functional role of thiol groups in protease-solubilized NADPH-cytochrome c reductase from pork-liver microsomes.
复制标题
硫醇基团在猪肝微粒体蛋白酶溶解的 NADPH-细胞色素 c 还原酶中的功能作用。
DOI：
--
复制
发表时间：
1977
期刊：
影响因子：
--
通讯作者：
L. Lumper
中科院分区：
文献类型：
--
作者：
T. Lazar;H. Ehrig;L. Lumper
测定了猪肝微粒体中蛋白酶溶解的nadph -细胞色素c还原酶（EC 1.6.2.4）的总-SH含量为每mol蛋白质5.9±0.3 mol硫醇组。蛋白酶溶解的NADPH -细胞色素c还原酶只有3个- sh基团可以在pH 7.5和+ 4℃下被0.34或1.0 mM的5,5′-二硫代双（2-硝基苯甲酸）修饰。在这种处理过程中，超过95%的原始酶活性丧失。在1 mM NADP+存在下，只有两个- sh基团的NADPH -细胞色素c还原酶与5,5 ' -二硫代双（2-硝基苯甲酸）反应。在去除竞争抑制剂NADP+后，得到了具有未修饰酶比活性的酶衍生物，含有两个半胱氨酸残基与2-硝基-5-硫苯甲酸酯混合二硫化物。用0.34或1.0 mM的5,5′-二硫代双（2-硝基苯甲酸）重新处理修饰后的还原酶，在完全失活的情况下，又有一个巯基被修饰（在0.34 mM Nbs2, pH 7.5， + 4°C下，修饰k = 0.027 min - 1，失活k = 0.039 min - 1）。动力学分析证实nadph -细胞色素c还原酶的单个巯基受到NADP+的保护，不受与5,5 ' -二硫代双（2-硝基苯甲酸酯）的反应的影响。细胞色素c （58 μM）明显增强NADP+的作用。1 mM 5,5′-二硫代双（2-硝基苯甲酸酯）修饰可达巯基不影响nadph -细胞色素c还原酶的半还原状态。
The total —SH content of protease-solubilized NADPH—cytochrome c reductase (EC 1.6.2.4) from pork liver microsomes was determined to be 5.9 ± 0.3 mol thiol groups per mol of protein. Only three —SH groups of the protease-solubilized NADPH – cytochrome c reductase could be modified by 0.34 or 1.0 mM 5,5′-dithio-bis(2-nitrobenzoate) at pH 7.5 and + 4°C. More than 95% of the original enzymatic activity was lost during this treatment. In the presence of 1 mM NADP+ only two —SH groups of the NADPH – cytochrome c reductase reacted with 5,5′-dithio-bis(2-nitrobenzoate). After removal of the competitive inhibitor NADP+, an enzyme derivative with the specific activity of the unmodified enzyme was obtained, containing two cysteinyl residues as mixed disulfides with 2-nitro-5-thiobenzoate. Renewed treatment of the modified reductase with 0.34 or 1.0 mM 5,5′-dithio-bis(2-nitrobenzoate) resulted in the modification of one additional thiol group under complete inactivation (k for modification = 0.027 min−1, k for inactivation = 0.039 min−1 at 0.34 mM Nbs2, pH 7.5, + 4°C). Kinetic analysis confirmed the suggestion that a single thiol group of the NADPH—cytochrome c reductase was protected by NADP+ against the reaction with 5,5′-dithio-bis(2-nitrobenzoate). Cytochrome c (58 μM) apparently enhanced the effect of NADP+. The modification of the accessible thiol groups by 1 mM 5,5′-dithio-bis(2-nitrobenzoate) did not affect the half-reduced state of the NADPH–cytochrome c reductase.