Dotan I, Shechter I
Biochim Biophys Acta. 1982 Nov 12;713(2):427-34. doi: 10.1016/0005-2760(82)90262-4.
The activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (hydroxymethylglutaryl-CoA reductase, EC 1.1.1.34) in preparations of thiol-deficient rat liver microsomes and microsomes containing thiols have been compared. Unlike microsomes containing thiols, which possess an active hydroxymethylglutaryl-CoA reductase (Ea), thiol-deficient microsomes contain an inactive, latent enzyme (E1) which can be activated by addition of thiols. Ea can be converted to E1 by dialysis. The maximal degree of activation of E1 depends on the activating thiol with the order of effectiveness: dithioerythritol = dithiothreitol greater than glutathione (GSH) greater than cysteine. Ea is inhibited by oxidized glutathione (GSSG). The degree of the inhibition of Ea by GSSG is proportional to the ratio GSSG/thiol in the reaction. E1 was solubilized from microsomes and purified. Its molecular weight is estimated to be 104 000 by gel filtration chromatography on Sepharose 6B. The reducing agents NaBH4, dithionite and ascorbate failed to activate E1. NaBH4 did not inhibit Ea whereas only partial inhibition was caused by ascorbate and dithionite. Soluble Ea binds to both blue dextran/Sepharose 4B and agarose/hexane-3-hydroxy-3-methylglutaryl Coenzyme A affinity resins at low-salt concentrations. By contrast, soluble E1 did not bind to agarose/hexane-hydroxymethylglutaryl-CoA whereas quantitative binding of E1 to blue dextran/Sepharose 4B was still observed at low salt concentrations. These results indicate that thiols are necessary cofactors for hydroxymethylglutaryl-CoA reductase reaction. Their effect on the activation of E1 is not caused by change in the state of aggregation of the enzyme. Rather, the reversible change of the enzyme from E1 to Ea is affected by increasing the affinity of the enzyme to the substrate hydroxymethylglutaryl-CoA.
已对硫醇缺乏型大鼠肝微粒体和含硫醇微粒体制剂中3-羟基-3-甲基戊二酰辅酶A还原酶（羟甲基戊二酰辅酶A还原酶，EC 1.1.1.34）的活性进行了比较。与含有具有活性羟甲基戊二酰辅酶A还原酶（Ea）的硫醇的微粒体不同，硫醇缺乏的微粒体含有一种无活性的潜伏酶（E1），可通过添加硫醇来激活。Ea可通过透析转化为E1。E1的最大激活程度取决于激活硫醇，其有效性顺序为：二硫赤藓糖醇 = 二硫苏糖醇大于谷胱甘肽（GSH）大于半胱氨酸。Ea被氧化型谷胱甘肽（GSSG）抑制。GSSG对Ea的抑制程度与反应中GSSG/硫醇的比例成正比。E1从微粒体中溶解并纯化。通过在Sepharose 6B上进行凝胶过滤色谱法估计其分子量为104000。还原剂硼氢化钠、连二亚硫酸盐和抗坏血酸未能激活E1。硼氢化钠不抑制Ea，而抗坏血酸和连二亚硫酸盐仅引起部分抑制。可溶性Ea在低盐浓度下与蓝色葡聚糖/琼脂糖4B和琼脂糖/己烷-3-羟基-3-甲基戊二酰辅酶A亲和树脂结合。相比之下，可溶性E1不与琼脂糖/己烷-羟甲基戊二酰辅酶A结合，而在低盐浓度下仍观察到E1与蓝色葡聚糖/琼脂糖4B的定量结合。这些结果表明硫醇是羟甲基戊二酰辅酶A还原酶反应所必需的辅因子。它们对E1激活的作用不是由酶聚集状态的改变引起的。相反，酶从E1到Ea的可逆变化受酶与底物羟甲基戊二酰辅酶A亲和力增加的影响。
