Luthman M, Holmgren A
Biochemistry. 1982 Dec 21;21(26):6628-33. doi: 10.1021/bi00269a003.
A reproducible scheme has been developed for the preparation of rat liver thioredoxin and thioredoxin reductase (EC 1.6.4.5) by using assays based on reduction of insulin and 5,5'-dithiobis(2-nitrobenzoic acid), respectively. Both proteins were purified to homogeneity, as judged from polyacrylamide gel electrophoresis. Thioredoxin had a molecular weight of 12 000 and contained about 110 amino acids including 4 half-cystines and an NH2-terminal valine. Peptide maps of reduced and carboxymethylated thioredoxin showed that the protein had the active center sequence -Cys-Gly-Pro-Cys-Lys-Met- characteristic of thioredoxins also from procaryotes. Prolonged air oxidation of fully reduced thioredoxin created inactive, aggregated disulfide-containing molecules. Thioredoxin reductase showed a subunit molecular weight of 58 000 and a native molecular weight of 116 000. The enzyme was highly specific for NADPH with a Km of 6 microM. It contained FAD as prosthetic group and was sensitive to inhibition by arsenite. Thioredoxin reductase had a Km of 2.5 microM for rat and calf liver thioredoxin and a Kcat of 3000 min-1.
已经开发出一种可重复的方案，用于制备大鼠肝脏硫氧还蛋白和硫氧还蛋白还原酶（EC 1.6.4.5），分别采用基于胰岛素还原和5,5'-二硫代双（2-硝基苯甲酸）还原的测定方法。从聚丙烯酰胺凝胶电泳判断，两种蛋白质均已纯化至同质。硫氧还蛋白的分子量为12000，含有约110个氨基酸，包括4个半胱氨酸和一个NH2末端缬氨酸。还原型和羧甲基化硫氧还蛋白的肽图表明，该蛋白质具有原核生物硫氧还蛋白特有的活性中心序列-Cys-Gly-Pro-Cys-Lys-Met-。完全还原的硫氧还蛋白长时间暴露于空气中会产生无活性的、聚集的含二硫键分子。硫氧还蛋白还原酶的亚基分子量为58000，天然分子量为116000。该酶对NADPH具有高度特异性，Km为6 microM。它含有FAD作为辅基，对亚砷酸盐抑制敏感。硫氧还蛋白还原酶对大鼠和小牛肝脏硫氧还蛋白的Km为2.5 microM，Kcat为3000 min-1。
