A Mixed Selenodisulphide Bond at the Active Site of Thioredoxin Reductase

硫氧还蛋白还原酶(TrxR)，这里命名为TrxR2， 不与先前发现的TrxR抗体发生反应 (现命名为TrxR1)是从大鼠肝脏中提纯的。像TrxR1一样， TrxR2在非变性条件下以二聚体形式存在 含有硒半胱氨酸(Secys)作为倒数第二个残基 COOH末端的Gly-Cys-Secys-Gly基序。用一种 巯基修饰试剂表明，赛赛斯残留物为 对TrxR2活动至关重要。克隆了编码TrxR2的基因 来自大鼠肝脏；开放阅读框预测多肽为526 具有COOH-末端Gly-Cys-Secys-Gly基序的氨基酸 假设帧内TGA密码子编码Secys。The 3‘ 该基因的非翻译区含有一个典型的秘密 插入序列元素。推导出的氨基酸序列 TrxR2与TrxR1有54%的同源性(62%的相似性)。这个 推导的序列包含36个额外的残基在上游 实验测定的纯化的氨基末端序列 TrxR2。该36个残基区域的序列是典型的 线粒体先导肽。事实上，免疫印迹分析表明 TrxR2几乎只定位于线粒体，而 TrxR1是一种胞质蛋白。与TrxR1不同，TrxR1在 所有大鼠每毫克总可溶性蛋白0.6-1.6克的水平 被检查的组织中，TrxR2相对丰富(0.3到0.6克/毫克) 仅限于肝脏、肾脏、肾上腺和心脏。 TrxR2在线粒体中的定位，以及以前的 线粒体特异性硫氧还蛋白的鉴定和鉴定 硫氧还蛋白依赖的过氧化物酶，提示这三种蛋白 提供了一条主要的防线，防止由 线粒体呼吸链。

A thioredoxin reductase (TrxR), named here TrxR2, that did not react with antibodies to the previously identified TrxR (now named TrxR1) was purified from rat liver. Like TrxR1, TrxR2 existed as a dimer under nondenaturing conditions and contained selenocysteine (Secys) as the penultimate residue in a Gly-Cys-Secys-Gly motif at the COOH-terminus. Analysis with a sulfhydryl-modifying reagent indicated that the Secys residue is essential for TrxR2 activity. A cDNA encoding TrxR2 was cloned from rat liver; the open reading frame predicts a polypeptide of 526 amino acids with a COOH-terminal Gly-Cys-Secys-Gly motif provided that an in-frame TGA codon encodes Secys. The 3' untranslated region of the cDNA contains a canonical Secys insertion sequence element. The deduced amino acid sequence of TrxR2 shows 54% identity (62% similarity) to that of TrxR1. The deduced sequence contained 36 additional residues upstream of the experimentally determined NH2-terminal sequence of purified TrxR2. The sequence of this 36-residue region is typical of that of a mitochondrial leader peptide. Indeed, immunoblot analysis indicated that TrxR2 is localized almost exclusively in mitochondria, whereas TrxR1 is a cytosolic protein. Unlike TrxR1, which was expressed at a level of 0.6 to1.6 ?g per milligram of total soluble protein in all rat tissues examined, TrxR2 was relatively abundant (0.3 to 0.6 ?g/mg) only in liver, kidney, adrenal gland, and heart.The specific localization of TrxR2 in mitochondria, together with the previous identification of mitochondria-specific thioredoxin and thioredoxin-dependent peroxidase, suggest that these three proteins provide a primary line of defense against H2O2 produced by the mitochondrial respiratory chain.