ROLE OF SELENOPROTEINS IN THE PROTECTION AGAINST OXCIDATIVE STRESS

硒是一种必需的微量元素，是谷胱甘肽过氧化物酶(GPX)的活性部位，催化过氧化氢还原。豚鼠肝脏和肾脏的细胞GPx(CGPx)活性极低。为了检测其他硒蛋白的表达，我们研究了在豚鼠体内注射放射性标记亚硒酸盐后，硒在肝脏蛋白质或多肽中的掺入情况。肝匀浆的SDS-PAGE分析表明，放射性Se的主带位于18 kDa，与磷脂过氧化氢GPx(PHGPx)的相对分子质量一致。经BSP-GSH亲和层析和Deab Sephacel柱层析分离，18 kDa含Se蛋白与PHGPx活性共洗脱。以大鼠PHGPx为模板，用RT-PCR方法从豚鼠肝组织中扩增出PHGPx基因，其序列与大鼠PHGPx基因有98%的同源性。因此，在cGPx表达极受抑制的豚鼠肝脏中，PHGPx基因被积极表达，形成与小鼠和大鼠相当的活性蛋白。另一方面，对肾脏蛋白质进行了SDS-PAGE分析，发现存在一个23 kDa的含Se蛋白，其大小与cGPx单体的大小相似，但该蛋白没有酶活性。肾脏蛋白质的双向凝胶分析表明，23 kDa含硒蛋白质的等电点与cGPx的等电点不同。这种不具有GPX活性的含Se蛋白的性质有待进一步阐明。

Se, an essential trace element, is known to constitute the active site of glutathione peroxidase (GPx) which catalyzes the reduction of hydroperoxides. Guinea pigs have extremely low activity of cellular GPx (cGPx) in the liver and kidney. To examine the expression of other selenoproteins, we investigated the incorporation of Se into proteins or peptides in the liver of guinea pigs after the intraperitoneal injection of radiolabeled selenite. SDS-PAGE analysis of the liver homogenate revealed that the major band of radioactive Se was found at 18 kDa, which coincided with the molecular weight of phospholipid hydroperoxide GPx (PHGPx). Upon the chromatographic separation by BSP-GSH affinity column and the subsequent DEAB Sephacel column, the 18 kDa Se-containing protein co-eluted with the activity of PHGPx. RT-PCR analysis of mRNA obtained from the liver of guinea pigs using primers constructed based on the rat PHGPx resulted in an amplification of PHGPx cDNA.The sequence of the amplified cDNA showed 98% homology to the rat PHGPx. Thus, in the liver of guinea pigs, in which the expression of cGPx is extremely suppressed, PHGPx gene is actively expressed to form the protein with the activity comparable to that of mice and rats. On the other hand, SDS-PAGE analysis of kidney proteins revealed an existence of a 23 kDa Se-containing protein, This size was similar to that of the monomer of cGPx, but this protein had no enzyme activity. 2-D gel analysis of kidney proteins demonstrated that the isoelectric points of the 23 kDa Se-containing protein were different from that of cGPx. The properties of this Se-containing protein with no GPx activity should be further clarified.