Yoshimura S, Watanabe K, Suemizu H, Onozawa T, Mizoguchi J, Tsuda K, Hatta H, Moriuchi T
Department of Cell Biology, Tokai University School of Medicine, Kanagawa.
J Biochem. 1991 Jun;109(6):918-23. doi: 10.1093/oxfordjournals.jbchem.a123480.
Rat plasma glutathione peroxidase (GSH-Px) was purified 1,400-fold from rat serum by a combination of phenyl Sepharose, DEAE Sephacel, blue Sepharose and Sephacryl S-200 column chromatographies. The purified GSH-Px migrated as a single band corresponding to a molecular weight of 22,500 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was used for the immunization of chickens to obtain a specific antibody and for determination of its amino acid sequence. Two overlapping cDNA clones for rat plasma GSH-Px were isolated from a placental cDNA library. The composite nucleotide sequence is 1,529 base-pairs long and encodes 226 amino acids. The deduced amino acid sequence completely coincided with the sequences of five individual peptide fragments derived from the purified plasma GSH-Px on digestion with lysyl endopeptidase. In order to identify the tissue(s) generating this plasma GSH-Px, immunoblot analysis was performed on homogenates prepared from 13 tissues. A single immunoreactive band of 22.5 kDa, corresponding to plasma GSH-Px, was detected for the kidney homogenate. A much fainter band was observed for the lung preparation, but liver, spleen, bone marrow, and other tissues examined were negative. Northern blot analysis further revealed that the expression level of the plasma GSH-Px gene was high in kidney and low in lung. No transcript was detected in liver or spleen. These results indicate that plasma GSH-Px is predominantly synthesized and secreted by renal cells.
通过苯基琼脂糖、二乙氨基乙基琼脂糖凝胶（DEAE Sephacel）、蓝色琼脂糖和Sephacryl S - 200柱层析相结合的方法，从大鼠血清中纯化出1400倍的大鼠血浆谷胱甘肽过氧化物酶（GSH - Px）。纯化后的GSH - Px在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上迁移为一条单一的条带，对应分子量为22,500。该酶用于免疫鸡以获得特异性抗体，并用于测定其氨基酸序列。从胎盘cDNA文库中分离出两个重叠的大鼠血浆GSH - Px cDNA克隆。复合核苷酸序列长1529个碱基对，编码226个氨基酸。推导的氨基酸序列与用赖氨酰内肽酶消化纯化的血浆GSH - Px得到的五个单独肽片段的序列完全一致。为了鉴定产生这种血浆GSH - Px的组织，对由13种组织制备的匀浆进行了免疫印迹分析。在肾匀浆中检测到一条对应血浆GSH - Px的22.5 kDa单一免疫反应条带。在肺匀浆中观察到一条较淡的条带，但肝脏、脾脏、骨髓和其他检测的组织均为阴性。Northern印迹分析进一步表明，血浆GSH - Px基因在肾脏中的表达水平高，在肺中的表达水平低。在肝脏或脾脏中未检测到转录本。这些结果表明，血浆GSH - Px主要由肾细胞合成和分泌。
