Novel human ocular glutathione S-transferases with high activity toward 4-hydroxynonenal.
Novel human ocular glutathione S-transferases with high activity toward 4-hydroxynonenal.
复制标题
DOI：
--
复制
发表时间：
1995
影响因子：
4.4
通讯作者：
S. Singhal;S. Awasthi;Satish K. Srivastava;P. Zimniak;Naseem H. Ansari;Yogesh C. Awasthi
中科院分区：
文献类型：
--
作者：
S. Singhal;S. Awasthi;Satish K. Srivastava;P. Zimniak;Naseem H. Ansari;Yogesh C. Awasthi
目的 研究参与脂质过氧化内源性有毒产物4-羟基壬烯醛（4-HNE）解毒的谷胱甘肽S-转移酶同工酶在人晶状体、视网膜、角膜、虹膜、睫状体中的分布和表达，并研究其动力学和结构特性。方法 作者之前已经克隆并测序了小鼠 mGSTA4-4 的 cDNA，该基因对 4-HNE 显示出高活性。他们在大肠杆菌中表达了它，并产生了针对重组 mGSTA4-4 的抗体。在本研究中，这些抗体用于蛋白质印迹分析和免疫亲和层析，以研究 mGSTA4-4 的表达并从眼组织中纯化 mGSTA4-4 的人类直系同源物。结果 人眼组织的蛋白质印迹分析表明，免疫学上与 mGSTA4-4 相似的谷胱甘肽 S-转移酶 (GST) 同工酶在角膜、视网膜、虹膜和睫状体中表达，但在晶状体中不表达。通过固定化抗 mGSTA4-4 抗体的免疫吸附，从人视网膜、角膜、虹膜和睫状体中纯化出同质的同工酶，称为 hGST 5.8。 mGSTA4-4 的人类直系同源物，命名为 hGST 5.8，从所有这些组织中纯化，pI 值为 5.8，亚基 Mr 值为 25 k，N 末端封闭。人眼组织hGST 5.8同工酶的CNBr片段的氨基酸序列与mGSTA4-4的相应区域表现出高度的一级结构同源性。角膜、视网膜、虹膜和睫状体的hGST 5.8 氨基酸序列存在显着差异，表明眼组织中存在几个密切相关的hGST 5.8 亚基。这种异质性是由于组织特异性表达而不是简单的等位基因多态性。 hGST 5.8 对 4-羟基壬烯醛的活性比 1-氯-2,4-二硝基苯 (CDNB) 高约六至八倍。眼部 hGST 5.8 对 4-HNE 的催化效率 (Kcat/Km) 比对 α、mu 或 pi 类 GST 的催化效率高约 100 倍。此外，hGST 5.8 表达针对磷脂氢过氧化物的谷胱甘肽过氧化物酶活性和针对 9,10-环氧硬脂酸的 GSH 结合活性。结论 结果表明，hGST 5.8 同工酶与 α、mu 和 pi 类 GST 不同，在人眼组织中差异表达，并且可能在针对脂质过氧化过程中产生的内源性毒物的保护机制中发挥重要作用。
PURPOSE To study the distribution and expression of glutathione S-transferase isozymes involved in detoxification of endogenously generated toxic products of lipid peroxidation, namely, 4-hydroxynonenal (4-HNE) in human lens, retina, cornea, iris, ciliary body and to study their kinetic and structural properties. METHODS The authors have previously cloned and sequenced cDNA of mouse mGSTA4-4, which shows high activity towards 4-HNE. They have expressed it in Escherichia coli and have raised antibodies against the recombinant mGSTA4-4. In the present study, these antibodies were used in Western blot analysis and immunoaffinity chromatography to study the expression and to purify the human ortholog(s) of mGSTA4-4 from ocular tissues. RESULTS Western blot analyses of human ocular tissues indicated that a glutathione S-transferases (GST) isozyme immunologically similar to mGSTA4-4 was expressed in cornea, retina, and iris and ciliary body, but not in lens. This isozyme designated as hGST 5.8 was purified to homogeneity from human retina, cornea, and iris and ciliary body by immunoabsorption on immobilized antibodies against mGSTA4-4. The human ortholog of mGSTA4-4, designated as hGST 5.8 purified from all these tissues and pI value of 5.8, subunit Mr value of 25 k and blocked N-terminal. Amino acid sequences of CNBr fragments of hGST 5.8 isozymes of human ocular tissues showed a high degree of primary structure homologies with the corresponding regions of mGSTA4-4. There were noticeable differences in the amino acid sequences of hGST 5.8 of cornea, retina, and iris and ciliary body, suggesting the presence of several closely related hGST 5.8 subunits in the ocular tissues. This heterogeneity was due to tissue-specific expression rather than simple allelic polymorphism. The hGST 5.8 had about sixfold to eightfold higher activity toward 4-hydroxynonenal than 1-chloro-2,4-dinitrobenzene, or CDNB. The catalytic efficiency (Kcat/Km) of ocular hGST 5.8 for 4-HNE was about 100-fold higher than those for the alpha, mu, or pi classes of GST. In addition, hGST 5.8 expressed glutathione peroxidase activity toward phospholipid hydroperoxides and GSH-conjugating activity toward 9,10-epoxy stearic acid. CONCLUSIONS The results indicate that hGST 5.8 isozyme(s) distinct from the alpha, mu, and pi classes of GSTs, are differentially expressed in human ocular tissues and may play an important role in protective mechanisms against endogenous toxicants generated during lipid peroxidation.