A simple technique to determine glutathione (GSH) levels and synthesis in ocular tissues as GSH-bimane adduct: application to normal and galactosemic guinea-pigs.
A simple technique to determine glutathione (GSH) levels and synthesis in ocular tissues as GSH-bimane adduct: application to normal and galactosemic guinea-pigs.
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一种测定谷胱甘肽 (GSH) 水平和眼组织中 GSH-bimane 加合物合成的简单技术：应用于正常和半乳糖血症豚鼠。
DOI：
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发表时间：
1993
影响因子：
3.4
通讯作者：
J. Fernández
中科院分区：
文献类型：
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作者：
Ram Kannan;Diana Tang;J. Mackic;B. Zlokovic;J. Fernández
先前描述的用于其他组织的荧光技术已应用于测定哈特利豚鼠眼组织中谷胱甘肽的水平及其合成率。在谷胱甘肽-S-转移酶催化的反应中，一氯代二甲烷与谷胱甘肽形成稳定的荧光加合物。随着时间的推移记录的荧光信号与谷胱甘肽的合成速率成正比。将晶状体、角膜和视网膜均质化，并将胞质部分透析过夜以耗尽内源性谷胱甘肽。谷胱甘肽合成率由谷胱甘肽前体和辅助因子的混合物确定，即。半胱氨酸+二硫苏糖醇、谷氨酸+甘氨酸、ATP 和 Mg++ 在一氯代二甲烷存在下。向混合物中补充谷胱甘肽-S-转移酶以催化荧光加合物的形成。在丁硫氨酸亚磺酰亚胺（γ-谷氨酰半胱氨酸合成酶的抑制剂）不存在和存在的情况下测定谷胱甘肽合成率。在存在和不存在丁硫氨酸亚磺酰亚胺的情况下荧光随时间变化的差异用于估计谷胱甘肽合成。晶状体、角膜和视网膜的透析前胞质部分中谷胱甘肽的基础水平分别为21.8+/-2.2、36.5+/-4.1和38.6+/-2.8nmol mg-1蛋白质。这些组织中的最大谷胱甘肽合成率分别为 0.52 +/- 0.04、2.25 +/- 0.67 和 3.35 +/- 0.65 nmol min-1 mg-1 蛋白质。当使用 γ-谷氨酰半胱氨酸代替半胱氨酸作为前体时，晶状体和视网膜的谷胱甘肽合成酶活性分别为 0.19 +/- 0.08 和 1.54 +/- 0.76 nmol-1 min mg-1 蛋白质。（摘要截断为 250 字）
A fluorimetric technique previously described for other tissues has been applied to determine levels of glutathione and its synthetic rates in ocular tissues of Hartley guinea-pigs. Monochlorobimane forms a stable, fluorescent adduct with glutathione in a reaction catalyzed by glutathione-S-transferase. The fluorescent signal recorded over time is directly proportional to the synthetic rate of glutathione. Lens, cornea and retina were homogenized and cytosolic fractions dialyzed overnight to deplete endogeneous glutathione. Glutathione synthetic rates were determined from a mixture of glutathione precursors and co-factors, viz. cysteine+dithiothreitol, glutamate+glycine, ATP and Mg++ in the presence of monochlorobimane. The mixture was supplemented with glutathione-S-transferase to catalyze the formation of the fluorescent adduct. Glutathione synthetic rates were determined in the absence and presence of buthionine sulfoximine, an inhibitor of gamma-glutamyl cysteine synthetase. The difference in fluorescence change over time in the presence and absence of buthionine sulfoximine was used to estimate glutathione synthesis. Basal levels of glutathione in pre-dialyzed cytosolic fractions of the lens, cornea, and retina were 21.8 +/- 2.2, 36.5 +/- 4.1 and 38.6 +/- 2.8 nmol mg-1 protein, respectively. The maximal glutathione synthetic rates in these tissues were 0.52 +/- 0.04, 2.25 +/- 0.67 and 3.35 +/- 0.65 nmol min-1 mg-1 protein, respectively. When gamma-glutamyl cysteine is used as a precursor instead of cysteine, the glutathione synthetase activities from lenses and retinas were 0.19 +/- 0.08 and 1.54 +/- 0.76 nmol-1 min mg-1 protein.(ABSTRACT TRUNCATED AT 250 WORDS)