Enzymatic formation of glutathione-citryl thioester by a mitochondrial system and its inhibition by (-)erythrofluorocitrate.
Enzymatic formation of glutathione-citryl thioester by a mitochondrial system and its inhibition by (-)erythrofluorocitrate.
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线粒体系统酶促形成谷胱甘肽-柠檬酰硫酯及其被 (-) 赤氟柠檬酸盐抑制。
DOI：
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发表时间：
1977
影响因子：
11.1
通讯作者：
M. Sharma
中科院分区：
文献类型：
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作者：
E. Kun;E. Kirsten;M. Sharma
由内膜和基质组成的线粒体隔室的可溶性提取物催化柠檬酸和谷胱甘肽的1：1加合物的酶促合成和水解。通过单同位素和双同位素标记（[（14）C]柠檬酸和[（35）S]谷胱甘肽）的分离以及通过转化为柠檬酸的单异羟肟酸盐并通过薄层色谱和高压电泳与合成产物进行比较，鉴定加合物为硫酯。硫酯的酶促形成（30 ℃时最佳pH值为7.39）需要氧化型谷胱甘肽和柠檬酸盐;两种底物均表现出Michaelis-Menten动力学。在酶促反应期间，形成等摩尔量的硫酯和谷胱甘肽亚磺酸。在凝胶过滤或盐分级分离之后，酶系统需要Mn（2+）（或Mg（2+），其不太有效）以获得最大活性。当检测有丝分裂体提取物时，由于硫酯形成系统（分子量171，000）快速合成产物，随后水解酶（分子量71，000）降解产物，反应的时间过程是双相的。用SephadexG-200分子筛和（NH 4）2SO 4沉淀法分离了两个体系。硫酯形成系统通过与0.5 mM mersalyl预孵育来抑制。其他抑制剂是1，2，3-丙烷三羧酸、10 mM Ca（2+）、200 mM K（+）和自由基捕获剂吩嗪硫酸甲酯。柠檬酸盐-谷胱甘肽硫酯的形成被（-）赤氟柠檬酸盐不可逆地特异性抑制（在每mg蛋白质添加25 pmol的氟柠檬酸盐时抑制50%），其与酶蛋白质形成三氯乙酸稳定的加合物（在50%抑制下，0.8 pmol与1 mg蛋白质结合）。（-）氟代红苹果酸酯选择性抑制内膜囊泡合成苹果酰谷胱甘肽硫酯。
A soluble extract of the mitochondrial compartment composed of the inner membrane and matrix catalyzes the enzymatic synthesis and hydrolysis of the 1:1 adduct of citric acid and glutathione. The adduct was identified as the thioester by isolation with single and double isotope labeling ([(14)C]citric acid and [(35)S]glutathione) and by conversion to the monohydroxamate of citric acid and comparison with the synthetic product by thin layer chromatography and high voltage electrophoresis. The enzymatic formation of the thioester (pH optimum 7.39 at 30 degrees ) requires oxidized glutathione and citrate; both substrates exhibit a Michaelis-Menten kinetics. During the enzymatic reaction equimolar quantities of thioester and glutathione sulfinic acid are formed. After gel filtration or salt fractionation the enzyme system requires Mn(2+) (or Mg(2+), which is less effective) for maximal activity. When extracts of mitoplast are tested, the time course of reaction is biphasic due to the rapid synthesis of the product by the thioester-forming system (molecular weight 171,000) followed by its decay by the hydrolase (molecular weight 71,000). The two systems were separated by molecular filtration on Sephadex G-200 and by precipitation with (NH(4))(2)SO(4). The thioester-forming system is inhibited by preincubation with 0.5 mM mersalyl. Other inhibitors are 1,2,3-propane tricarboxylic acid, 10 mM Ca(2+), 200 mM K(+), and the free radical trapping agent, phenazine methosulfate. The citrate-glutathione thioester formation is irreversibly and specifically inhibited by (-)erythrofluorocitrate (50% inhibition at 25 pmol of added fluorocitrate per mg of protein), which forms a trichloroacetic acid-stable adduct with the enzyme protein (at 50% inhibition, 0.8 pmol is bound to 1 mg of protein). Synthesis of malyl-glutathione thioester by inner membrane vesicles is selectively inhibited by (-)erythrofluoromalate.