二酰胺处理大鼠红细胞和血浆中半胱氨酸通过谷氨酰转肽酶和巯基交换对 S-巯基化的调控。

The control of S-thiolation by cysteine via gamma-glutamyltranspeptidase and thiol exchanges in erythrocytes and plasma of diamide-treated rats.

机构信息

Department of Neuroscience, Pharmacology Unit, University of Siena, Via A. Moro no. 4, 53100 Siena, Italy.

出版信息

Toxicol Appl Pharmacol. 2010 Feb 1;242(3):333-43. doi: 10.1016/j.taap.2009.11.003. Epub 2009 Nov 10.

DOI:10.1016/j.taap.2009.11.003

PMID:19909765

Abstract

Protein thiol modifications including cysteinylation (CSSP) and glutathionylation (GSSP) in erythrocytes of rat treated with diamide have been reported, but mechanism and origin of CSSP formation are unknown. Experiments were performed to relate CSSP formation to GSH hydrolysis via gamma-glutamyltranspeptidase (gamma-GT) and know whether cysteine may act as deglutathionylation factor. Time-dependent variations of redox forms of glutathione and cysteine were investigated in erythrocytes, plasma, liver and kidney of diamide-treated rats (0.4 mmol/kg by infusion for 45 min followed by 135 min of washout) in the presence and absence of acivicin (10 mg/kg administered twice 1 h before diamide) a known gamma-GT inhibitor. Diamide-treated rats showed decreased concentrations of erythrocyte GSH and increased levels of GSSP and CSSP. The rate of CSSP formation was slower than that of GSSP. Besides the entity of CSSP accumulation of erythrocytes was high and equivalent to approximately 3-fold of the normal plasma content of total cysteine. The result was paradoxically poorly related to gamma-GT activity because the gamma-GT inhibition only partially reduced erythrocyte CSSP. After gamma-GT inhibition, a large concentration fluctuation of glutathione (increased) and cysteine (decreased) was observed in plasma of diamide-treated rats, while little changes were seen in liver and kidney. There were indications from in vitro experiments that the CSSP accumulation in erythrocytes of diamide-treated rats derives from the coexistence of GSH hydrolysis via gamma-GT and production of reduced cysteine via plasma thiol exchanges. Moreover, reduced cysteine was found to be involved in deglutathionylation processes. Mechanisms of protein glutathionylation by diamide and deglutathionylation by cysteine were proposed.

摘要

已有报道称，二酰胺处理的大鼠红细胞中存在蛋白质巯基修饰，包括半胱氨酰化（CSSP）和谷胱甘肽化（GSSP），但 CSSP 形成的机制和来源尚不清楚。本实验旨在通过γ-谷氨酰转肽酶（γ-GT）将 CSSP 形成与 GSH 水解相关联，并研究半胱氨酸是否可以作为脱谷胱甘肽作用因子。在二酰胺（0.4 mmol/kg 静脉输注 45 分钟，随后洗脱 135 分钟）处理的大鼠（预先 1 小时两次给予 10 mg/kg 阿卡西坦，一种已知的 γ-GT 抑制剂）的红细胞、血浆、肝和肾中，研究了谷胱甘肽和半胱氨酸的氧化还原形式的时间依赖性变化。二酰胺处理的大鼠红细胞中 GSH 浓度降低，GSSP 和 CSSP 水平升高。CSSP 形成的速度比 GSSP 慢。除了红细胞 CSSP 积累的程度很高，相当于正常血浆总半胱氨酸含量的约 3 倍外。结果与 γ-GT 活性的相关性很差，因为 γ-GT 抑制仅部分降低了红细胞 CSSP。γ-GT 抑制后，在二酰胺处理的大鼠的血浆中观察到谷胱甘肽（增加）和半胱氨酸（减少）浓度的大幅波动，而在肝和肾中变化很小。体外实验表明，二酰胺处理的大鼠红细胞中 CSSP 的积累来源于γ-GT 介导的 GSH 水解和通过血浆巯基交换产生还原型半胱氨酸的共同作用。此外，还发现还原型半胱氨酸参与脱谷胱甘肽作用过程。提出了二酰胺的蛋白质谷胱甘肽化和半胱氨酸的脱谷胱甘肽作用机制。