Zhang Hongqiao, Forman Henry Jay, Choi Jinah
School of Natural Sciences, University of California Merced, USA.
Methods Enzymol. 2005;401:468-83. doi: 10.1016/S0076-6879(05)01028-1.
Glutathione (GSH) is the most abundant nonprotein thiol in cells and has multiple biological functions. Glutathione biosynthesis by way of the gamma-glutamyl cycle is important for maintaining GSH homeostasis and normal redox status. As the only enzyme of the cycle located on the outer surface of plasma membrane, gamma-glutamyl transpeptidase (GGT) plays key roles in GSH homeostasis by breaking down extracellular GSH and providing cysteine, the rate-limiting substrate, for intracellular de novo synthesis of GSH. GGT also initiates the metabolism of glutathione S-conjugates to mercapturic acids by transferring the gamma-glutamyl moiety to an acceptor amino acid and releasing cysteinylglycine. GGT is expressed in a tissue-, developmental phase-, and cell-specific manner that may be related to its complex gene structure. In rodents, there is a single GGT gene, and several promoters that generate different mRNA subtypes and regulate its expression. In contrast, several GGT genes have been found in humans. During oxidative stress, GGT gene expression is increased, and this is believed to constitute an adaptation to stress. Interestingly, only certain mRNA subtypes are increased, suggesting a specific mode of regulation of GGT gene expression by oxidants. Here, protocols to measure GGT activity, relative levels of total and specific GGT mRNA subtypes, and GSH concentration are described.
谷胱甘肽（GSH）是细胞中含量最丰富的非蛋白质硫醇，具有多种生物学功能。通过γ-谷氨酰循环进行的谷胱甘肽生物合成对于维持GSH稳态和正常的氧化还原状态至关重要。作为该循环中唯一位于质膜外表面的酶，γ-谷氨酰转肽酶（GGT）通过分解细胞外GSH并为细胞内从头合成GSH提供限速底物半胱氨酸，在GSH稳态中发挥关键作用。GGT还通过将γ-谷氨酰部分转移到受体氨基酸并释放半胱氨酰甘氨酸，启动谷胱甘肽S-共轭物向巯基尿酸的代谢。GGT以组织、发育阶段和细胞特异性的方式表达，这可能与其复杂的基因结构有关。在啮齿动物中，有一个单一的GGT基因，以及几个产生不同mRNA亚型并调节其表达的启动子。相比之下，在人类中发现了几个GGT基因。在氧化应激期间，GGT基因表达增加，这被认为是对压力的一种适应。有趣的是，只有某些mRNA亚型增加，这表明氧化剂对GGT基因表达有特定的调节模式。在此，描述了测量GGT活性、总GGT和特定GGT mRNA亚型的相对水平以及GSH浓度的方法。
