Gamma-glutamyl transpeptidase in glutathione biosynthesis

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.