Suresh S. Tate

γ-Glutamyl transpeptidase: catalytic, structural and functional aspects

Summaryγ-Glutamyl transpeptidase catalyzes transfer of the γ-glutamyl moiety of glutathione to amino acids, dipeptides, and to glutathione itself; the enzyme also catalyzes the hydrolysis of glutathione to glutamate and cysteinyl-glycine. This review deals with the tissue distribution and localization of the enzyme in mammals, the catalytic properties of the enzyme (including its inhibition by reversible and irreversible inhibitors), structural studies on the enzyme, and new findings about its physiological function.

[30] γ-Glutamyl transpeptidase

Publisher Summary This chapter presents a procedure for the preparation of γ- glutamyl transpeptidase. In the assay of γ- glutamyl transpeptidase the most widely used substrate is L-y-glutamyl-p-nitroanilide. p -Nitroaniline, released during transpeptidation and hydrolysis, is readily determined from the increase in absorbance at 410 nm. A variety of other procedures has been used to determine the activity of γ-glutamyl transpeptidase. Other chromogenic substrates include L-γ- glutamylanilide and L-γ-glutamylnaphthylamides. Assay procedures in which glutathione is used are complicated by its oxidation to glutathione disulfide; oxidation can be retarded by including ethylenediaminetetraacetic acid in the reaction mixtures. Convenient spectrophotometric assays involving the use of S -substituted glutathione derivatives are also available. Such derivatives include S -pyruvoylglutathione and S -acetophenoneglutathione . For purification procedures, various methods of solubilization are employed including the treatment of the particulate enzyme with detergents, organic solvents, and proteinases. The rat kidney enzyme can be purified following its solubilization with either proteinases (e.g., papain and bromelain) or detergents (e.g., Triton X-100 and Lubrol WX).

[50] γ-Glutamyl transpeptidase from kidney

Publisher Summary This chapter focuses on mammalian γ-glutamyl transpeptidase, emphasizing the rat kidney enzyme, the most extensively studied transpeptidase. γ-glutamyl transpeptidase plays a key role in the γ-glutamyl cycle, a pathway for the synthesis and degradation of glutathione. Glutathione is synthesized from its constituent amino acids within the cells by the successive actions of γ-glutamylcysteine and glutathione synthetases. The reactions allow the storage of cysteine as glutathione and γ-glutamyl transpeptidase catalyzes the first step in the pathway that leads to release of the cysteine moiety from the tripeptide. Homogenates of animal tissues exhibit a wide range of transpeptidase activity. Low transpeptidase activity in a tissue homogenate is often misleading because specific localization studies indicate that there are specific regions of intense enzyme activity in many tissues. In most mammals, the kidney exhibits by far the highest activity.

Membrane-Bound γ-Glutamyl Transpeptidase

γ-Glutamyl transpeptidase is a membrane-bound enzyme that catalyzes the transfer of the γ-glutamyl moiety of glutathione (and of other γ-glutamyl compounds) to amino acid (and peptide) acceptors as indicated in reaction (1):

Decrease in glutathione levels of kidney and liver after injection of methionine sulfoximine into rats

glutathione + a\min o\,acid \to \gamma - glutamyl\,a\min o\,acid + cysteinyl\,glycine

Formation of the COOH-terminal amide group of thyrotropin-releasing-factor

(1) This reaction is the first step in a quantitatively significant pathway of glutathione metabolism, and it has been postulated that this or analogous reactions are involved in amino acid transport. Thus, γ-glutamyl transpeptidase mediates the translocation of the amino acid across the cell membrane by interacting with both extracellular amino acid and intracellular glutathione (or other compounds that contain the γ-glutamyl carrier); according to this idea, the amino acid enters the cell as a γ-glutamyl amino acid.

γ-glutamyl transpeptidase and related enzyme activities in the reproductive system of the male rat

y-Glutamyl transpeptidase, a membrane-bound enzyme, catalyzes the initial step in the degradative metabolism of glutathione by transferring the y-glutamyl moiety to a number of acceptors such as amino acids, peptides and water. High activities of this enzyme are found in epithelial cells of the renal proximal tubules, jejunal villi, choroid plexus, bile ducts, seminal vesicles, epididymis, and ciliary body, consistent with its proposed role in transport processes [ 1,2]. Histochemical studies at light microscopic level were suggestive of the enzyme’s localization in the brush border of renal and jejunal epithelial cells [2]. This inference has been supported by subcellular fractionation studies which showed that the enzyme activity was enriched in brush border membranes [3-lo]. However, these conclusions have been challenged by claims that the enzyme is neither localized in the renal brush border membranes nor in the basallateral membranes [ 11 ,121. We report here the ultrastructural localization of y-glutamyl transpeptidase in tissue slices that provides direct evidence that the enzyme is indeed primarily located in the brush border membranes of the rat kidney proximal tubules and of the tip cells of the jejunal villi. Activity is also seen in the membranes of the foot processes of glomerular podocytes. Furthermore, immunocytochemical studies using ferritin-antibody conjugates and results from papain treatment of intact membrane vesicles show that the enzyme is located on the outer (luminal) surface of the brush border membranes. Such localization is consistent with the enzyme’s ability to metabolize both extracellular glutathione as well as intracellular glutathione translocated to membrane

γ-Glutamyl transpeptidase of rat seminal vesicles; Effect of orchidectomy and hormone administration on the transpeptidase in relation to its possible role in secretory activity

After rats were injected with the convulsant methionine sulfoximine, there was a rapid decrease in the glutathione concentrations of the kidney and liver, but there was no measurable effect (within 5 hours) on brain glutathione. The maximum decreases in the glutathione concentrations of kidney and liver were observed 1 hr after injection and were about 60 and 40%, respectively, of the control levels. The findings suggest that there may be at least two pools of tissue glutathione. Studies in which other amino acids were injected, and earlier in vitro studies, are consistent with the conclusion that methionine sulfoximine affects glutathione synthesis in vivo by inhibiting γ-glutamylcysteine synthetase. Injection of glycylglycine also decreased glutathione levels, an effect probably mediated by γ-glutamyltranspeptidase.