Membrane-bound Gamma-glutamyl Transpeptidase Research Articles

Enhanced gamma-glutamyl transpeptidase expression and superoxide production in Mpv17-/- glomerulosclerosis mice.

Recently, gamma-glutamyl transpeptidase, which initiates cleavage of extracellular glutathione, has been shown to promote oxidative damage to cells. Here we examined a murine disease model of glomerulosclerosis, involving loss of the Mpv17 gene coding for a peroxisomal protein. In Mpv17-/- cells, enzyme activity and mRNA expression (examined by quantitative RT-PCR) of membrane-bound gamma-glutamyl transpeptidase were increased, while plasma glutathione peroxidase and superoxide dismutase levels were lowered. Superoxide anion production in these cells was increased as documented by electron spin resonance spectroscopy. In the presence of Mn(III)tetrakis(4-benzoic acid)porphyrin, the activities of gamma-glutamyl transpeptidase and plasma glutathione peroxidase were unchanged, suggesting a relationship between enzyme expression and the amount of reactive oxygen species. Inhibition of gamma-glutamyl transpeptidase by acivicin reverted the lowered plasma glutathione peroxidase and superoxide dismutase activities, indicating reciprocal control of gene expression for these enzymes.

Biochemical characteristics of gamma-glutamyl transpeptidase in capillaries from entorhinohippocampal complex of quinolinate-lesioned rat brain.

Quinolinic acid (QUIN) is an endogenous excitotoxic agonist of the N-methyl-D-aspartate (NMDA) type of glutamate receptor, which causes slowly progressing degeneration of vulnerable neurons in some brain regions. Using changes in the activity of membrane-bound gamma-glutamyl transpeptidase (GGT) as a marker of cell damage, we found a significant decrease of this enzyme activity, which was preferentially located in the ipsilateral hippocampal formation and entorhinal cortex, 4 d after the unilateral intracerebroventricular (icv) injection of 0.5 mumol QUIN. The dose of QUIN divided into two half-doses injected bilaterally led to a symmetrical decline of GGT activity in hippocampal areas. The lesion was characterized by a suppression of GGT activity in hippocampal and entorhinal capillaries, corresponding to 60 and 81% of their initial value, respectively, but no significant changes were ascertained in synaptosomal membranes. The changes in the activity of capillary GGT were associated with the decrease of apparent maximal velocity Vmaxapp, whereas apparent Michaelis constant K(m)app (0.69-0.79 mM) remained unaffected. In the nonlesioned brain, concanavalin A (Con A) affinity chromatography revealed five glycoforms of synaptosomal GGT in contrast to only one found in hippocampal and entorhinal capillaries. The results document that neither the saccharide moiety of GGT nor the value of enzyme K(m)app is significantly affected by the QUIN-induced lesion of the rat brain. However, the suppression of GGT activity, which is accompanied by a decrease in the value of Vmaxapp in brain microvessels, may suggest dysfunction of the blood-brain barrier (BBB) in the QUIN-injured rat brain.

Role of extracellular glutathione and gamma-glutamyltranspeptidase in the disposition and kidney toxicity of inorganic mercury in rats.

The role of extracellular glutathione (GSH) and membrane-bound gamma-glutamyltranspeptidase (gamma-GT) as contributory factors in the disposition and toxicity of inorganic mercury (HgCl2, 1 mg kg-1, i.p.) was investigated in rats pretreated with acivicin (AT-125, 10 mg kg-1), a gamma-GT inhibitor. A high degree of gamma-GT inhibition (75%) and of protection (90%) against HgCl2-induced nephrotoxicity was obtained in gamma-GT-inhibited rats 24 h post-treatment. Pretreatment with acivicin affected the fractional distribution profile of 203 Hg, resulting in a twofold decrease in the renal incorporation of mercury 4 h post-treatment and a threefold increase in the 24-h urinary excretion of mercury. Plasma radioactivity remained constant over 24 h in rats dosed with 203Hg alone, whereas it decreased by 60% between 4 h and 24 h in gamma-GT-inhibited rats. In gamma-GT-inhibited rats treated with HgCl2 the renal and plasma reduced glutathione (GSH) content increased by 68% and 330% respectively, as compared to controls. The gamma-GT inhibition affected the distribution profile of mercury within urinary proteins, shifting the binding of mercury from the high-molecular-weight fraction (3% against 80%) to the low-molecular-weight fraction (72% against 10%). A significant but less impressive shift of mercury from the high- to the low-molecular-weight fraction also arose in the plasma. These results taken together support the pivotal role of extracellular GSH and membrane-bound gamma-GT in the renal incorporation, toxicity and excretion of inorganic mercury in rats.

Cortisol regulation of gamma-glutamyl transpeptidase in liver, choroid plexus, blood plasma and cerebrospinal fluid of developing chick embryo.

A relationship between membrane-bound gamma-glutamyl transpeptidase (GGT) in liver and choroid plexus and the soluble GGT in blood plasma and cerebrospinal fluids is indicated by a decrease of the former activity in the organs and a concomitant increase of the latter in the body fluids of the embryonic chick between day 11 and hatching. Starting on day 15, the administration of cortisol caused a marked increase in the activity of soluble GGT in blood plasma and cerebrospinal fluid which might be related to an increase of the membrane-bound activity of this enzyme by the glucocorticoid in liver, brain and choroid plexus during the same developmental period. However, before day 15 cortisol reduced GGT activity in the liver and choroid plexus suggesting a biphasic effect of the steroid on the activity of membrane-bound GGT.

Transport of gamma-glutamyl amino acids: role of glutathione and gamma-glutamyl transpeptidase.

This work relates to the hypothesis that one of the mechanisms that mediates amino acid translocation across cell membranes involves the action of membrane-bound gamma-glutamyl transpeptidase on intracellular glutathione and extracellular amino acids to form gamma-glutamyl amino acids. According to this idea, the latter are translocated into the cell where the gamma-glutamyl moiety is removed to yield free amino acids. Previous studies in this laboratory showed that intracellular glutathione is translocated out of many cells. We have now directly examined the transport of gamma-glutamyl amino acids into tissues in the mouse by use of the model substrate L-gamma-glutamyl-L-[14C]methionine sulfone. Of 11 tissues examined, only the kidney showed strong and preferential uptake of the substrate. A substantial amount of the administered L-gamma-glutamyl-L-[14C]methionine sulfone was found intact in the kidney; the total uptake of this compound was greater (by about 2-fold) than that of free L-methionine sulfone. Studies with a number of other gamma-glutamyl amino acids and gamma-glutamyl compounds indicate that the kidney has a relatively specific transport system for gamma-glutamyl amino acids. Small but significant amounts of gamma-glutamylmethionine sulfone were found in the liver and pancreas, suggesting that other tissues may also have this system. Transport of gamma-glutamylmethionine sulfone into the kidney was inhibited by inhibitors of glutathione synthesis and of gamma-glutamyl transpeptidase. The results suggest that both the transpeptidase and glutathione may be involved in transport of gamma-glutamyl amino acids.

Glutathione: interorgan translocation, turnover, and metabolism.

Glutathione is translocated out of cells; cells that have membrane-bound gamma-glutamyl transpeptidase can utilize translocated glutathione, whereas glutathione exported from cells that do not have appreciable transpeptidase enters the blood plasma. Glutathione is removed from the plasma by the kidney and other organs that have transpeptidase. Studies in which mice and rats were treated with buthionine sulfoximine, a selective and potent inhibitor of gamma-glutamylcysteine synthetase and therefore of glutathione synthesis, show that glutathione turns over at a significant rate in many tissues, especially kidney, liver, and pancreas; the rate of turnover in mouse skeletal muscle is about 60% of that in the kidney. Experiments on rats surgically deprived of one or both kidneys and treated with the gamma-glutamyl transpeptidase inhibitor D-gamma-glutamyl-(o-carboxy)phenylhydrazide establish that extrarenal gamma-glutamyl transpeptidase activity accounts for the utilization of about one-third of the total blood plasma glutathione. Normal animals treated with the transpeptidase inhibitor excrete large amounts of glutathione in their urine. They also excrete gamma-glutamylcysteine, suggesting that cleavage of glutathione at the cysteinylglycine bond may be of metabolic significance. The present and earlier findings lead to a tentative scheme (presented here) for the metabolism and translocation of glutathione, gamma-glutamyl amino acids, and related compounds.

Translocation of glutathione from lymphoid cells that have markedly different gamma-glutamyl transpeptidase activities.

Translocation of intracellular glutathione to the medium was studied in lymphoid cells (grown in tissue culture) that have very high, very low, or intermediate levels of membrane-bound gamma-glutamyl transpeptidase, in the absence and presence of various inhibitors of this enzyme. The data show that glutathione is translocated to the medium by all of the cell lines studied, but that glutathione does not accumulate in the medium unless the cellular transpeptidase activity is either very low or substantially inhibited. Translocation of glutathione does not seem to be directly related to the activity of gamma-glutamyl transpeptidase. The present and previous [Griffith, O.W. & Meister, A. (1979) Proc. Natl. Acad. Sci. USA 76, 268--272] findings suggest that translocation of intracellular glutathione is a general property of many mammalian cells. Glutathione exported from cells that have membrane-bound transpeptidase may be recovered by the cell in the form of transpeptidation or degradation products. Translocation of glutathione may also reflect operation of a rather general mechanism that protects and maintains the integrity of cell membranes.