Significance of γ-glutamyl transpeptidase and thiols in amino acid uptake by rat pancreatic islets: Studies with glutamine and leucine

The importance of γ-glutamyl transpeptidase, the key enzyme of the γ-glutamyl cycle and of thiols for the uptake of amino acids into rat pancreatic islets was investigated. Both serine–borate, an inhibitor of γ-glutamy transpeptidase, and serine which does not inhibit this enzyme, but probabaly is a competitive inhibitor of amino acid uptake, inhibited of glutamine. The inhibitory effect of serine-borate was not greater than that of serine alone. The uptake of glutamine was not affected by either GSH (reduced glutathione) or diamide (a thiol oxidant). Niether substances affected the uptake of leucine. The results indicate that the uptake of glutamine by rat pancreatic islets is not dependent on the functioning of γ-glutamyl transpeptidase and that thiols are not important for the uptake of the amino acids glutamine and leucine.     

Stimulation of intralysosomal proteolysis by cysteinyl-glycine,a product of the action of γ-glutamyl transpeptidase on glutathione

The mechanism of the stimulatory effect of glutathione on proteolysis in mouse kidney lysosomes and a lack of an effect in lysomes from the liver was investigated. The stimulation in kidney lysosomes was inhibited by serine plus borate, a reversible inhibitor of γ-glutamyl transpeptidase. Treatment of mouse kidney lysosome suspensions with $\text{l}\text{-(αS,5S)-α-}\text{amino}\text{-3-}\text{chloro}\text{-4,5-}\text{dihydro}\text{-5-}\text{isoxazoleacetic}$ acid (acivicin), an irreversible inhibitor of the transpeptidase, also inhibited the effect of glutathione, but this inhibition was completely relieved by washing and addition of freshly prepated kidney membranes or purified γ-glutamyl transpeptidase to the incubation mixtures. Cysteinyl-glycine, a product of the action of γ-glutamyl transpeptidase, stimulated proteolysis in acivicin-inhibited kidney lysosome preparations similarly to glutathione, and cysteine had no effect at equivalent concentrations. Glutathione also stimulated proteolysis in liver lysosomes in the presence of washed kidney membranes or γ-glutamyl transpeptidase, but the effect was similar to that produced by equivalent concentrations of cysteine. These results suggest that the stimulatory effect of glutathione was mediated by the action of γ-glutamyl transpeptidase present in contaminating cell membrane fragments in the lysosome preparations, and that glutathione does not take part in intralysosomal proteolysis. However, the possibility that cysteinyl-glycine is a physiological intralysosomal disulfide reductant in kidney lysosomes has not been excluded.     

ENZYMES OF THE γ-GLUTAMYL CYCLE IN THE CHOROID PLEXUS AND BRAIN

—The presence of enzymes of the γ-glutamyl cycle in the bovine and rabbit brain and choroid plexus is described. The activities of γ-glutamyl transpeptidase, γ-glutamyl cyclotransferase and γ-glutamyl-cysteine synthetase in the choroid plexus were found to be higher than in the brain. The activity of γ-glutamyl transpeptidase in the choroid plexus was many times higher than the activity of the other enzymes. Brain and choroid plexus γ-glutamyl transpeptidase were activated by Na⁺ and K⁺. Both brain and choroid plexus showed only a very limited capacity to metabolize [¹⁴C]5-oxoproline to ¹⁴CO₂.     

Kinetics of the conversion of leukotriene C by gamma-glutamyl transpeptidase

γ-Glutamyl transpeptidase (EC 2.3.2.2) converts leukotriene C to leukotriene D by removal of a glutamyl residue. The Michaelis constant for leukotriene C₄ hydrolysis was found to be 5.6 μM. Under the same conditions the K_m value for hydrolysis of reduced glutathione was 5.7 μM. This suggests that leukotriene C₄ and glutathione may be competing substrates for γ-glutamyl transpeptidase under physiological conditions. The apparent K_I for inhibition of leukotriene C₄ hydrolysis by equimolar amounts of L-serine and sodium borate was 0.8 mM.     

Mechanism and significance of the mammalian pathway for elimination of D-glutamate; inhibition of glutathione synthesis by D-glutamate

Administration of a tracer dose of L-[¹⁴C] glutamate to rats led to rapid labeling of tissue 5-oxoproline, a finding in accord with the γ-glutamyl cycle. After giving the same dose of D-[¹⁴C] glutamate, the labeling of kidney 5-oxoproline was about 400 times greater than found after giving L-[¹⁴C] glutamate; this reflects the activity of D-glutamate cyclase, which catalyzes cyclization of D-glutamate to 5-oxo-D-proline. The mammalian pathway for detoxication of D-glutamate, which does not require energy or utilization of other metabolites, may have evolved to protect L-amino acid-specific systems against inhibition by D-glutamate; D-glutamate cyclase seems to account for the presence of 5-oxo-D-proline in normal blood plasma and urine. Administration of D-glutamate decreased the glutathione level in the kidney markedly, reflecting inhibition of γ-glutamylcysteine synthetase; D-glutamate may be useful in various studies as an inhibitor of glutathione synthesis.     

A fluorescence-based microtiter plate assay for γ-glutamylcyclotransferase

γ-Glutamylcyclotransferase (GGCT) is a component of the γ-glutamyl cycle and specifically cleaves γ-glutamyl-amino acid dipeptides to release the free amino acid and 5-oxoproline. The action of GGCT in glutathione synthetase-deficient patients results in the accumulation of high levels of 5-oxoproline. In addition, GGCT may be a biomarker in some cancers. GGCT inhibitors, therefore, may be of value in the treatment of glutathione synthetase deficiency or in the elucidation of the role of GGCT in cancer cells. Published GGCT assays are not suitable for high-throughput screening for inhibitory molecules. We have developed a fluorescence-based 96-well plate assay for the determination of the rate of γ-glutamylcysteine cleavage by GGCT. After the reaction, the residual γ-glutamylcysteine is determined fluorometrically after derivatization with 2,3-naphthalenedicarboxaldehyde. The method has sufficient sensitivity to detect low-affinity competitive inhibitors and, as a result of its simplicity and microtiter plate format, can be readily used in high-throughput inhibitor screens.     

Comparative effects of antioxidants on enzymes involved in glutathione metabolism

The present study was designed to examine changes in glutathione metabolism in the liver of mice as influenced by supplementation of their diet with 1 of 4 antioxidants: butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), vitamin E and selenium. In addition to determination of the acid-soluble thiol levels, 5 different enzymes involved with glutathione utilization and synthesis were measured: glutathione transferase, γ-glutamyl transpeptidase, selenium-dependent glutathione peroxidase, γ-glutamylcysteine synthetase and glutathione reductase. All 4 antioxidants produced significant increases in glutathione transferase activity, with BHA and BHT being much more effective than the other two. With the exception of vitamin E, BHA, BHT and selenium all resulted in a slight enhancement in the activity of glutathione reductase as well as in the acid-soluble thiol level. On the other hand, the induction of γ-glutamyl transpeptidase and γ-glutamylcysteine synthetase was responsive to only vitamin E and selenium supplementation, respectively. Although the influence of each of these antioxidants in glutathione metabolism appears to be specific and somewhat compartmentalized, the overall impression is that of an increased capacity for glutathione-conjugate formation and recovery of reduced glutathione. These biochemical changes in glutathione metabolism may be relevant to the anticarcinogenic effects observed with BHA, BHT and selenium.     

MODULATION OF γ-GLUTAMYL TRANSPEPTIDASE ACTIVITY FROM BOVINE CHOROID PLEXUS

Abstract— The catalytic activity of γ-glutamyl transpeptidase (γ-GTP) from bovine choroid plexus has been shown to be subject to modulation by a variety of effectors. L-Alanine and L-serine not only functioned as acceptor substrates to which γ-glutamyl moieties could be transferred, but also as noncom-petitive inhibitors of the reaction in the presence of the dipeptide acceptor substrate glycylglycine. In contrast, D-alanine does not function as an acceptor substrate, but does noncompetitively inhibit the transfer of γ-glutamyl groups to glycylglycine. Similarly, borate ions inhibited y-GTP noncompetitively, while a mixture of L-serine and borate were potent uncompetitive inhibitors of the reaction with a K _i of 0.6 mM. Several dicarboxylic acids, including maleate, maleylglycine, and malonate, inhibited γ-GTP; this inhibition was acceptor substrate-dependent. The inhibition of γ-GTP by maleate was competitive with respect to the acceptor substrate glycylglycine.     

Inhibition of muscle actomyosin ATPase activity by mitochondrial ATPase inhibitor.

Ascites hepatoma cell line AH-130 was tested for the ability to transport various amino acids and glutathione before and after γ-glutamyl transpeptidase of the cells was affinity-labeled and inactivated by 6-diazo-5-oxo-L-norleucine, a glutamine analog. The rate of uptake of alanine, glycine, leucine and glutamine by the cells remained unchanged after γ-glutamyl transpeptidase was inactivated by this affinity label. This indicated that γ-glutamyl transpeptidase of the cell was not involved in the transport process of these amino acids tested. The uptake of glutathione was also tested before and after affinity labeling the enzyme. The total amount of the radioactivity incorporated into the cells was not significantly affected by the enzyme inactivation. However, the relative amount of incorporated intact glutathione was found to be slightly but significantly increased after membraneous γ-glutamyl transpeptidase was inactivated by the affinity label, while that of component amino acid, glycine, was found to decrease. This indicated that glutathione was taken up by the cell in its intact form as well as in degraded forms into its component amino acids, and γ-glutamyl transpeptidase in the ascites tumor cell AH-130 seemed to be involved in the metabolic process via the latter system.     

Inactivation of gamma-glutamyl transpeptidase by phenylmethanesulfonyl fluoride, a specific inactivator of serine enzymes.

Rat kidney γ-glutamyl transpeptidase was found to be inactivated by phenylmethanesulfonyl fluoride, a specific inactivator of serine enzymes. The inactivation occurred only in the presence of maleate which was known to enhance the hydrolytic activity of this enzyme. The concentration of phenylmethanesulfonyl fluoride giving a half maximum rate of inactivation was 1.1 mM. The presence of S-methyl glutathione, a substrate for this enzyme, prevented the inactivation in a competitive fashion. These findings indicate that phenylmethanesulfonyl fluoride acts as an active site directed reagent for γ-glutamyl transpeptidase. A possible identity of the labeled site with that for 6-diazo-5-oxo-L-norleucine, another affinity label for this enzyme, was discussed.     

Ammonia production by IMR-90 fibroblast cultures: effects of ammonia on glutathione, gamma-glutamyl transpeptidase, lysosomal enzymes, and cell division

gamma-Glutamyl transpeptidase has multi-catalytic activities. It degrades glutathione and can produce ammonia from glutamine. The present study was designed to examine whether the decreased cell proliferation, cellular glutathione content and concurrent increase in ammonia production in senescent cells in culture are the result of increased gamma-glutamyl transpeptidase activity. We used IMR-90 fibroblast and 3T3 LI preadipocyte cultures. The cellular glutathione content depended upon cell proliferation and cell density. The glutathione content was higher in cells at logarithmic growth, and lower at stationary growth or post confluency; dead cells had no detectable glutathione by the method currently used. The glutathione content was minimal in "old" IMR-90 cells, regardless of cell density. On the other hand, an increase occurred in the unit number of molecules of bound 5-iodoacetoamidofluorescein, an active-site directed stoichiometric inhibitor of transpeptidase. That result corresponded favorably with the increased enzyme activity, suggesting that the number of enzyme molecules per cell was increased. The inhibition of ammonia production of the cultures by inhibition of gamma-glutamyl transpeptidase by 5-iodoacetoamidofluorescein and reversible inhibition of ammonia production by a serine-borate mixture were consistent with our postulate. Addition of NH4Cl (0.1 mM) to IMR-90 cultures caused increased activities of transpeptidase and some of the lysosomal enzymes; concurrently, the amount of cellular glutathione and the number of cell divisions decreased. This suggests that the increased ammonia production presumably resulting from glutaminase activity of the observed increase of transpeptidase may profoundly affect certain cellular functions.     

棉酚对大鼠肾脏γ-谷氨酰转肽酶动力学的影响

本文应用动力学分析观察了棉酚对大鼠肾脏γ-谷氨酰转肽酶(γ-GT)的抑制作用。实验结果证实了棉酚在体外是大鼠肾脏γ-GT的抑制剂,而且抑制常数远小于r-GT的天然抑制剂——马尿酸。在不同浓度的棉酚作用下,改变双底物浓度,测定其活力并应用Lineweaver-Burk双倒数作图法,测得棉酚在两种底物情况下,对γ-GT的抑制作用均呈非竞争性抑制。     

Localization of gamma-glutamyl transpeptidase in the retinal pigment epithelium and visual receptor cells.

The activities of γ-glutamyl transpeptidase and other enzymes of the γ-glutamyl cycle, a series of reactions that catalyzes the synthesis and utilization of glutathione, were studied in the rabbit retina. Histochemical studies demonstrated that γ-glutamyl transpeptidase is localized in the visual receptor cells and the retinal pigment epithelium. Rat and mouse retinas revealed similar localizations of transpeptidase. These findings are in accord with the view that γ-glutamyl transpeptidase is involved in the transport of amino acids between the retinal pigment epithelium and the avascular visual receptor cells.     

A futile cycle,formed between two ATP-dependant <Emphasis Type="Italic">γ</Emphasis>-glutamyl cycle enzymes, <Emphasis Type="Italic">γ</Emphasis>-glutamyl cysteine synthetase and 5-oxoprolinase: the cause of cellular ATP depletion in nephrotic cystinosis?

Cystinosis, an inherited disease caused by a defect in the lysosomal cystine transporter (CTNS), is characterized by renal proximal tubular dysfunction. Adenosine triphosphate (ATP) depletion appears to be a key event in the pathophysiology of the disease, even though the manner in which ATP depletion occurs is still a puzzle. We present a model that explains how a futile cycle that is generated between two ATP-utilizing enzymes of the γ-glutamyl cycle leads to ATP depletion. The enzyme γ-glutamyl cysteine synthetase (γ-GCS), in the absence of cysteine, forms 5-oxoproline (instead of the normal substrate, γ-glutamyl cysteine) and the 5-oxoproline is converted into glutamate by the ATP-dependant enzyme, 5-oxoprolinase. Thus, in cysteine-limiting conditions, glutamate is cycled back into glutamate via 5-oxoproline at the cost of two ATP molecules without production of glutathione and is the cause of the decreased levels of glutathione synthesis, as well as the ATP depletion observed in these cells. The model is also compatible with the differences seen in the human patients and the mouse model of cystinosis, where renal failure is not observed.     

Evidence for the gamma-glutamyl cycle in yeast.

Many previous studies have shown that yeast contains high concentrations of glutathione and enzymes needed for its synthesis. We report here that yeast also contains γ-glutamyl transpeptidase, γ-glutamyl cyclotransferase, dipeptidase, and 5-oxoprolinase activities, suggesting that the γ-glutamyl cycle may be operative in yeast. The presence of the cycle enzymes in yeast offers a simple free-cell system which can probably be adapted to studies on the function of this cycle.     

Glutathione metabolism in normal and cystinotic fibroblasts

Intracellular concentrations of glutathione and activities of the enzymes gamma-glutamylcysteine synthetase, glutathione synthetase, and gamma-glutamyl transpeptidase were measured in confluent cultured human fibroblasts cell lines from 14 normal cell lines and four cystinotic cell lines. gamma-Glutamyl transpeptidase had a wide range of variability while the glutathione synthetic enzymes, gamma-glutamylcysteine synthetase and glutathione synthetase, had narrower variations and also exhibited no apparent relationship to glutathione content. No differences in the activities of these enzymes were found between normal and cystinotic cells in confluent cell cultures. The activities of the above enzymes and the cell number and content of glutathione, cystine, DNA, and total protein in two normal and two cystinotic fibroblast cell lines were measured during growth. The following growth-dependency patterns were observed: (1) gamma-glutamylcysteine synthetase activity increased markedly in lag and early log phases in both normal and cystinotic cells and decreased rapidly to low confluent levels thereafter. (2) gamma-Glutamyl transpeptidase showed the same wide range of activity noted at confluency but activities decreased in the log phase of growth, a pattern also seen in cystinotic cells. (3) Glutathione synthetase activity remained relatively constant during growth of normal cells but exhibited a peak of activity during lag and early growth of cystinotic cells. (4) Comparative glutathione levels of normal and cystinotic cells were not significantly different and exhibited similar fluctuations with time. (5) The cystine content of normal and cystinotic cells unexpectedly rose to high levels in the lag phase, then decreased to 0.1 nmol 1/2 cystine/mg protein in normal cells and to 0.3 to 1.2 nmol 1/2 cystine/mg protein in cystinotic cells during the log phase. As confluency was approached, normal cell cystine remained at low levels while cystinotic cell cystine rose to characteristically high levels of 50- to 100-fold greater than normal cells at late confluency. These studies extend our understanding of the regulation of glutathione and cystine content in cultured fibroblasts and suggest that glutathione content is closely controlled throughout the cell cycle in the face of varying activities of its anabolic and catabolic enzymes.     

gamma-Glutamyl transpeptidase: relation to immunoglobulin A and free secretory component.

The experiments reported show that bovine γ-glutamyl transpeptidase can be separated from free secretory component. An ion-exchange Chromatographic procedure was developed to analyze the incubation mixtures of the enzyme with glutathione or S-(2-acetamido)-glutathione and glycylglycine. Using this system or the γ-glutamyl p-nitroanilide assay, no significant transpeptidase activity could be detected in the free secretory component-containing fractions of DEAE-cellulose chromatography. Gel filtration on Biogel A-5M showed that the bovine whey transpeptidase chromatographed in the void volume suggesting an aggregate of a minimum molecular weight of about 5 × 10⁶. The transpeptidase could be separated from all immunoglobulins in bovine whey and human colostrum by a combination of agarose gel filtration and immunoadsorption. Concentrated samples of human and sheep saliva showed normal amounts of secretory component, but no detectable γ-glutamyl transpeptidase activity. These experiments show that (1) the transpeptidase and secretory component are two different proteins, and (2) the transpeptidase is present in bovine and human milk as a high molecular weight aggregate which does not include any of the immunoglobulins.     

gamma-Glutamyl transpeptidase in Hydra littoralis.

$\text{Hydra}$$\text{littoralis}$ exhibits high γ-glutamyl transpeptidase activity, i.e., about 12% of the activity (determined with glutathione) of rat kidney. Histochemical studies show that the enzyme is located mainly in the gastric and sub-hypostome regions; the enzyme is also present in the tentacles and basal disc. These results and the presence of other enzymes of the γ-glutamyl cycle suggest that the cycle plays a role in the metabolism of glutathione in hydras and that γ-glutamyl transpeptidase may function in their digestive and absorptive processes and possibly also in the behavioral response to glutathione.     

Influence of estrogen on glutathione levels and glutathione-metabolizing enzymes in uteri and R3230AC mammary tumors of rats

The glutathione content and the activities of several enzymes in its metabolism, glutathione reductase, glutathione peroxidase and γ-glutamyl transpeptidase, were assayed in uteri obtained from estrogen-treated rats and in R3230AC mammary adenocarcinomas obtained from ovariectomized, intact and estrogen-treated hosts. Normal mammary glands, obtained 10–12 days post-partum, were also examined for these parameters.A daily pharmacological dose of 0.4 μg of estradiol-17β induced a maximal increase in uterine weight and in reduced glutathione (GSH); higher doses of estrogen did not significantly increase either of these parameters. Levels of oxidized glutathione (GSSG) were comparable in both estrogen-treated and untreated rats. The time course of the estrogen-induced uterotrophic response was associated with increases in glutathione reductase, glutathione peroxidase and γ-glutamyl transpeptidase activities with the increased GSH level preceding the increase in uterine weight. Compared to neoplasms from intact or ovariectomized animals, tumors from estrogen-treated hosts exhibited significant decreases in levels of GSSG and GSH, as well as in glutathione reductase and glutathione peroxidase activities, but demonstrated a significant elevation of γ-glutamyl transpeptidase activity. Normal glands from lactating rats had decreased GSH levels, lower activities of glutathione reductase and glutathione peroxidase, but elevated γ-glutamyl transpeptidase activity versus tumors from intact rats. Tumors from estrogen-treated rats more closely resembled mammary glands during lactation. The divergent growth responses elicited by estrogen in the uterus and mammary tumor are correlated with the observed changes in GSH levels and enzymes involved in glutathione metabolism.     

Dissection of glutathione conjugate turnover in yeast

Xenobiotics are widely used as pesticides. The detoxification of xenobiotics frequently involves conjugation to glutathione prior to compartmentalization and catabolism. In plants, degradation of glutathione-S-conjugates is initiated either by aminoterminal or carboxyterminal amino acid cleavage catalyzed by a γ-glutamyl transpeptidase and phytochelatin synthase, respectively. In order to establish yeast as a model system for the analysis of the plant pathway, we used monochlorobimane as a model xenobiotic in Saccharomyces cerevisiae and mutants thereof. The catabolism of monochlorobimane is initiated by conjugation to form glutathione-S-bimane, which is then turned over into a γ-GluCys-bimane conjugate by the vacuolar serine carboxypeptidases CPC and CPY. Alternatively, the glutathione-S-bimane conjugate is catabolized by the action of the γ-glutamyl transpeptidase Cis2p to a CysGly-conjugate. The turnover of glutathione-S-bimane was impaired in yeast cells deficient in Cis2p and completely abolished by the additional inactivation of CPC and CPY in the corresponding triple knockout. Inducible expression of the Arabidopsis phytochelatin synthase AtPCS1 in the triple knockout resulted in the turnover of glutathione-S-bimane to the γ-GluCys-bimane conjugate as observed in plants. Challenge of AtPCS1-expressing yeast cells with zinc, cadmium, and copper ions, which are known to activate AtPCS1, enhanced γ-GluCys-bimane accumulation. Thus, initial catabolism of glutathione-S-conjugates is similar in plants and yeast, and yeast is a suitable system for a study of enzymes of the plant pathway.