Subcellular Localization and Possible Functions of γ-Glutamyltransferase in the Radish (Raphanus sativus L.) Plant

Previously we reported the purification of soluble γ-glutamyltransferases (GGTs) from radish cotyledon. Subcellular fractionation of radish cells revealed that soluble GGT is a vacuolar enzyme. Acivicin, a GGT inhibitor, mediated the in vivo catabolism inhibition of the glutathione S-conjugate generated from endogenous glutathione and exogenously supplied monochlorobimane. Thus soluble GGT is possibly involved in the catabolism of glutathione S-conjugates.     

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.     

A kinetic study of gamma-glutamyltransferase (GGT)-mediated S-nitrosoglutathione catabolism

S-Nitrosoglutathione (GSNO) is a nitric oxide (NO) donor compound which has been postulated to be involved in transport of NO in vivo. It is known that γ-glutamyl transpeptidase (GGT) is one of the enzymes involved in the enzyme-mediated decomposition of GSNO, but no kinetics studies of the reaction GSNO-GGT are reported in literature.In this study we directly investigated the kinetics of GGT with respect to GSNO as a substrate and glycyl-glycine (GG) as acceptor co-substrate by spectrophotometry at 334 nm. GGT hydrolyses the γ-glutamyl moiety of GSNO to give S-nitroso-cysteinylglycine (CGNO) and γ-glutamyl-GG. However, as both the substrate GSNO and the first product CGNO absorb at 334 nm, we optimized an ancillary reaction coupled to the enzymatic reaction, based on the copper-mediated decomposition of CGNO yielding oxidized cysteinyl-glycine and NO. The ancillary reaction allowed us to study directly the GSNO/GGT kinetics by following the decrease of the characteristic absorbance of nitrosothiols at 334 nm. A K_m of GGT for GSNO of 0.398 ± 31 mM was thus found, comparable with K_m values reported for other γ-glutamyl substrates of GGT.     

Phytochelatins are synthesized by two vacuolar serine carboxypeptidases in Saccharomyces cerevisiae

Phytochelatins (PCs) are cysteine-rich peptides that chelate heavy metal ions, thereby mediating heavy metal tolerance in plants, fission yeast, and Caenorhabditis elegans. They are synthesized from glutathione by PC synthase, a specific dipeptidyltransferase. While Saccharomyces cerevisiae synthesizes PCs upon exposure to heavy metal ions, the S. cerevisiae genome does not encode a PC synthase homologue. How PCs are synthesized in yeast is unclear. This study shows that the vacuolar serine carboxypeptidases CPY and CPC are responsible for PC synthesis in yeast. The finding of a PCS-like activity of these enzymes in vivo discloses another route for PC biosynthesis in eukaryotes.     

Transport of the large-neutral amino acids by the gamma-glutamyl cycle: a proposal.

The γ-glutamyl cycle has been proposed by Meister (1973) as one possible mechanism for the mediation of amino acid transport. The high energy requirement of the pathway, the very low specificity of γ-glutamyl transpeptidase and the inability to account for trans membrane stimulation of amino acid entry are but three criticisms of this hypothesis. It is proposed that the various objections can be overcome by postulating that the soluble form of γ-glutamyl transpeptidase transfers the γ-glutamyl moiety from gluthathione to glutamine (in the case of brain) and that the membrane sequestered form of this enzyme catalyzes the exchange of the γ-glutamyl group between γ-glutamyl glutamine and an entering neutral amino acid. The released glutamine leaves the cell. The γ-glutamyl amino acid then passes into the cytoplasm where it is acted upon by either γ-glutamyl cyclotransferase or the soluble γ-glutamyl transpeptidase which transfers the γ-glutamyl group to another molecule of glutamine. It is postulated that access to the membrane-bound enzyme is dependent on the relative lipophilia of the entering large-neutral amino acids. The available data support this mechanism. By regeneration of γ-glutamyl glutamine, a low expenditure of energy is required for the transport process. Specificity of transpeptidation is attained by the constraints of access to the membrane bound enzyme site.     

Modification of the acceptor binding site of gamma-glutamyl transpeptidase by the diazonium derivatives of p-aminohippurate and p-aminobenzoate

Hippurate and maleate have been shown to bind to the aminoacylglycine (acceptor) binding site of γ-glutamyl transpeptidase, thereby stimulating the hydrolysis of γ-glutamyl compounds at the expense of transpeptidation (Thompson, G. A., and Meister, A. (1979) J. Biol. Chem.254, 2956–2960; Thompson, G. A., and Meister, A. (1980) J. Biol. Chem.255, 2109–2113). It has now been found that a number of benzoate derivatives also bind and modulate rat kidney transpeptidase, as indicated by their ability to enhance the rate of inactivation of transpeptidase by the glutamine antagonist l-(αS, 5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125). Furthermore, rapid loss of transpeptidase activity results upon preincubation of the enzyme with the diazonium derivatives of p-aminohippurate and p-aminobenzoate. The modified enzyme can still hydrolyze γ-glutamyl substrates but is no longer modulated by hippurate and maleate. Loss of transpeptidase activity was not associated with incorporation of radioactive label from diazotized [¹⁴C]p-aminohippurate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the modified enzyme revealed a nondissociable species, M_r 68,000, shown to result from crosslinking of the two subunits of transpeptidase (M_r 46,000 and 22,000, respectively). The crosslinking of the subunits paralleled the extent of inactivation of transpeptidation activity and both crosslinking and inactivation were prevented by treatment with the diazotized derivatives in the presence of either hippurate or maleate. These and other data indicate that the diazonium derivatives of p-aminohippurate and p-aminobenzoate interact with the acceptor binding site and produce a stable bond between amino acid residues in the vicinity of this site which, thus, appears to be located in the intersubunit contact region.     

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.     

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.     

Function of phytochelatin synthase in catabolism of glutathione-conjugates

Detoxification of xenobiotic compounds and heavy metals is a pivotal capacity of organisms, in which glutathione (GSH) plays an important role. In plants, electrophilic herbicides are conjugated to the thiol group of GSH, and heavy metal ions form complexes as thiolates with GSH-derived phytochelatins (PCs). In both detoxification processes of plants, phytochelatin synthase (PCS) emerges as a key player. The enzyme is activated by heavy metal ions and catalyzes PC formation from GSH by transferring glutamylcysteinyl residues (gamma-EC) onto GSH. In this study with Arabidopsis, we show that PCS plays a role in the plant-specific catabolism of glutathione conjugates (GS-conjugates). In contrast to animals, breakdown of GS-conjugates in plants can be initiated by cleavage of the carboxyterminal glycine residue that leads to the generation of the corresponding gamma-EC-conjugate. We used the xenobiotic bimane in order to follow GS-conjugate turnover. Functional knockout of the two PCS of Arabidopsis, AtPCS1 and AtPCS2, revealed that AtPCS1 provides a major activity responsible for conversion of the fluorescent bimane-GS-conjugate (GS-bimane) into gamma-EC-bimane. AtPCS1 deficiency resulted in a gamma-EC-bimane deficiency. Transfection of PCS-deficient cells with AtPCS1 recovered gamma-EC-bimane levels. The level of the gamma-EC-bimane conjugate was enhanced several-fold in the presence of Cd2+ ions in the wild type, but not in the PCS-deficient double mutant, consistent with a PCS-catalyzed GS-conjugate turnover. Thus AtPCS1 has two cellular functions: mediating both heavy metal tolerance and GS-conjugate degradation.     

gamma-Glutamyl transpeptidase from WI-38 fibroblasts: purification and active site modification studies

γ-Glutamyl transpeptidase has been purified to homogeneity from WI-38 human fetal lung fibroblasts, following extraction with Triton X-100 in the absence of added proteases. The specific activity of the purified enzyme is 16 units/mg protein at the optimum of pH 8.0. Although this activity value is low, the WI-38 enzyme is very similar to previously described γ-glutamyl transpeptidases in its molecular properties. The native molecule (apparent molecular weight of 82,000) is composed of one light and one heavy subunit (apparent molecular weights of 20,000 and 62,000, respectively). Papain digestion reduces the native molecular weight to an apparent value of 73,000 by proteolysis of the heavy chain. The known active site modifying agent and glutamine analog 6-diazo-5-oxo-l-nor-leucine, completely inactivates the enzyme, coincident with its stoichiometric incorporation into the light subunit. This inactivation is accelerated by maleate and prevented by S-methylglutathione. The WI-38 γ-glutamyl transpeptidase is also inactivated by the fluorescent alkylating agent, 5-iodoacetamidofluorescein. Selective reaction of this reagent with an active site residue is suggested by prevention of the inactivation by S-methylglutathione, the stoichiometric incorporation of the fluorescein moiety, and the loss of one methionine residue per molecule of protein accompanying inactivation.     

A new chromogenic substrate for gamma-glutamyl transpeptidase.

A benzfurazan derivative of glutathione l-γ-glutamyl-(S-4-nitrobenz-2-oxa-1,3-diazole)-l-cysteinylglycine (GS-NBD) with an absorption maximum at 419 nm is readily acted upon by γ-glutamyl transpeptidase to yield the S-benzfurazan derivative of cysteinylglycine. An internal S→N shift occurs immediately to yield the N-benzfurazan derivative, which in turn reacts with the sulfhydryl reagent 4,4′-dithiodipyridine to produce the mixed disulfide with an intense absorption at 461 nm. The maximum difference in molar extinction coefficient is 13,200 and occurs at 470 nm. This general device should be applicable to the assay of many other peptidases.     

Overexpression of Arabidopsis phytochelatin synthase in tobacco plants enhances Cd2+ tolerance and accumulation but not translocation to the shoot

Phytochelatins (PCs) are metal binding peptides involved in heavy metal detoxification. To assess whether enhanced phytochelatin synthesis would increase heavy metal tolerance and accumulation in plants, we overexpressed the Arabidopsis phytochelatin synthase gene (AtPCS1) in the non-accumulator plant Nicotiana tabacum. Wild-type plants and plants harbouring the Agrobacterium rhizogenes rolB oncogene were transformed with a 35S AtPCS1 construct. Root cultures from rolB plants could be easily established and we demonstrated here that they represent a reliable system to study heavy metal tolerance. Cd²⁺ tolerance in cultured rolB roots was increased as a result of overexpression of AtPCS1, and further enhanced when reduced glutathione (GSH, the substrate of PCS1) was added to the culture medium. Accordingly, HPLC analysis showed that total PC production in PCS1-overexpressing rolB roots was higher than in rolB roots in the presence of GSH. Overexpression of AtPCS1 in whole seedlings led to a twofold increase in Cd²⁺ accumulation in the roots and shoots of both rolB and wild-type seedlings. Similarly, a significant increase in Cd²⁺ accumulation linked to a higher production of PCs in both roots and shoots was observed in adult plants. However, the percentage of Cd²⁺ translocated to the shoots of seedlings and adult overexpressing plants was unaffected. We conclude that the increase in Cd²⁺ tolerance and accumulation of PCS1 overexpressing plants is directly related to the availability of GSH, while overexpression of phytochelatin synthase does not enhance long distance root-to-shoot Cd²⁺ transport.     

Immobilization of γ-glutamyl transpeptidase,a membrane enzyme,in gel beads via liposome entrapment

Bovine kidney γ-glutamyl transpeptidase, a membrane enzyme, was immobilized in gel beads by application of the method of Wallstén et al. (Biochim. Biophys. Acta, 982, 47–52, 1989). The gel beads were equilibrated with a dispersion of the enzyme, phospholipids, and cholate and subsequently dialyzed against a buffer for reconstitution and immobilization of enzyme-bound liposomes in the pores of the beads. From the standpoints of the immobilized contents of protein and phospholipids and of the reactivity of γ-glutamyl transpeptidase, a dialysis buffer of Tris-HCl (pH 7.5), a phospholipid concentration of 45 mg/ml in the enzyme-phospholipid-cholate dispersion, and the use of Sepharose CL-6B as the support gel were found to be most appropriate for the immobilization of γ-glutamyl transpeptidase, γ-Glutamyl transpeptidase was activated and stabilized by reconstitution in liposomes. In operation with a packed bed reactor, liposome-bound γ-glutamyl transpeptidase immobilized in Sepharose CL-6B exhibited relatively stable and constant activity for 12 h. In addition, it was found that enzyme substrates were able to pass through the pores of the gel beads to interact with the enzyme present on the outer surface of the liposome membrane in the gel beads. These results thus indicated that a novel support made up of liposomes and Sepharose CL-6B would permit efficient immobilization of lipid-requiring and/or membrane enzymes.     

Isolation from bovine brain of a fraction containing capillaries and a fraction containing membrane fragments of the choroid plexus

Combined differential and density gradient centrifugation was used for the isolation of a capillary-rich fraction from the cerebral cortex and a brush border containing fraction from the bovine choroid plexus. The activities of γ-glutamyl transpeptidase and several other marker enzymes were monitored during the fractionation procedure. Electron microscopic examination showed a membrane-rich fraction in the choroid plexus high in γ-glutamyl transpeptidase and 5'-nucleotidase activities. From the brain cortex, a capillary-rich fraction was obtained which was high in γ-glutamyl transpeptidase and alkaline phosphatase activities. A histochemical examination showed γ-glutamyl transpeptidase activity localized in the capillary walls.     

Molecular and kinetic properties of purified γ-glutamyl transpeptidase from yeast (Saccharomyces cerevisiae)

The enzyme γ-glutamyl transpeptidase was purified from the yeast Saccharomyces cerevisiae by a procedure involving protamine sulfate treatment, chromatography on DEAE-Sephadex A 50, salt fractionation, successive chromatography on Sephadex G 150 and lentil lectin sepharose 6B. The procedure achieves 25 % yield and 4200-fold purification. The final preparation is a glycoprotein (M, 90 000) containing 31.4 % carbohydrates and composed of two non-identical subunits (M, 64 000 and 23 000). The specificity patterns of the yeast enzyme are rather similar to those of mammalian and higher plant transpeptidases. The enzyme mechanism might be of the double displacement (ping-pong) type.     

Chloroplast targeting of phytochelatin synthase in Arabidopsis: effects on heavy metal tolerance and accumulation

The enzymatically synthesized thiol peptide phytochelatin (PC) plays a central role in heavy metal tolerance and detoxification in plants. In response to heavy metal exposure, the constitutively expressed phytochelatin synthase enzyme (PCS) is activated leading to synthesis of PCs in the cytosol. Recent attempts to increase plant metal accumulation and tolerance reported that PCS over-expression in transgenic plants paradoxically induced cadmium hypersensitivity. In the present paper, we investigate the possibility of synthesizing PCs in plastids by over-expressing a plastid targeted phytochelatin synthase (PCS). Plastids represent a relatively important cellular volume and offer the advantage of containing glutathione, the precursor of PC synthesis. Using a constitutive CaMV 35S promoter and a RbcS transit peptide, we successfully addressed AtPCS1 to chloroplasts, significant PCS activity being measured in this compartment in two independent transgenic lines. A substantial increase in the PC content and a decrease in the glutathione pool were observed in response to cadmium exposure, when compared to wild-type plants. While over-expressing AtPCS1 in the cytosol importantly decreased cadmium tolerance, both cadmium tolerance and accumulation of plants expressing plastidial AtPCS1 were not significantly affected compared to wild-type. Interestingly, targeting AtPCS1 to chloroplasts induced a marked sensitivity to arsenic while plants over-expressing AtPCS1 in the cytoplasm were more tolerant to this metalloid. These results are discussed in relation to heavy metal trafficking pathways in higher plants and to the interest of using plastid expression of PCS for biotechnological applications.     

gamma-Glutamyl transpeptidase in Hydra.

γ-Glutamyl transpeptidase (EC 2.3.2.2) activity is described in the coelenterate, $\text{Hydra}$$\text{attenuata}$, using the substrate γ-glutamyl-p-nitroanilide. The properties of the γ-glutamyl donor required for binding to the transpeptidase were investigated by measuring the ability of GSH analogs to inhibit the release of p-nitroaniline. Whereas no binding was observed when the γ-glutamyl moiety was altered, analogs with substitution in the Cys residue were capable of binding to the enzyme. A specificity for the Gly residue was indicated because analogs containing Leu or Tyr in place of Gly exhibited decreased binding capacities for the hydra transpeptidase. A comparison of these data with those obtained using the same analogs in the GSH induced feeding response bioassay shows that γ-glutamyl transpeptidase activity and the GSH receptor for the hydra feeding response have different specificities.     

Isolation of anthglutin, an inhibitor of gamma-glutamyl transpeptidase from Penicillum oxalicum.

Anthglutin, a new inhibitor of γ-glutamyl transpeptidase, has been isolated from the cultured medium of Penicillium oxalicum and its structure established as l-γ-l-glutamyl-2-(2-carboxyphenyl)hydrazine. The isolation of anthglutin was achieved by ion-exchange chromatography. Anthglutin inhibited γ-glutamyl transpeptidase specifically and the kinetic analysis of the inhibition showed that anthglutin inhibited the enzyme competitively with regard to the glutamyl donor, γ-glutamyl-p-nitroanilide, and noncompetitively with regard to the glutamyl acceptor, glycylglycine. K₁ values were 5.7 μm for the hog kidney enzyme, 18.3 μm for the human kidney enzyme, 13.6 μm for the human liver soluble enzyme, and 10.2 μm for the bound enzyme. After oral administration of [¹⁴C]methionine and anthglutin to rats, no effect of anthglutin was observed on the absorption of methionine in the intestine.     

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.     

Isolation and identification of l-cystathionine and l-selenocystathionine from the foliage of Astragalus pectinatus

l-Cystathionine and l-selenocystathionine have been isolated from the foliage of Astragalus pectinatus. In addition to these two amino acids, some S-methylcysteine and trace amounts of Se-methyl-selenocysteine were also detected in the foliage extracts. The seeds of A pectinatus were found to contain significant amounts of all four of these amino acids plus the γ-glutamyl peptides of S-methylcysteine and Se-methylselenocysteine.