γ-Glutamyl transpeptidase of the ackee plant

The enzyme γ-glutamyl transpeptidase was purified from seeds of immature ackee fruit (Blighia sapida; Sapindaceae) by salt fractionation and gel filtration on Biogel P-10 and P-200. The procedure, which differs from an earlier one applied to kidney bean fruit, achieves 9.8% yield and 577-fold purification. The enzyme is also present in other parts of the fruit and in leaves. A MW of 12 500 was found by SDS-polyacrylamide gel electrophoresis, a value much lower that that reported for the enzyme from kidney bean fruit. Neutral or amino sugar accounts for 10% of the dry weight. In vitro, the enzyme catalysed synthesis of an unusual γ-glutamyl dipeptide which occurs in ackee seeds, using glutathione as glutamyl group donor. The enzyme mechanism was of the double displacement (ping-pong) type.     

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.     

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.     

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.     

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.     

Hydrolysis and transfer reactions catalyzed by gamma-glutamyl transpeptidase; evidence for separate substrate sites and for high affinity of L-cystine.

γ-Glutamyl transpeptidase was studied with L- and D-γ-glutamyl-p-nitroanilide as γ-glutamyl donors. No autotranspeptidation occurred with the D-γ-glutamyl donor or when the L-γ-glutamyl donor was used at concentrations lower than 10 μM. The K_m values for hydrolysis were 5 and 31 μM for the L- and D-γ-glutamyl donors, respectively; the corresponding V_max values were identical. The γ-glutamyl donor site of the enzyme thus exhibits low stereospecificity (but high affinity), while the acceptor site exhibits absolute L-specificity. The K_m value for L-cystine as acceptor is very low (30 μM); the same value was obtained with L- and D-γ-glutamyl donors. The data are consistent with a ping pong mechanism and the existence of separate donor and acceptor enzyme sites. The present findings thus extend the usefulness of γ-glutamyl-p-nitro-anilide as a substrate in probing the catalytic behavior of this enzyme.     

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.     

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.     

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.     

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.     

beta-Adrenergic receptor induction in HeLa cells: synergistic effect of 5-azacytidine and butyrate

³H-Labeled leukotriene C₃ was efficiently taken up by the isolated, perfused rat liver and excreted into the bile. The isolated, perfused kidney eliminated leukotriene C₃ from the perfusate slower and excreted only a fraction of the radioactivity into the urine. Isolated hepatic, intestinal and renal cells also took up leukotriene C₃, the renal cells being the most effective in accumulating the label. Anthglutin, an inhibitor of γ-glutamyl transferase, decreased the uptake by kidney cells but had no effect on the uptake by the other cell types. In liver cells, the uptake rate was sensitive to temperature and to cellular ATP content. Chromatographic analyses indicated that renal cells metabolized leukotriene C₃ more rapidly than hepatic and intestinal cells. Leukotriene D₃ and E₃ were formed during the incubations with kidney cells, whereas intestinal cells produced mainly more polar metabolites.     

Glutamine synthetases from rice: purification and preliminary characterization of two forms in leaves and one form in roots

A relatively rapid five-step procedure was used in purifying to apparent homogeneity the glutamine synthetase from roots and one form of the enzyme (GS_I) from leaves of rice. The steps were: preparation of crude extracts, ammonium sulfate precipitation, filtration on Sepharose 4B, fractionation on DEAE-Sephadex A25, and affinity chromatography on ADP-Sepharose 4B. The purified protein appeared as a single band on polyacrylamide gel electrophoresis. Leaf GS_I and the second type of leaf glutamine synthetase (GS_II) formed distinct peaks when eluted from DEAE-Sephadex (step 4). The root enzyme and leaf GS_I were similar in all the properties which were examined. Both enzymes bound to ADP-Sepharose, had similar biosynthetic (18 μmol P/_img protein/min) and transferase (1324 and 1156 μmol γ-glutamyl hydroxamate/mg protein/min) activities, and the same or nearly the same K_m values for glutamate (2.17 mm), Mg²⁺ (4.5 and 5.0 mm), ATP (286 μm), NH₄⁺ (210 and 135 μm), and ADP (3.8 and 5.3 μm). In contrast, leaf GS_II did not bind to ADP-Sepharose and had much higher K_m values for glutamate (8.3 mm), Mg²⁺ (15 mm), NH₄⁺ (684 μm), and ADP (33 μm).     

Gamma-glutamyl transpeptidase of rat seminal vesicles; effect of orchidectomy and hormone administration on the transpeptidase in relation to its possible role in secretory activity.

γ-Glutamyl transpeptidase was prepared from rat seminal vesicles by two methods and was found to be similar to rat kidney γ-glutamyl transpeptidase with respect to substrate specificity, stimulation of “glutaminase” activity by maleate, and apparent molecular weight. Histochemical studies demonstrated that γ-glutamyl transpeptidase is concentrated in the secretory epithelium of the seminal vesicle. Like the epithelium itself, the enzyme responds to the presence or absence of testosterone. The content and specific activities of γ-glutamyl transpeptidase and γ-glutamyl cyclotransferase in rat seminal vesicles are low in orchidectomized animals, an effect which is reversed by administration of testosterone but accentuated by estradiol administration. These enzymes may be involved in the secretory functions of the seminal vesicles.     

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.     

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.     

gamma-Glutamyl transpeptidase of human seminal fluid.

γ-Glutamyl transpeptidase, which is present in high levels in human seminal fluid plasma, was purified about 870-fold from this source. The enzyme is present in seminal fluid plasma in particulate form. Purification by a procedure involving treatment with bromelain gave a protein (apparent molecular weight, about 70,000), which exhibited catalytic properties characteristic of γ-glutamyl transpeptidase preparations isolated from rat kidney and other mammalian tissues. The physiological significance of seminal fluid γ-glutamyl transpeptidase and its potential clinical value are considered.     

Properties of γ-Glutamyltransferase from Lentinus edodes

An enzyme preparation catalyzing p-nitroaniline release from γ-glutamyl-p-nitroanilide was obtained in a 200-fold purified state from fruit bodies of an edible mushroom, Lentinus edodes. Analysis of the final preparation by differential centrifugation revealed that the enzyme was still bound with subcellular particles. The enzyme catalyzed both the hydrolysis and transfer of the γ-glutamyl moiety from γ-glutamyl-p-nitroanilide, but exhibited essentially no activity of glutaminase, glutamine aminotransferase, glutamine synthetase or γ-glutamyl cyclotransferase. With γ-glutamyl-p-nitroanilide the activity was maximal at about pH 7.6. The enzyme activity increased with an increasing concentration of Tris-HCl buffer, but not with phosphate buffer which was inhibitory. An apparent Michaelis constant of 4 mm was obtained in 0.5 m Tris-HCl buffer at pH 7.6. S-Alkylcysteine sulfoxide served as the best glutamyl acceptor. A serine-borate mixture, pCMB, Cu²⁺, Hg²⁺ and Zn²⁺ were potent inhibitors. All the experimental results, including the insoluble nature of the enzyme, allowed us to classify the Lentinus enzyme in the family of γ-glutamyl transferase.     

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.     

Biochemical and structural properties of gamma-glutamyl transpeptidase from Geobacillus thermodenitrificans: An enzyme specialized in hydrolase activity

Gamma-glutamyltranspeptidases (γ-GTs) catalyze the transfer of the gamma-glutamyl moiety of glutathione and related gamma-glutamyl amides to water (hydrolysis) or to amino acids and peptides (transpeptidation) and play a key role in glutathione metabolism. Recently, γ-GTs have been considered attractive pharmaceutical targets for cancer and useful tools to produce γ-glutamyl compounds. To find out γ-GTs with special properties we have chosen microorganisms belonging to Geobacillus species which are source of several thermostable enzymes of potential interest for biotechnology. γ-GT from Geobacillus thermodenitrificans (GthGT) was cloned, expressed in Escherichia coli, purified to homogeneity and characterized. The enzyme, synthesized as a precursor homotetrameric protein of 61-kDa per subunit, undergoes an internal post-translational cleavage of the 61 kDa monomer into 40- and 21-kDa shorter subunits, which are then assembled into an active heterotetramer composed of two 40- and two 21-kDa subunits. The kinetic characterization of the hydrolysis reaction using l-glutamic acid γ-(4-nitroanilide) as the substrate reveals that the active enzyme has K_m 7.6 μM and V_max 0.36 μmol min/mg. The optimum pH and temperature for the hydrolysis activity are 7.8 and 52 °C, respectively. GthGT hydrolyses the physiological antioxidant glutathione, suggesting an involvement of the enzyme in the cellular defense mechanism against oxidative stress. Unlike other γ-GTs, the mutation of the highly conserved catalytic nucleophile, Thr353, abolishes the post-translational cleavage of the pro-enzyme, but does not completely block the hydrolytic action. Furthermore, GthGT does not show any transpeptidase activity, suggesting that the enzyme is a specialized γ-glutamyl hydrolase. The GthGT homology-model structure reveals peculiar structural features, which should be responsible for the different functional properties of the enzyme and suggests the structural bases of protein thermostability.