PARTIAL PURIFICATION AND KINETICS OF γ-GLUTAMYL TRANSPEPTIDASE FROM BOVINE CHOROID PLEXUS

Abstract— γ-Glutamyl transpeptidase from bovine choroid plexus has been shown to be a membrane-bound enzyme. Partial purification of the enzyme has been accomplished using detergent extraction and ammonium sulfate fractionation. Important determinants of enzymatic activity with acceptor substrates included chain length, stereoisomerism, and amino acid composition of the acceptors. L-Methionine was the best amino acid substrate and its corresponding peptides L-methionylmethionine and L-methionyl-L-serine were also good γ-glutamyl acceptors. L-Alanine and glycine were poor acceptor substrates; whereas, some peptides containing these amino acids were excellent substrates. Glycylglycine was significantly more effective as a γ-glutamyl acceptor than glycine, triglycine, or tetraglycine. L-Alanylglycine was a superior acceptor to glycine, L-alanine, or L-alanylglycylglycine, while the D-isomer of alanylglycine was only minimally effective as an acceptor substrate. In general glycyl peptides were the best acceptor substrates examined. Our findings that γ-glutamyl transpeptidase could catalyze the transfer of γ-glutamyl groups to glycylglycyl-L-alanine and L-alanylglycylglycine are of special interest, since few examples of tripeptide acceptors for the enzyme have been found. It is suggested that γ-glutamyl transpeptidase might play a role in the inactivation and/or transport of biologically active peptides.     

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.     

γ-Glutamyl compounds and their enzymatic production using bacterial γ-glutamyltranspeptidase

Summary. Some amino acids and peptides, which have low solubility in water, become much more soluble following γ-glutamylation. Compounds become more stable in the blood stream with γ-glutamylation. Several γ-glutamyl compounds are known to have favorable physiological effects on mammals. γ-Glutamylation can improve taste and can stabilize glutamine in aqueous solution. Because of such favorable features, γ-glutamyl compounds are very attractive. However, only a small number of γ-glutamyl amino acids have been studied although many other γ-glutamyl compounds may have characteristics that will benefit humans. This is mainly because γ-glutamyl compounds have not been readily available. An efficient and simple method of producing various γ-glutamyl compounds, especially γ-glutamyl amino acids, using bacterial γ-glutamyltranspeptidase has been developed. With this method, modifications of reactive groups of the substrate and energy source such as ATP are not required, and a wide-range of γ-glutamyl compounds can be synthesized. Moreover, bacterial γ-glutamyltranspeptidase, a catalyst for this method, is readily available from the strain over-producing this enzyme. The superiority of producing γ-glutamyl compounds with bacterial γ-glutamyltranspeptidase over other methods of production is discussed.     

Increased Intra- and Extracellular Concentrations of γ-Glutamylglutamate and Related Dipeptides in the Ischemic Rat Striatum: Involvement of γ-Glutamyl Transpeptidase

Abstract: The present work relates to the possibility that the ATP-independent enzyme γ-glutamyl transpeptidase (EC 2.3.2.2), which has been postulated to be part of an amino acid uptake system, is active during cerebral ischemia. This was evaluated in the ischemic rat striatum by determination of intra- and extracellular concentrations of γ-glutamyl dipeptides (the products of the transpeptidation) and glutathione (the physiological γ-glutamyl donor). An ischemic period (0–30 and 31–60 min) resulted in prominent increases in the respective concentration of extracellular γ-glutamylglutamate (24- and 67-fold), γ-glutamyltaurine + γ-glutamylglycine (5.8- and 19-fold), and γ-glutamylglutamine (2.6- and 6.8-fold) as revealed using in vivo microdialysis. The changes coincided with increased respective extracellular concentrations of glutamate (83- and 115-fold), taurine (17- and 25-fold), glycine (4.6- and 6.1-fold), and glutamine (1.7- and 2.1-fold). Furthermore, under anoxic conditions in vitro (0–30 and 0–60 min), respective striatal tissue concentrations were increased for γ-glutamylglutamate (20- and 17-fold), γ-glutamyltaurine (6.7- and 11-fold), γ-glutamylglutamine (1.7- and 1.2-fold), and γ-glutamylglycine (14- and 18-fold), whereas glutathione levels were, on an average, decreased by ∼350 µ M . In summary, γ-glutamyl transpeptidase is involved in de novo dipeptide synthesis in the mammalian brain during anoxic conditions, indicating transport of amino acids such as glutamate.     

FORMATION OF γ-GLUTAMYLHISTAMINE FROM HISTAMINE IN RAT BRAIN

Abstract– γ-Glutamyl amides of histamine, serotonin and dopamine were formed from these amines by the transfer of the γ-glutamyl moiety from γ-glutamyl peptides in the presence of γ-glutamyl transpeptidase. [^I4C]Histamine was injected intraventricular into rats, and the formation of γ-glutamyl-[¹⁴C] histamine in the brain was confirmed by purification and identification with the authentic compound. The radioactivity was highest 30 min after the injection. The possible significance of γ-glutamyl amides in nerve transmission is discussed.     

Ontogeny of the enzymes related to γ-glutamyl cycle in the human fetal system

Measurements have been made of the enzymes associated with γ-glutamyl cycleviz, γ-glutamyl transpeptidase, γ-glutamyl cysteine synthetase, and 5-oxoprolinase in human fetal brain, liver and kidney over 12–36 weeks of gestation. γ-Glutamyl transpeptidase activity increases gradually with age. γ-Glutamyl cysteine synthetase and 5-oxoprolinase show biphasic pattern of development in human fetal brain. The data presented in this communication may indicate a relationship between γ-glutamyl cycle and amino acid transport.     

γ-Glutamylcyclotransferase in tobacco suspension cultures: Catalytic properties and subcellular localization

Steinkamp, R., Schweihofen, B. and Rennenberg, H. 1987. γ-Glutamylcyclotransfer-ase in tobacco suspension cultures: Catalytic properties and subcellular localization.
γ-Glutamylcyclotransferase activity (EC 2.3.2.4) in ammonium sulfate precipitates (40–70% saturation) of extracts from cultured tobacco cells ( Nicoliana tabacum L. cv. Samsun) was analyzed as liberation of 5-oxo-proline from L-γ-glutamyl dipeptides. The enzyme was highly specific for the sulfur containing γ-glutamyl dipeptides γ-glutamyl-L-methionine and γ-glutamyl-i.-cysteine. Maximum activity was obtained at pH 8.7 and 35°C. As also observed with animal γ-glutamylcyclotransferase, the tobacco enzyme exhibited a relatively low substrate affinity (γ-glutamyl-i.-methionine: K_m 2.2 ± 0.4 mM ). In contrast to animal γ-glutamylcyclotransferase, the tobacco enzyme was not inhibited by the D-isomerof the substrate D-γ-glutamyl-i.-methionine; it also did not use the D isomer as a substrate. γ-Glutamylcyclotransferase of tobacco cells was shown to be a soluble enzyme entirely localized in the cytoplasm.     

Enzymatic synthesis of γ-glutamyl-l-histidine by γ-glutamyltranspeptidase from Escherichia coli K-12

γ-Glutamyl- l -histidine was synthesized from l -glutamine and l -histidine by γ-glutamyltranspeptidase (EC 2.3.2.2) from Escherichia coli K-12. The optimum conditions for the production of γ-glutamyl- l -histidine are determined. The yield of γ-glutamyl- l -histidine synthesized under the optimum conditions was 48% with both substrates (41.2 g/l). The product was isolated and then identified by an amino acid analyser, PMR spectrum and secondary ion mass spectrum analyses.     

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.     

The human gamma-glutamyltransferase gene family

Assays for gamma-glutamyl transferase (GGT1, EC 2.3.2.2) activity in blood are widely used in a clinical setting to measure tissue damage. The well-characterized GGT1 is an extracellular enzyme that is anchored to the plasma membrane of cells. There, it hydrolyzes and transfers γ-glutamyl moieties from glutathione and other γ-glutamyl compounds to acceptors. As such, it has a critical function in the metabolism of glutathione and in the conversion of the leukotriene LTC4 to LTD4. GGT deficiency in man is rare and for the few patients reported to date, mutations in GGT1 have not been described. These patients do secrete glutathione in urine and fail to metabolize LTC4. Earlier pre-genome investigations had indicated that besides GGT1, the human genome contains additional related genes or sequences. These sequences were given multiple different names, leading to inconsistencies and confusion. Here we systematically evaluated all human sequences related to GGT1 using genomic and cDNA database searches and identified thirteen genes belonging to the extended GGT family, of which at least six appear to be active. In collaboration with the HUGO Gene Nomenclature Committee (HGNC) we have designated possible active genes with nucleotide or amino acid sequence similarity to GGT1, as GGT5 (formerly GGL, GGTLA1/GGT-rel), GGT6 (formerly rat ggt6 homologue) and GGT7 (formerly GGTL3, GGT4). Two loci have the potential to encode only the light chain portion of GGT and have now been designated GGTLC1 (formerly GGTL6, GGTLA4) and GGTLC2. Of the five full-length genes, three lack of significant nucleotide sequence homology but have significant (GGT5, GGT7) or very limited (GGT6) amino acid similarity to GGT1 and belong to separate families. GGT6 and GGT7 have not yet been described, raising the possibility that leukotriene synthesis, glutathione metabolism or γ-glutamyl transfer is regulated by their, as of yet uncharacterized, enzymatic activities. In view of the widespread clinical use of assays that measure γ-glutamyl transfer activity, this would appear to be of significant interest. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of the B Subtype of γ-Phospholipase A2 Inhibitor from Protobothrops flavoviridis Serum and Molecular Evolution of Snake Serum Phospholipase A2 Inhibitors

A cDNA encoding a novel phospholipase A₂ (PLA₂) inhibitor (PLI) was isolated from a Protobothrops flavoviridis snake (Tokunoshima island, Japan) liver cDNA library. This cDNA encoded a signal peptide of 19 amino acids followed by a mature protein of 181 amino acids. Its N-terminal amino acid sequence was completely in accord with that of a PLI, named PLI-II, previously found in P. flavoviridis serum. PLI-II showed a high similarity in sequence to the B subtype of γPLI, denoted γPLI-B, isolated from Agkistrodon blomhoffii siniticus serum. Thus, PLI-II is P. flavoviridis serum γPLI-B. Since PLI-I, previously isolated from P. flavoviridis serum, can be assigned as γPLI-A, P. flavoviridis serum contains both A and B subtypes of γPLI. Phylogenetic analysis of γPLIs from the sera of various kinds of snakes, Elapinae, Colubrinae, Laticaudinae, Acanthophiinae, Crotalinae, and Pythonidae, based on the amino acid sequences revealed that A and B subtypes of γPLIs are clearly separated from each other. It was also found that phylogenetic topologies of γPLIs are in good agreement with speciation processes of snakes. The BLAST search followed by analyses with particular Internet search engines of proteins with Cys/loop frameworks similar to those of PLI-II and PLI-I revealed that γPLI-Bs, including PLI-II and PLI-II-like proteins from mammalian sources, form a novel PLI-II family which possesses the common Cys/loop frameworks in the anterior and posterior three-finger motifs in the molecules. Several lines of evidence suggest that PLI-II is evolutionarily ancestral to PLI-I. The nucleotide sequence reported in this paper is available from the GenBank/EMBL/DDBJ databases under accession number AB290845.     

Light-dependent modulation of foliar glutathione synthesis and associated amino acid metabolism in poplar overexpressing γ-glutamylcysteine synthetase

Glutathione (GSH), γ-glutamylcysteine (γ-EC) and major free amino acids were measured in darkened and illuminated leaves from untransformed poplars (Populus tremula × P. alba) and poplars expressing Escherichia coli genes for γ-glutamylcysteine synthetase (γ-ECS; EC 3.2.3.3) and glutathione reductase (GR; EC 1.6.4.2). In poplars overexpressing γ-ECS, foliar γ-EC contents and GSH contents were markedly enhanced compared to poplars lacking the bacterial gene for the enzyme. However, the quantitative relationship between the foliar pools of γ-EC and GSH in these transformants was markedly dependent on light. In the dark, GSH content was relatively low and γ-EC content high, the latter being higher than the foliar GSH contents of untransformed poplars in all conditions. Hence, this transformation appears to elevate γ-EC from the ranks of a trace metabolite to one of major quantitative importance. On illumination, however, γ-EC content decreased fourfold whereas GSH content doubled. Glutathione was also higher in the light in untransformed poplars and in those overexpressing GR. In these plants, γ-EC was negligible in the light but increased in the dark. Cysteine content was little affected by light in any of the poplar types. No light-dependent changes in the extractable activities of γ-ECS, glutathione synthetase (EC 3.2.3.2) or GR were observed. In contrast, both the activation state and the maximum extractable activity of nitrate reductase (EC 1.6.6.1) were increased by illumination. In all poplar types, glutamate and aspartate were the major amino acids. The most marked light-induced increases in individual amino acids were observed in the glutamine, asparagine, serine and glycine pools. Illumination of leaves from poplars overexpressing γ-ECS at elevated CO₂ or low O₂ largely abolished the inverse light-dependent changes in γ-EC and GSH. Low O₂ did not affect foliar contents of cysteine or glutamate but prevented the light-induced increase in the glycine pool. It is concluded that light-dependent glycine formation through the photorespiratory pathway is required to support maximal rates of GSH synthesis, particularly under conditions where the capacity for γ-EC synthesis is augmented. Received: 17 December 1996 / Accepted: 28 January 1997     

IN VIVO INHIBITION OF γ-GLUTAMYLCYSTEINE SYNTHETASE BY l-METHIONINE-RS-SULFOXIMINE; INFLUENCE ON INTERMEDIATES OF THE γ-GLUTAMYL CYCLE

—The inhibition of γ-glutamylcysteine synthetase and its influence on the concentration of intermediates associated with the metabolism of glutathione was studied in mice receiving methionine sulfoximine, a convulsant agent. The activity of the enzyme decreased significantly in the liver and kidney 1-4 h after administration of methionine sulfoximine; the activity of the enzyme in the brain was unchanged after 1 and 2 h but decreased significantly after 4 h. There was a rapid and sharp decrease in the concentration of glutathione in the kidney and a slower decrease in the liver. Brain glutathione concentrations were unaffected. Methionine sulfoximine in vivo, inhibited the synthesis of l -γ-glutamyl-l -α-aminobutyrate after administration of l -α-aminobutyrate, a reaction catalyzed by γ-glutamylcysteine synthetase. The inhibitor also lowered the concentration of pyrrolidone carboxylate in mouse tissues and prevented the accumulation of this intermediate after administration of l -α-aminobutyrate. The results show that methionine sulfoximine in vivo affects the metabolism of glutathione and that this action may contribute to its convulsive properties.     

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.     

Purification and characterization of cystathionine γ-lyase from Lactobacillus fermentum DT41

A homo-tetrameric ca. 140-kDa cystathionine γ-lyase was purified to homogeneity from Lactobacillus fermentum DT41 by four chromatographic steps. This was the first enzyme responsible for amino acid catabolism purified from lactobacilli. The activity is pyridoxal-5'-phosphate dependent and the enzyme catalyzes the α,γ-elimination reaction of l -cystathionine producing l -cysteine, ammonia and α-ketobutyrate. The cystathionine γ-lyase produced a free thiol group, a keto acid component and ammonia from several amino acids, including l -cysteine and methionine, and amino acid derivatives. l -Cystine was the best substrate. The enzyme was stable in the conditions of cheese ripening and may contribute to the biosynthesis of sulfur-containing compounds.     

Gene cloning and protein expression of γ-glutamyltranspeptidases from <Emphasis Type="Italic">Thermus thermophilus</Emphasis> and <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>: comparison of molecular and structural properties with mesophilic counterparts

γ-Glutamyltranspeptidase (γ-GT) is an ubiquitous enzyme that catalyzes the hydrolysis of γ-glutamyl bonds in glutathione and glutamine and the transfer of the released γ-glutamyl group to amino acids or short peptides. γ-GTs from extremophiles, bacteria adapted to live in hostile environments, were selected as model systems to study the molecular underpinnings of their adaptation to extreme conditions and to find out special properties of potential biotechnological interest. Here, we report the cloning, expression and purification of two members of γ-GT family from two different extremophilic species, Thermus thermophilus (TtGT) and Deinococcus radiodurans (DrGT); the first is an aerobic eubacterium, growing at high temperatures (50–82°C), the second is a polyextremophile, as it tolerates radiations, cold, dehydration, vacuum, and acid. TtGT and DrGT were both synthesized as precursor proteins of 59–60 kDa, undergoing an intramolecular auto-cleavage to yield two subunits of 40 and 19–20 kDa, respectively. However, like the γ-GT from Geobacillus thermodenitrificans, but differently from the other characterized bacterial and eukaryotic γ-GTs, the two new extremophilic enzymes displayed γ-glutamyl hydrolase, but not transpeptidase activity in the 37–50°C temperature range, pH 8.0. The comparison of sequences and structural models of these two proteins with experimental-determined structures of other known mesophilic γ-GTs suggests that the extremophilic members of this protein family have found a common strategy to adapt to different hostile environments. Moreover, a phylogenetic analysis suggests that γ-GTs displaying only γ-glutamyl hydrolase activity could represent the progenitors of the bacterial and eukaryotic counterparts.     

Enzymatic synthesis of glutathione using yeast cells in two-stage reaction

In the present study, permeated yeast cells were used as the catalyst to synthesize glutathione. When waste cells of brewer’s yeast were incubated with the three precursor amino acids and glucose for 36 h, 899 mg/L of glutathione were produced. To release the feedback inhibition of γ-glutamylcysteine synthetase caused by glutathione, two-stage reaction was adopted. In the first stage, glycine was omitted from the reaction mixture and only γ-glutamylcysteine was formed. Glycine was then added in the second stage, and 1,569 mg/L of glutathione were produced. The conditions of the two-stage reaction were optimized using Plackett–Burman design and response surface methodology. Under the optimized condition, commercially available baker’s yeast produced 3,440 mg/L of glutathione in 30 h, and most of the produced glutathione was in the medium. The two-stage reaction could effectively reduce the feedback inhibition caused by glutathione, but degradation of glutathione was significant.     

Effect of isoniazid on glutathione biosynthesis and degradation inMycobacterium smegmatis

The mechanism of glutathione (GSH) depletion by isoniazid (INH) was studied inM. smegmatis. INH increased the activity of γ-glutamyl transferase (GGT) whether added before medium inoculation or to actively growing cells. The activity of GGT in cells grown from the beginning in INH-containing medium increased significantly on growth days 2–6. Three-day oldM. smegmatis cells treated with INH exhibited a 30–65 % increase in the activity of GGT. The activities of γ-glutamyl-cysteine synthase (GGCS) and GSH synthase (GS) were lowered by 50 and 56 % respectively on the second day of growth whenM. smegmatis was grown in a medium supplemented with 1.5 mg INH per L. In 3-day oldM. smegmatis, INH significantly inhibited the activities of GSH biosynthetic enzymes. The results demonstrate that the increased activity of GGT and decreased activities of GSH biosynthetic enzymes are responsible for GSH depletion by INH inM. smegmatis.     

THE γ SUBUNITS OF PHYCOERYTHRIN FROM A RED ALGA: POSITION IN PHYCOBILISOMES AND SEQUENCE CHARACTERIZATION

Aglaothamnion neglectum Feldman-Mazoyer has two γ subunits, γ³¹ and γ³³, that are associated with phycoerythrin in the light-harvesting phycobilisomes. We demonstrate that these subunits are spatially separated within the phycobilisome, with the γ³¹ subunit present at the distal end of phycobilisome rods and the γ³³ subunit present on the proximal end. These subunits are thought to link phycoerythrin hexamers together in the rod substructure, serving a role analogous to that of linker polypeptides of cyanobacteria (although unlike the cyanobacterial linker polypeptides they are chromophorylated). The sequencing of tryptic polypeptides of the γ subunits enabled us to prepare oligonucleotides encoding different regions of γ³¹. These oligonucleotides were used as primers to generate a probe for isolating a γ³¹ cDNA clone. Characterization of the cDNA clone predicts a polypeptide of 280 amino acids with a 42 amino acid presequence that is characteristic of a transit peptide, the peptide that targets proteins to chloroplasts of vascular plants. The γ³¹ subunit has 50% similarity to the previously characterized γ³³ subunit but has no identifiable similarity to functionally related polypeptides present in cyanobacterial phycobilisomes or to any other polypeptides in the databases. A repeat of 95 amino acids is present in the red algal γ subunit sequences, suggesting that these proteins were generated by a gene duplication followed by fusion of the duplicate sequences.     

OCCURRENCE OF γ-GLUTAMYLHISTIDINE IN BOVINE BRAIN

γ-Glutamylhistidine was purified from 7.5 kg of bovine brain. ldentification was based on the comparison with the synthetized compound. γ-Glutamyllysine and γ-glutamylarginine were not detected. The present study added another γ-glutamyl amino acid 10 the list of the previously known 10 γ-glutamyl compounds.