Hydrolysis ofγ-glutamyl linkages byFusobacterium nucleatum

The cell extracts of two human oral strains (FN2 and FN3) ofFusobacterium nucleatum displayed exceptionally high-glutamylpeptidase activity as determined withN--l-glutamyl-2-naphthylamine as substrate. This activity was so dominant that the hydrolysis of otherN-aminoacyl-2-naphthylamines progressed at a rate <10% of the former. Two major enzymes (I and II) were partially purified from FN2. I had a molecular weight of 115,000 and did not hydrolyze-glutamylcysteinylglycine (glutathione). II had a molecular weight of 70,000 and rapidly liberated only glutamic acid from glutathione. Strain FN3 contained several enzymes hydrolyzing-glu-2NA. Direct anion exchange chromatography of FN3 cell extracts separated one enzyme that liberated both glutamic acid and glycine from glutathione, one that was inactive against glutathione (but hydrolyzed-glu-2NA), and one that liberated only glutamic acid. Although-glu-2NA was a good synthetic substrate, glutathione was hydrolyzed at least 500 times faster by an enzyme present in both strains. These results indicate that the presence of-glutamylpeptidase activity is very characteristic of theseF. nucleatum strains.     

Detection of ε(γ-glutamyl) lysine

Summary Detection of (-glutamyl) lysine crosslinks is not only necessary for establishing the importance of the dipeptide as a post-translational modification of proteins, but provides information as to the importance of the transglutaminase enzyme in a biological system. The crosslink may be detected using both indirect and direct methodology. Indirect methods for its detection include measurement of masked lysines within a protein, detection of polymer formation by gel-electrophoresis and the inhibition of crosslinking by the incorporation of small molecular weight amines into the substrate protein. Direct methods for the detection of (-glutamyl) lysine require the actual isolation of the dipeptide following its release from the sample protein by exhaustive proteolytic digestion. Separation of the dipeptide from other components of the digest may be achieved by either ion-exchange chromatography or gel filtration and its qualitative identification achieved by techniques such as paper-electrophoresis or thin layer chromatography. Quantitative estimation of (-glutamyl) lysine normally involves its further separation by ion-exchange chromatography and its post-column detection following derivatisation with ninhydrin. More recent techniques include pre-column derivatisation of the dipeptide with fluorogenic reagents such as -pthalaldehyde and separation by reverse phase HPLC. With the recent advances in liquid chromatography resulting in the improved resolution of amino acids, increased sensitivity, rapid analysis times, and small sample sizes, it appears likely that direct quantitation of (-glutamyl) lysine will be the preferred method for the future.     

Bioluminescence probe for γ-glutamyl transpeptidase detection in vivo

To detect γ-Glutamyl Transpeptidase (GGT) activity in vitro and in vivo, a bioluminescence probe with high sensitivity and specificity was well designed and synthesized. This probe can be recognized by GGT and release strong bioluminescence with its further reaction with luciferase. The performance of this probe was demonstrated in vitro and in cells. Finally, we applied the probe for detection of GGT activity in xenograft model.     

Serum γ-glutamyl Transferase Levels,Insulin Resistance and Liver Fibrosis in Patients with Chronic Liver Diseases

Background and Aims

Serum levels of γ-glutamyl-transpeptidase(γ-GT) were associated with liver disease severity and metabolic alterations, which in turn are able to affect hepatic damage. In patients with nonalcoholic fatty liver disease (NAFLD), genotype 1 chronic hepatitis C (G1CHC) and chronic hepatitis B (CHB), we assessed the link between liver fibrosis and γ-GT serum levels, and we evaluated if normal or high γ-GT serum levels affect the association between insulin resistance (IR) and severity of liver fibrosis.

Methods

843 consecutive patients with chronic liver disease (CLD)(193 NAFLD, 481 G1CHC, 169 CHB) were evaluated by liver biopsy (Kleiner and Scheuer scores) and clinical and metabolic measurements. IR was diagnosed if HOMA>3. A serum γ-GT concentration of >36 IU/L in females and >61 IU/L in males was considered the threshold value for identifying high levels of γ-GT.

Results

By multivariate logistic regression analysis, abnormal γ-GT serum levels were independently linked to severe liver fibrosis in patients with NAFLD (OR2.711,CI1.120–6.564,p = 0.02), G1CHC (OR3.461,CI2.138–5.603,p<0.001) and CHB (OR2.778,CI1.042–7.414,p = 0.04), together with IR and liver necroinflammation, and with a negative predictive value>80%. Interestingly, among patients with high or normal γ-GT values, even if IR prevalence was significantly higher in patients with severe fibrosis compared to those without, IR remained significantly associated with severe fibrosis in patients with abnormal γ-GT values only (OR4.150,CI1.079–15.970,p = 0.03 for NAFLD; OR2.250,CI1.211–4.181,p = 0.01 for G1CHC; OR3.096,CI2.050–34.220,p = 0.01 for CHB).

Conclusions

In patients with CLD, IR is independently linked to liver fibrosis only in patients with abnormal γ-GT values, without differences according to liver disease etiology, and suggesting a role of γ-GT as a marker of metabolic-induced liver damage. These data could be useful for the clinical and pharmacologic management of patients with CLD.     

Subcellular and regional localization ofγ-glutamyl transpeptidase in sheep brain

Studies of the subcellular distribution of-glutamyl transpeptidase from sheep brain by discontinuous sucrose density gradient centrifugation showed that 40% of the transpeptidase activity associated with the mitochondrial-synaptosomal fraction was localized with the synaptosomal-enriched fraction. The microsomal fraction was found to have the highest specific activity when-glutamylp-nitroanalide was used as substrate. This activity, however, represented only 5% of the total-glutamyl transpeptidase activity. Approximately 90% of the total enzyme activity was apparently associated with the fraction containing cell debris and membrane fragments.The 160,000g supernatant fluid (soluble supernatant fraction) represented the least total activity, with only 1.2% recovery; however, this fraction contained two apparent forms of the enzyme. One form had a highK _mand the other a lowK _m for the substrate,-glutamylp-nitroanilide.It was observed that the enzyme-glutamyl transpeptidase was not evenly distributed in all areas of brain when the homogenate was used as the enzyme source. The brain region with the highest enzyme activity was the thalamus, which was able to form 1.10 molp-nitroanaline/min/g wet brain tissue. The cortex was found to have the lowest activity. The 40,000g supernatant fluid from each region, however, exhibited only slight distribution differences.     

Regional distribution of membrane-bound γ-glutamyl transpeptidase activity in mouse brain

Activity of membrane-bound -glutamyl transpeptidase (-GTP) was examined in various regions of mouse brain, in capillaries of the cerebral cortex and in telencephalic choroid plexuses. The level of activity in the capillaries was double and that of the choroid plexus nine times that of the -GTP activity found in the brain, septum, hippocampus, hypothalamus, thalamus, cerebellum, frontal cortex, pons, medulla oblongata, and amygdala. Histochemically the -GTP activity was demonstrated in the surface membranes of choroidal cells and in the endothelium of small capillaries.The activities of -GTP of cerebral cortex, choroid plexus, and capillaries from rabbit were 5–17 times greater than those from corresponding areas of mouse brain. While 30 mM methionine stimulated (in vitro) the enzyme from mouse brain, no such effect was observed with the enzyme activity from rabbit brain. The -GTP activity from the capillaries of cerebral cortex of both mouse and rabbit was not effected by the presence of methionine.These findings suggest existence of differences in the specificity of -GTP activity in these two species.     

Proteomic identification of Drosophila melanogaster male accessory gland proteins, including a pro-cathepsin and a soluble γ-glutamyl transpeptidase

Background  

In Drosophila melanogaster, the male seminal fluid contains proteins that are important for reproductive success. Many of these proteins are synthesised by the male accessory glands and are secreted into the accessory gland lumen, where they are stored until required. Previous studies on the identification of Drosophila accessory gland products have largely focused on characterisation of male-specific accessory gland cDNAs from D. melanogaster and, more recently, Drosophila simulans. In the present study, we have used a proteomics approach without any sex bias to identify proteins in D. melanogaster accessory gland secretions.     

Free amino acids and γ-glutamyl peptides in seeds of Fagus silvatica

The contents of amino acids and peptides have been investigated in seeds of Fagus silvatica L. (beechnuts). In addition to the common amino acids, the following compounds have been isolated and identified: 4-hydroxyproline (probably the cis-l-isomer), N⁵-acetylornithine, 3-(2-furoyl)-l-alanine, methionine sulfoxide (probably an artefact), pipecolic acid (probably partially racemized d-isomer), l-willardiine (with a small amount of the d-isomer), N-(3-amino-3-carboxypropyl)azetidine-2-carboxylic acid, N-[N-(3-amino-3-carboxypropyl)-3-amino-3-carboxypropyl]azetidine-2-carboxylic acid, 2(S),5(S),6(S)-5-hydroxy-6-methylpipecolic acid, 2(S),5(R),6(S)-5-hydroxy-6-methylpipecolic acid, γ-glutamylalanine, γ-glutamylglutamic acid, γ-glutamylisoleucine, γ-glutamylleucine, γ-glutamylmethionine sulfoxide (probably an artefact), γ-glutamylphenylalanine, γ-glutamyltyrosine, γ-glutamylvaline, glutathione, γ-glutamylwillardiine, and γ-glutamylphenylalanylwillardiine. γ-Glutamylphenylalanine and willardiine are the dominating components of the amino acid fraction.The isolations were performed by use of ion exchange chromatography, taking advantage of the different pK-values of the amino acids, mainly on acid resins in the 3-chloropyridinium form with aq. 3-chloropyridine as eluant and on basic resins in the acetate form with aqueous acetic acid as eluant. These methods in combination with preparative paper chromatography have permitted the isolation and identification of compounds present in amounts as low as 1/6000 of the dominant ninhydrin-reactive component. The implications of the occurrence of this large variety of compounds in the Fagaceae are briefly discussed.     

Probing the donor and acceptor substrate specificity of the γ-glutamyl transpeptidase

γ-Glutamyl transpeptidase (GGT) is a two-substrate enzyme that plays a central role in glutathione metabolism and is a potential target for drug design. GGT catalyzes the cleavage of γ-glutamyl donor substrates and the transfer of the γ-glutamyl moiety to an amine of an acceptor substrate or water. Although structures of bacterial GGT have revealed details of the protein-ligand interactions at the donor site, the acceptor substrate site is relatively undefined. The recent identification of a species-specific acceptor site inhibitor, OU749, suggests that these inhibitors may be less toxic than glutamine analogues. Here we investigated the donor and acceptor substrate preferences of Bacillus anthracis GGT (CapD) and applied computational approaches in combination with kinetics to probe the structural basis of the enzyme's substrate and inhibitor binding specificities and compare them with human GGT. Site-directed mutagenesis studies showed that the R432A and R520S variants exhibited 6- and 95-fold decreases in hydrolase activity, respectively, and that their activity was not stimulated by the addition of the l-Cys acceptor substrate, suggesting an additional role in acceptor binding and/or catalysis of transpeptidation. Rat GGT (and presumably HuGGT) has strict stereospecificity for L-amino acid acceptor substrates, while CapD can utilize both L- and D-acceptor substrates comparably. Modeling and kinetic analysis suggest that R520 and R432 allow two alternate acceptor substrate binding modes for L- and D-acceptors. R432 is conserved in Francisella tularensis, Yersinia pestis, Burkholderia mallei, Helicobacter pylori and Escherichia coli, but not in human GGT. Docking and MD simulations point toward key residues that contribute to inhibitor and acceptor substrate binding, providing a guide to designing novel and specific GGT inhibitors.     

Culture of mouse brain capillary endothelial cell lines that express factor VIII, γ-glutamyl transpeptidase,and form junctional complexes in vitro

Summary The isolation and culture of cell lines from mouse brain capillary endothelium (MBE) is described. Cells migrating from collagenase-treated capillary fragments proliferated rapidly in the 1st wk of culture forming large epithelioid cobblestonelike colonies. The cells showed only marginal proliferation after 2 to 3 wk in culture, until peripheral cells migrated away from the colony which exhibited a marked degree of proliferation. These cells were trypsinized and subcultured to confluence. The cells can be maintained for well over 40 passages and seem to retain their endothelial morphology. The endothelial origin of these cells was demonstrated by positive immunoperoxidase reactivity with Factor VIII-related antigen, specific binding ofBandeiraea simplicifolia lectin and γ-glutamyl transpeptidase activity. Electron microscopic examination of the MBE cells showed junctional complexes including intermediate junctions, but no tight junctions. The overall ultrastructure indicates that a degree of dedifferentiation has occurred, the cells ultrastructurally resembling immature endothelium. An earlier investigation of cultured mouse brain endothelial cells reported a cell line that had lost many functional and structural characteristics. Our study demonstrates, as the previous one, that a certain degree of dedifferentiation needs to occur if MBE cells are to be maintained for long-term culture. However, the degree of dedifferentiation seems to be variable, depending in part on the culture conditions employed. This work was supported in part by grants from the National Health and Medical Research Council of Australia and the Telethon and Royal Perth Hospital Research Foundations.     

Structural analysis and taste evaluation of γ-glutamyl peptides comprising sulfur-containing amino acids

The structures, flavor-modifying effects, and CaSR activities of γ-glutamyl peptides comprising sulfur-containing amino acids were investigated. The chemical structures, including the linkage mode of the N-terminal glutamic acid, of γ-L-glutamyl-S-(2-propenyl)-L-cysteine (γ-L-glutamyl-S-allyl-L-cysteine) and its sulfoxide isolated from garlic were established by comparing their NMR spectra with those of authentic peptides prepared using chemical methods. Mass spectrometric analysis also enabled determination of the linkage modes in the glutamyl dipeptides by their characteristic fragmentation. In sensory evaluation, these peptides exhibited flavor-modifying effects (continuity) in umami solutions less pronounced but similar to that of glutathione. Furthermore, the peptides exhibited intrinsic flavor due to the sulfur-containing structure, which may be partially responsible for their flavor-modifying effects. In CaSR assays, γ-L-glutamyl-S-methyl-L-cysteinylglycine was most active, which indicates that the presence of a medium-sized aliphatic substituent at the second amino acid residue in γ-glutamyl peptides enhances CaSR activity.     

Isolation and purification of multiple forms of γ-glutamyl transpeptidase from rat brain

Four different forms of the enzyme -glutamyl transpeptidase were isolated from rat brain by chromatography on concanavalin A. An approximate 1500-fold purification was achieved. The four forms were characterized with respect to molecular weight,K _m for -glutamyl-p-nitroanilide, mobility on polyacrylamide gels, and inhibitory effects of borate-serine. The multiple forms of the enzyme were found to have molecular weights ranging from 74,000 to 234,000 andK _ms of 0.07 to 8.6 mM. It was determined that in brain, the major portion of the enzyme activity is associated with plasma membrane fragments and endoplasmic reticulum.     

Production of theanine and other γ-glutamyl derivatives by Camellia sinensis cultured cells

The maximum theanine production by Camellia sinensis cultured cells was achieved by culturing in the modified MS medium containing 2 mg/l indole-3-butyric acid, 0.1 mg/l kinetin, 0 mM NH₄NO₃ and 39.6 mM KNO₃ with 40 mM ethylamine hydrochloride or 20 mM ethylamine hydrochloride and 10 mM L-glutamic acid. Other primary amines, such as methylamine, n-butylamine, 2-hydroxyethylamine, 2cyanoethylamine, aniline, benzylamine and phenylethylamine, were also biotransformed to N⁵-alkyl-L-glutamine derivatives by C. sinensis cultured cells.Abbreviations IBA indole-3-butyric acid - K kinetin - MS medium Murashige and Skoog's basal medium (1962) - NMR nuclear magnetic resonance Part 70 in the series Studies on Plant Tissue Culture. For Part 69 see Furuya et al. (1990).     

Effect of γ-glutamyl cycle inhibitors on brain amino acid transport and utilization

Two inhibitors of the -glutamyl cycle, methionine sulfoximine (MSO) and 2-imidazolidone-4-carboxylic acid (ICA) were administered to C57BL/6J mice. Both agents resulted in a reduced rate of transport of tyrosine from blood to brain and a decreased rate of incorporation of tyrosine from plasma into brain protein. MSO administration also diminished the concentrations of brain tyrosine, dopamine, and norepinephrine. MSO decreased the transport rate of valine by brain as well as the rate of its incorporation into protein when expressed in relation to the plasma specific activity. The results demonstrate a significant role for the -glutamyl cycle in the transport of large neutral amino acids from blood to brain.Presented in part in the April 1977 meeting of the American Academy of Neurology, Atlanta, Georgia.     

Characterization of γ-glutamyl transpeptidase activity of cultured endothelial cells from porcine brain capillaries

Summary Endothelial cells were isolated with high viability (>93%) from porcine brain capillaries by Percoll gradient centrifugation after purely enzymatic digestion. Primary cultures were grown to confluent cell monolayers and quantitated for the activity of -glutamyl transpeptidase. The -glutamyl transpeptidase activity starts from a high enzymatic level, decreases with time in culture to about 15% of the initial value, and remains constant at this level after day 10 in culture. The activity progression depends on surface conditions. In the presence of collagen, an exponential decrease starts immediately after seeding, with a time constant of 70±10h. In the absence of collagen, -glutamyl transpeptidase activity first decreases on day 1 after plating, recovers to the initial value on day 2 and 3 and afterwards declines exponentially to a low and constant activity level. Ethanol added to the cell culture at a time when low constant activity is reached, reactivates the -glutamyl transpeptidase to 30% of the initial value.     

Glutathione and γ-glutamyl transpeptidase are differentially distributed in the olfactory mucosa of rats

Summary Components of the -glutamyl cycle, including thiols, glutathione (GSH) and -glutamyl transpeptidase (-GT), were localized in the nasal mucosae of rats using histochemical and immunohistochemical methods. In olfactory mucosa, thiols were widely distributed, with intense staining in the mucociliary complex (MC), basal cells, acinar cells of Bowman's glands (BG), and olfactory nerve bundles, and with moderate staining in olfactory receptor neurons (ORNs). GSH was localized in MC, BG acinar cells, nerve bundles and, to a lesser extent, in ORNs. -GT immunoreactivity was restricted to the MC and to basolateral and apical membranes of BG acinar and duct cells. The basolateral membrane of BG acinar cells, located in close association with blood vessels and connective tissue, showed granule-like immunoreactivity. Inrespiratory mucosa, all three compounds were localized in the MC and acinar cells of respiratory glands (RG). In the MC, -GT immunoreactivity was associated primarily with brush borders of ciliated cells. Granular immunoreactivity was also apparent in the supranuclear region of RG acinar cells. These results demonstrate that components of the -glutamyl cycle are localized in olfactory and respiratory glands, and that they are secreted into the mucus, where they may mediate perireceptor events such as detoxification and/or solubilization of air-borne xenobiotics, toxicants and odorants.     

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.     

Divergent effects of compounds on the hydrolysis and transpeptidation reactions of γ-glutamyl transpeptidase

A novel class of inhibitors of the enzyme γ-glutamyl transpeptidase (GGT) were evaluated. The analog OU749 was shown previously to be an uncompetitive inhibitor of the GGT transpeptidation reaction. The data in this study show that it is an equally potent uncompetitive inhibitor of the hydrolysis reaction, the primary reaction catalyzed by GGT in vivo. A series of structural analogs of OU749 were evaluated. For many of the analogs, the potency of the inhibition differed between the hydrolysis and transpeptidation reactions, providing insight into the malleability of the active site of the enzyme. Analogs with electron withdrawing groups on the benzosulfonamide ring, accelerated the hydrolysis reaction, but inhibited the transpeptidation reaction by competing with a dipeptide acceptor. Several of the OU749 analogs inhibited the transpeptidation reaction by slow onset kinetics, similar to acivicin. Further development of inhibitors of the GGT hydrolysis reaction is necessary to provide new therapeutic compounds.     

Functional analysis of the C-terminal region of γ-glutamyl kinase of Saccharomyces cerevisiae

γ-Glutamyl kinase (GK) is the rate-limiting enzyme in proline synthesis in microorganisms. Most microbial GKs contain an N-terminal kinase domain and a C-terminal pseudouridine synthase and archaeosine transglycosylase (PUA) domain. In contrast, higher eukaryotes possess a bifunctional Δ(1)-pyrroline-5-carboxylate synthetase, which consists of a PUA-free GK domain and a γ-glutamyl phosphate reductase (GPR) domain. Here, to examine the role of the C-terminal region, including the PUA domain of Saccharomyces cerevisiae GK, we constructed a variety of truncated yeast GK and GK/GPR fusion proteins from which the C-terminal region was deleted. A complementation test in Escherichia coli and S. cerevisiae and enzymatic analysis of recombinant proteins revealed that a 67-residue linker sequence between a 255-residue kinase domain and a 106-residue PUA domain is essential for GK activity. It also appeared that 67 or more residues of the C-terminal region, not the PUA domain itself, are required for the full display of GK activity. Further, the GK/GPR fusion protein was functional in E. coli, but decreased stability and Mg-binding ability as compared to wild-type GK. These results suggest that the C-terminal region of S. cerevisiae GK is involved in the folding and/or the stability of the kinase domain.