Essential role of Helicobacter pylori gamma-glutamyltranspeptidase for the colonization of the gastric mucosa of mice

Constitutive expression of gamma-glutamyltranspeptidase (GGT) activity is common to all Helicobacter pylori strains, and is used as a marker for identifying H. pylori isolates. Helicobacter pylori GGT was purified from sonicated extracts of H. pylori strain 85P by anion exchange chromatography. The N-terminal amino acid sequences of two of the generated endo-proteolysed peptides were determined, allowing the cloning and sequencing of the corresponding gene from a genomic H. pylori library. The H. pylori ggt gene consists of a 1681 basepair (bp) open reading frame encoding a protein with a signal sequence and a calculated molecular mass of 61 kDa. Escherichia coli clones harbouring the H. pylori ggt gene exhibited GGT activity at 37 degrees C, in contrast to E. coli host cells (MC1061, HB101), which were GGT negative at 37 degrees C. GGT activity was found to be constitutively expressed by similar genes in Helicobacter felis, Helicobacter canis, Helicobacter bilis, Helicobacter hepaticus and Helicobacter mustelae. Western immunoblots using rabbit antibodies raised against a His-tagged-GGT recombinant protein demonstrated that H. pylori GGT is synthesized in both H. pylori and E. coli as a pro-GGT that is processed into a large and a small subunit. Deletion of a 700 bp fragment within the GGT-encoding gene of a mouse-adapted H. pylori strain (SS1) resulted in mutants that were GGT negative yet grew normally in vitro. These mutants, however, were unable to colonize the gastric mucosa of mice when orally administered alone or together (co-infection) with the parental strain. These results demonstrate that H. pylori GGT activity has an essential role for the establishment of the infection in the mouse model, demonstrating for the first time a physiological role for a bacterial GGT enzyme.     

Measurement of beta-galactosyltransferase activity in cell extracts with an ELISA-based assay

An enzyme-linked immunosorbent assay (ELISA)-based glycosyltransferase assay has been used to measure UDP-Gal:N-acetylglucosamine beta-1,4-galactosyl-transferase (EC 2.4.1.38) activity in detergent extracts of chinese hamster ovary (CHO) cells. LEC11 cells (a mutant of the CHO cell line, Pro -5), which are known to express a complex array of carbohydrate structures, were used to develop the assay for use with whole cell extracts. A detergent-solubilized preparation of the enzyme from whole cells was used to convert the substrate, lactotriglycosylceramide, to the product, neolactotetraglycosylceramide. The monoclonal antibody, 1B2, which specifically binds to the Gal beta 1-4GlcNAc epitope, was used in an ELISA to identify and quantify the product. The enzyme activity in the preparations was found to be similar to that obtained by conventional radioactive assay methods. The beta-galactosyltransferase found in LEC11 cell detergent extracts exhibited an absolute requirement for the nucleotide sugar and MnCl2. The activity of the enzyme was also strictly dependent on the presence of exogenous glycolipid acceptor. When Triton X-114 was used to solubilize the LEC11 beta-galactosyltransferase, activity was found in both the hydrophilic and the hydrophobic phases, suggesting the presence of two forms of the enzyme. The ELISA-based assay was used to compare beta-1,4-galactosyltransferase activity in detergent extracts of four CHO cell lines: Pro-5, Lec1, LEC11, and LEC12 and in detergent-solubilized microsomes from human leukemia cells. The results from this study demonstrate the utility of the ELISA-based assay for measuring glycosyltransferase activity in detergent-solubilized whole cells and microsome preparations.     

Characterization of the extracellular gamma-glutamyl transpeptidases, GGT1 and GGT2, in Arabidopsis

gamma-Glutamyl transpeptidase (GGT) is the only enzyme known that can cleave the gamma-peptide bond between glutamate and cysteine in glutathione, and is therefore a key step in glutathione degradation. There are three functional GGT genes in Arabidopsis, two of which are considered here. GGT1 and GGT2 are apoplastic, associated with the plasma membrane and/or cell wall. RNA blots and analysis of enzyme activity in knockout mutants suggest that GGT1 is expressed most strongly in leaves but is found throughout the plant. A GGT1::GUS fusion construct showed expression only in vascular tissue, specifically the phloem of the mid-rib and minor veins of leaves, roots and flowers. This localization was confirmed in leaves by laser microdissection. GGT2 expression is limited to embryo, endosperm, outer integument, and a small portion of the funiculus in developing siliques. The ggt2 mutants had no detectable phenotype, while the ggt1 knockouts were smaller and flowered sooner than wild-type. In ggt1 plants, the cotyledons and older leaves yellowed early, and GSSG, the oxidized form of glutathione, accumulated in the apoplastic space. These observations suggest that GGT1 is important in preventing oxidative stress by metabolizing extracellular GSSG, while GGT2 might be important in transporting glutathione into developing seeds.     

The effect of ammonia and pH on brain γ-glutamyl transpeptidase in young rats

Acute hyperammonemia, induced by two consecutive injections of ammonium acetate (550 and 450 mg per kg b.wt.), decreased the activity of γ-glutamyl transpeptidase (GGT) in most brain regions of 18- and 30-day-old rats. This decrease in the brain GGT activity was more pronounced in younger than in older rats. After the addition or NH₄Cl to the incubation medium, the inhibitory action of NH₄⁺ on this enzyme activity was also demonstrated in crude synaptosomal membranes at pH 7.4, but in a range of NH₄⁺ concentrations many-times higher then those found in the plasma or brains of young hyperammonemic rats. Because similar concentrations of NH₄⁺ stimulated the activity of the purified enzyme from rat kidney (mainly at pH 9.0), the inhibition of GGT activity in the young rat brain is probably mediated indirectly and not by a direct interaction of ammonia with the enzyme molecules.     

Presence of gamma glutamyl transferase in Mycobacterium smegmatis

The presence of gamma glutamyl transferase (GGT) has been established in Mycobacterium smegmatis. The 10,000 x g supernatant demonstrated only hydrolase activity and did not exhibit any transpeptidase activity. Most of the transferase activity was recovered in 100,000 x g supernatant demonstrating that GGT is a cytosolic enzyme. Maximum activity of GGT was observed at two days of growth and the activity decreased significantly till the seventh day of growth when mycobacteria was grown as stationary culture. The km for gamma glutamyl-p-nitroanilide was found to be 0.074 mM and Vmax for the reaction approached 11.9 nmol per min per mg protein. L-serine + borate was found to be a competitive inhibitor (Ki 12.05 mM) for GGT activity. The pH optimum for GGT activity was observed between 7.5 to 8.5 and temperature above 35 degrees C rapidly inactivated the enzyme activity. To the best of our knowledge, this is the first report which unequivocally establishes the presence of GGT activity in 10,000 x g supernatant of M. smegmatis.     

Histochemical localization of gamma glutamyl transpeptidase in the paranodal region of peripheral nerve

Gamma glutamyl transpeptidase (GGT), an amino acid transport enzyme, was investigated in normal and degenerated sciatic nerve of rat. The enzyme activity, which is considered to be a marker for cerebrovascular endothelium, was found to be absent in microvessels of normal and degenerated nerves. In the perineurium of normal nerve, GGT activity was faint, while in degenerated nerve, it increased. The most striking finding of this study was the observation of GGT activity at the paranode of each normal myelinated axon. It is interesting that after axotomy (8 weeks), no GGT activity was observed in the Schwann cells of degenerated nerve. Thus, Schwann cell plasmalemma contributed to GGT staining only when this cell was in contact with an axon mature enough to cause it to produce myelin. We conclude that, in peripheral nerve, transmembrane amino acid transport is apparently regional and associated with the paranodal region of myelinated nerve fibers.     

Lipid bilayer stabilization of the Na,K-ATPase reconstituted in DPPC/DPPE liposomes

Different subunit aggregates of the Na,K-ATPase may be formed depending on the method used to solubilize and purify the enzyme. We have studied the thermal unfolding of detergent-solubilized and dipalmitoylphosphatidylcholine/ dipalmitoylphosphatidylethanolamine liposome-reconstituted forms of the Na,K-ATPase by circular dichroism (CD) spectroscopy and p-nitrophenylphosphatase activity. The ellipticity at 222 nm of the solubilized and reconstituted forms showed a sigmoid decrease in the absolute value of the signal of 36 and 31% with T(50%) of 44 and 42 degrees C, respectively. The catalytic activity was reduced in two steps with T(50%) of 32 and 52 degrees C in the detergent-solubilized enzyme and T(50%) of 25 and 53 degrees C in the reconstituted enzyme. The reduction in catalytic activity of the detergent-solubilized enzyme was bi-exponential with t(1/2) of 8.3 and 67.9 min, resulting in the total loss of activity after 120 min. However, under the same conditions, the ATPase activity of the reconstituted enzyme was reduced by approx 35% with a t(1/2) of 145 min. The results suggest that the alpha- and beta-subunits present different thermal stability that may be modulated by the nature of the co-solvent (detergent or lipid) used in the preparations of the Na,K-ATPase. In addition, distinct processes of beta-subunit displacement and alpha-alpha-subunit aggregate formation may also contribute to the changes in both the CD spectra and the enzyme activity. Furthermore, we have demonstrated the protective role of the phospholipid bilayer in the reconstituted enzyme compared with the detergent-solubilized enzyme.     

Secretion of gamma-glutamyltranspeptidase by the pancreas: evidence for a membrane shedding process during exocytosis

Gamma Glutamyltranspeptidase (GGT) is a membrane-bound enzyme involved in glutathione metabolism. It is present in rat exocrine pancreas at a level which is only exceeded by the kidney. It has been previously shown that most of the enzyme activity is located in the apical area of the acinar cell, more precisely at the level of zymogen granules and plasma membrane. The aim of the present study was to examine the secretory behavior of that enzyme. Under resting conditions, in vivo, high levels of GGT were found in pancreatic juice and its level was not related to protein concentration. Under secretin infusion, a relatively constant level of GGT was released, and again, there was no correlation between enzyme activity and protein concentration. Following a bolus injection of caerulein, an analog of cholecystokinin, marked and concomitant rises in protein and GGT levels were observed. Ultracentrifugation, as well as gel filtration on Sepharose 4B, demonstrated that the enzyme was not released in a soluble form. This observation is in agreement with in vitro determinations on isolated zymogen granules showing that GGT is totally associated with the ZG membrane and undetect-able in the content of these organelles. The present data show that 1 degree GGT is released from the rat pancreas acinar cells in a particulate form; 2 degree GGT release is elicited by hormonal stimulation coinciding with the exocytotic release of secretory proteins. Our observations lead us to propose that in rat pancreas, ZG membrane fragments are released along with secretory proteins during exocytosis.     

Gamma-glutamyl transpeptidase from two plant growth promoting rhizosphere fluorescent pseudomonads

Glutathione is the most abundant non-protein thiol compound present in many cells. Because this molecule is involved in many physiological processes, each cell maintains a critical level of glutathione. Gamma-glutamyl transpeptidase (GGT, E.C.2.3.2.2) is the key enzyme involved in the glutathione cycle. In the present study, GGT was isolated from two plant growth promoting rhizosphere isolates, Pseudomonas protegens strain Pf-5 and Pseudomonas fluorescens strain PfT-1. GGT in these strains is located in the periplasm and possessed good hydrolytic activity at pH 8.0. Strains Pf-5 and PfT-1 showed maximum enzyme activity when grown at 30–35 °C. The ggt gene from both the strains was cloned in pGEM-T cloning vector and sequenced. Subsequently, GGT expressed in Escherichia coli BL21(DE3) using the pET-28a(+) expression vector was purified and characterized. The enzymes are active in a wide range of pH and some divalent cations significantly enhanced the hydrolytic activity. These enzymes showed higher thermal stability as compared to those of other mesophilic strains, as they retained ~50 % of activity at 50 °C even after 12 h of incubation. The enzymes could also tolerate up to 3.0 M NaCl.     

Gamma-glutamyl transpeptidase activity in isolated cells and in various regions of the mouse brain during early development and aging

Differences in the distribution of gamma-glutamyl transpeptidase (GGT) activity during early and late postnatal development of the mouse brain were studied at the cellular and regional level. From the 7th to the 25th day of brain development, GGT activity rose evenly in the neuronal perikarya, glial cells and in the neuropil. At 25 days and 5 weeks of age, a 1.5-fold enrichment was observed in nerve cell bodies and in neuroglia as compared with the neuropil and the original cell suspension. During the same period, an abrupt increase in GGT activity was observed in brain capillaries, and between the 10th day and the 5th week, this was 1.5-5 times higher than the enzyme activity in cellular elements and in the neuropil. This increase in enzyme activity in brain capillaries continued up to the age of 12 months, when it was succeeded by a decrease in GGT activity to the level found at 3 months. GGT activity in the choroid plexi rose significantly (more than double) between the age of 5 weeks and 18 months and was a whole order higher than GGT activity in 10 other regions of the mouse brain, whose development displayed no pronounced changes in the activity of the enzyme. A single dose of dexamethasone, or adrenalectomy, were used to demonstrate any participation by steroid hormones in the regulation of GGT activity in the brain tissue. Of the two techniques, only adrenalectomy led to a statistically significant decrease in activity in brain and liver tissue, while the apparent increase in GGT activity after a single dose of dexamethasone was not statistically significant.     

pH-dependent interconversion of two forms of tyrosinase in human skin.

1. We have shown that the characteristic lag in cresolase activity of human skin tyrosinase at inhibitory concentration of tyrosine was absent at all pH values studied, i.e. pH 5.2, 5.7, 6.2 and 6.8, if the enzyme solubilized at low pH was used as the source of enzyme, but the same enzyme when dialysed against buffers of various pH values showed linear activity only at pH 5.2 and was not inhibited by excess tyrosine, whereas at higher pH values it exhibited a lag and inhibition by excess tyrosine. 2. However, the enzyme solubilized in buffer/detergent, pH 6.8, when dialysed against buffer of the same pH showed linear activity at pH 5.2 and non-linear activity at pH 6.8. 3. The water/detergent-solubilized enzyme from human skin melanosomes showed linear activity even at inhibitory concentrations of tyrosine at pH 5.2 and 6.8 up to 2 h, but acceleration of rate was observed after 2 h for the enzyme measured at pH 6.8. 4. After dialysis of the water/detergent-solubilized enzyme against double-glass-distilled water, it still exhibits linear activity at inhibitory concentration of tyrosines at pH 6.8 for the first 2 h, but the same enzyme when dialysed against 0.02 M-sodium phosphate buffer, pH 6.8, exhibits negligible activity up to 1/2 h, in contrast with considerable activity before dialysis during the same interval of time, but without any loss of activity at later intervals of incubation time. 5. On the basis of these results, it is concluded that the enzyme exists in at least two interconvertible forms, one without lag and inhibition by excess tyrosine and the other with lag and inhibition by excess tyrosine. These two forms are interconvertible only by gradual change in pH over a period of hours.     

Effects of short-term and prolonged aerogenic hypoxia on gamma-glutamyl transpeptidase activity in the brain,liver, and biological fluids of young rats

Posthypoxic fluctuations in the levels of two excitatory amino acids, glutamate and aspartate, may be related to changes in mechanisms(s) which are responsible for their reuptake. As gamma-glutamyl transpeptidase (GGT) plays a role in mediating the uptake of glutamate and aspartate into various compartments of the brain, we studied changes in the activity of this enzyme in main regions of the brain in young and adult rats. We found a posthypoxic increase in bound GGT activity in some brain regions of 18-day-old animals after acute exposure, but no changes were observed after prolonged altitude hypoxia, with the exception of a decrease in cortical GGT activity. In contrast, acute hypoxia decreased GGT activity in the cortical capillaries to 59%, but prolonged hypoxic exposure was ineffective. However, the activity of soluble GGT in the cerebrospinal fluid of both groups of rats was several-times elevated in comparison with controls. At the same time, bound GGT activity was increased in the liver after acute or prolonged altitude hypoxia. The soluble GGT activity in plasma was only increased after prolonged exposure. Ninety days after prolonged hypoxic exposure the bound GGT activity was reduced in all brain regions to about 60–70% of controls (significantly higher in females than in males) as long-term developmental sequel from early postnatal hypoxia.     

Nitric Oxide Exposure of CC531 Rat Colon Carcinoma Cells Induces γ-glutamyltransferase which May Counteract Glutathione Depletion and Cell Death

&#110 -Glutamyltransferase (GGT) has a central role in glutathione homeostasis by initiating the breakdown of extracellular GSH. We investigated in the present study whether nitric oxide exposure of CC531 rat colon carcinoma cells modulates GGT and how the activity of the enzyme affects the level of intracellular GSH. The data show that GGT activity was induced in a dose-related manner by two NO-donors (spermineNONOate and nitrosoglutathione) and that antioxidants partly inhibited the induction. SpermineNONOate lowered intracellular GSH and induced apoptosis. Cultivating the cells in cystine-depleted medium also resulted in a 50% lowering of GSH, but this was avoided when GSH was added to the medium. This effect was mediated by the activity of GGT and shown after inhibiting GGT activity with acivicin and cyst(e)ine transporters with alanine and homocysteic acid. This shows that the cells benefit from GGT in maintaining the intracellular GSH level. Cells with induced GGT activity obtained after NO incubation showed a higher uptake rate of cysteine (2-fold), measured by incubating the cells with 35 S-radiolabeled GSH. The enzyme was also induced by interferon- &#110 and tumor necrosis factor- &#102, but this induction was not connected to activation of the endogenous nitric oxide synthase, as the addition of aminoguanidine, a NO-synthase inhibitor, did not affect the induction. The present study shows that the activity of GGT is upregulated by NO-donors and that the colon carcinoma cells, when cultivated in cystine-depleted medium, benefit from the enzyme in maintaining the intracellular level of GSH. Thus, the enzyme will add to the protective measures of the tumor cells during nitrosative stress.     

Nitric oxide exposure of CC531 rat colon carcinoma cells induces gamma-glutamyltransferase which may counteract glutathione depletion and cell death

Gamma-glutamyltransferase (GGT) has a central role in glutathione homeostasis by initiating the breakdown of extracellular GSH. We investigated in the present study whether nitric oxide exposure of CC531 rat colon carcinoma cells modulates GGT and how the activity of the enzyme affects the level of intracellular GSH. The data show that GGT activity was induced in a dose-related manner by two NO-donors (spermineNONOate and nitrosoglutathione) and that antioxidants partly inhibited the induction. SpermineNONOate lowered intracellular GSH and induced apoptosis. Cultivating the cells in cystine-depleted medium also resulted in a 50% lowering of GSH, but this was avoided when GSH was added to the medium. This effect was mediated by the activity of GGT and shown after inhibiting GGT activity with acivicin and cyst(e)ine transporters with alanine and homocysteic acid. This shows that the cells benefit from GGT in maintaining the intracellular GSH level. Cells with induced GGT activity obtained after NO incubation showed a higher uptake rate of cysteine (2-fold), measured by incubating the cells with 5S-radiolabeled GSH. The enzyme was also induced by interferon-gamma and tumor necrosis factor-alpha, but this induction was not connected to activation of the endogenous nitric oxide synthase, as the addition of aminoguanidine, a NO-synthase inhibitor, did not affect the induction. The present study shows that the activity of GGT is upregulated by NO-donors and that the colon carcinoma cells, when cultivated in cystine-depleted medium, benefit from the enzyme in maintaining the intracellular level of GSH. Thus, the enzyme will add to the protective measures of the tumor cells during nitrosative stress.     

Cytochemical evaluation of gamma-glutamyl-transpeptidase activity in cervical smears

The cervicovaginal smears of 43 patients attending an outpatient service for early cancer detection were cytochemically studied for the presence of gamma-glutamyl-transpeptidase (GGT) in epithelial cells. This was done in order to evaluate such an enzyme phenotype as a marker for cancer development. The results showed that 70% of the 38 patients with a cytologic diagnosis of "inflammatory" or preneoplastic/neoplastic conditions had GGT-positive cells in their smears. None of the five cytologically normal cases showed any epithelial cells with GGT activity. Although most of the GGT-positive cells were metaplastic, some morphologically normal, dysplastic or neoplastic cells also expressed the enzyme. The data suggest that cytochemically detectable transpeptidase activity appears whenever alterations of the normal epithelial microenvironment occurs, but is not necessarily linked to the carcinogenic process. Therefore, cytochemically GGT-positive cells should not be used as an indicator of neoplastic transformation of the cervical epithelium.     

Contribution by polymorphonucleate granulocytes to elevated gamma-glutamyltransferase in cystic fibrosis sputum

Background

Cystic fibrosis (CF) is an autosomal recessive disorder characterized by a chronic neutrophilic airways inflammation, increasing levels of oxidative stress and reduced levels of antioxidants such as glutathione (GSH). Gamma-glutamyltransferase (GGT), an enzyme induced by oxidative stress and involved in the catabolism of GSH and its derivatives, is increased in the airways of CF patients with inflammation, but the possible implications of its increase have not yet been investigated in detail.

Principal Findings

The present study was aimed to evaluate the origin and the biochemical characteristics of the GGT detectable in CF sputum. We found GGT activity both in neutrophils and in the fluid, the latter significantly correlating with myeloperoxidase expression. In neutrophils, GGT was associated with intracellular granules. In the fluid, gel-filtration chromatography showed the presence of two distinct GGT fractions, the first corresponding to the human plasma b-GGT fraction, the other to the free enzyme. The same fractions were also observed in the supernatant of ionomycin and fMLP-activated neutrophils. Western blot analysis confirmed the presence of a single band of GGT immunoreactive peptide in the CF sputum samples and in isolated neutrophils.

Conclusions

In conclusion, our data indicate that neutrophils are able to transport and release GGT, thus increasing GGT activity in CF sputum. The prompt release of GGT may have consequences on all GGT substrates, including major inflammatory mediators such as S-nitrosoglutathione and leukotrienes, and could participate in early modulation of inflammatory response.     

Light form of Morris hepatoma gamma-glutamyltransferase

Using solubilization with bromelain, the light form of gamma-glutamyltransferase was purified from Morris hepatoma 5123D. Some properties of this enzyme were compared to those of the light form rat kidney GGT. Anthglutin and its isomer inhibit competitively the former enzyme but non-competitively the latter. On zymograms of rat control sera, five GGT fractions were noted, but in sera of rats with hepatoma 5123D also the light form and the increase of GGT activity ain region of fraction II were observed. Only these two enzyme fractions react with antibody anti heavy form of Morris hepatoma GGT.     

Phorbol ester regulation of the human gamma-glutamyltransferase gene promoter

In the present study the molecular mechanisms underlying tetradecanoylphorbol-13-acetate (TPA) mediated regulation of the human gamma-glutamyltransferase (GGT) gene were examined. TPA challenge of HeLa cells resulted in an increase of GGT mRNA and enzyme activity. Deletion analysis of the promoter revealed that the -348 to +60 fragment was able to mediate TPA induced expression. Gel shift and supershift analyses showed that TPA treatment increased nuclear protein binding to a putative AP-1 site (-225 to -214) and that c-Jun was part of the complex. This AP-1 element, when cloned either in its native arrangement or as tandem repeat 5' of the minimal thymidine kinase promoter, mediated a significant increase of luciferase activity after TPA treatment of transfected HeLa cells, while its mutated counterpart abolished the induction. The same AP-1 element was able to mediate TPA induced expression in HepG2 cells. Collectively these results indicate that like other GSH metabolising enzymes, GGT too is a target for AP-1 mediated regulation.     

Tissue difference in gamma-glutamyl transpeptidase attributed to sialic acid content.

Gamma-glutamyl transpeptidase (GGT) was extracted from the microsomal fraction of various bovine tissues and partially purified. Purified enzymes demonstrated different mobilities toward the anode in polyacrylamide gel electrophoresis in 0.5% Emulphogene BC720, pH 7.5. The ciliary-body GGT migrated fastest, while the brain enzyme was electrophoresed most slowly. The apparent Km values (Km′) of GGT for L-gamma-glutamyl-p-nitroanilide were 1.4–2.0 mM when assayed with glycylglycine as the gamma-glutamyl acceptor. After neuraminidase treatment, electrophoretic mobility was decreased considerably for all enzyme preparations, compatibly with the removal of negatively charged sialic-acid residues. The Km′ values of the enzyme were not affected by the hydrolytic treatment. Electrophoresis of digested enzymes showed essentially identical mobilities. From these results we conclude that tissue differences in GGT are attributable to the varying extent of sialylation of enzyme.