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Journal of Dairy Science Feb 2004The membrane spanning enzyme gamma-glutamyl transpeptidase (gamma-GT; EC 2.3.2.2) catalyses the breakdown of the tripeptide glutathione and uses free amino acids (AA) to...
The membrane spanning enzyme gamma-glutamyl transpeptidase (gamma-GT; EC 2.3.2.2) catalyses the breakdown of the tripeptide glutathione and uses free amino acids (AA) to form gamma-glutamyl transpeptidase (GT) AA that become transported into cells and converted back into free AA. gamma-Glutamyl transpeptidase activity has been shown to be important for mammary AA uptake in rodent systems, and while gamma-GT activity is high in lactating bovine mammary tissue, the role of this enzyme in milk protein synthesis of the ruminant has not been defined. The present study shows that gamma-GT activity in the ovine mammary gland, like that of rodents, increases during pregnancy and peaks early in lactation. Acivicin, a well-known inhibitor of gamma-GT, decreased gamma-GT activity in acini isolated from the ovine mammary gland and did not have secondary toxicity effects on cell viability or the uptake of radiolabeled amino-isobutyric acid. Isolated ovine acini were incubated in the presence of radiolabeled leucine, and incorporation of label into secreted protein increased during incubation. Incubation of acini with acivicin decreased milk protein secretion by 75%, indicating that gamma-GT plays an important role in milk protein production in the ruminant. Acivicin did not inhibit secretion of specific caseins but caused a global decrease in individual proteins suggesting that gamma-GT may be responsible for providing a complement of AA for milk protein synthesis.
Topics: Animals; Enzyme Inhibitors; Female; Isoxazoles; Lactation; Mammary Glands, Animal; Milk Proteins; Pregnancy; Sheep; gamma-Glutamyltransferase
PubMed: 14762075
DOI: 10.3168/jds.S0022-0302(04)73171-9 -
Kidney International Jul 1988Analysis with radiotracer and high performance liquid chromatography techniques showed that glutathione (GSH) is transported intact into cells primarily of proximal...
Analysis with radiotracer and high performance liquid chromatography techniques showed that glutathione (GSH) is transported intact into cells primarily of proximal tubule origin. Characteristics of GSH uptake were the same as previously reported for basal-lateral membrane vesicles, namely, uptake was Na+-dependent, inhibited by gamma-glutamylglutamate and/or probenecid, and not inhibited by cysteinylglycine or the constituent amino acids. Studies with inhibitors of gamma-glutamyltransferase (acivicin) and gamma-glutamylcysteine synthetase (buthionine sulfoximine) showed that GSH uptake, degradation and resynthesis are independent processes. The GSH uptake rate with 1 mM GSH was approximately three-fold greater than the GSH synthetic rate with 1 mM amino acids. To examine whether uptake of GSH can supplement synthesis to protect against injury, we incubated cells with a toxic concentration of t-butylhydroperoxide with or without GSH or its constituent amino acids. Although amino acids provided significant protection, GSH provided greater protection (cells with t-butylhydroperoxide plus GSH were not significantly different from cells alone). This protection by GSH was eliminated by gamma-glutamylglutamate or probenecid, indicating that GSH uptake was required for the protection seen. Protection was also eliminated when the GSSG reductase/GSH peroxidase system was inhibited by bischloronitrosourea (BCNU), indicating that GSH transport affords protection by maintaining GSH levels in the cell. Thus, intact GSH is transported into isolated proximal tubule cells by a Na+-dependent system, and this transported GSH can be used to supplement endogenous synthesis and GSSG reduction to protect cells against oxidative injury.
Topics: Amino Acids; Animals; Cell Survival; Cells, Cultured; Epithelial Cells; Epithelium; Glutathione; Kidney Tubules, Proximal; Male; Oxidation-Reduction; Peroxides; Probenecid; Rats; Rats, Inbred Strains; tert-Butylhydroperoxide
PubMed: 3172638
DOI: 10.1038/ki.1988.147 -
The Journal of Pharmacology and... Jan 2002Cisplatin is nephrotoxic. The mechanism underlying this organ-specific toxicity is unknown. We hypothesize that cisplatin is metabolized via a gamma-glutamyl...
Cisplatin is nephrotoxic. The mechanism underlying this organ-specific toxicity is unknown. We hypothesize that cisplatin is metabolized via a gamma-glutamyl transpeptidase (GGT) and cysteine S-conjugate beta-lyase-dependent pathway that has been shown to activate several haloalkenes to nephrotoxins. To test this hypothesis, we inhibited GGT and cysteine S-conjugate beta-lyase in C57BL/6 mice and analyzed the effect of the inhibitors on the nephrotoxicity of cisplatin. GGT was inhibited by pretreating the mice with acivicin. Cysteine S-conjugate beta-lyase was inhibited by aminooxyacetic acid (AOAA). Male C57BL/6 mice were treated with 15 mg/kg cisplatin (i.p.) and sacrificed on day 5. Half the mice treated with cisplatin alone died before sacrifice. The cisplatin-treated mice sacrificed at 5 days had significantly elevated levels of blood urea nitrogen (BUN). Histologic analysis revealed severe damage to the renal proximal tubules. Pretreatment with acivicin or AOAA protected the mice from the nephrotoxicity of cisplatin. None of the pretreated animals died before sacrifice. BUN levels and quantitative histologic analysis of the kidneys confirmed the protective effect of acivicin and AOAA. Platinum levels in the kidneys were not altered by acivicin or AOAA, indicating that neither affected the uptake of cisplatin into the kidney. Likewise, cisplatin-induced weight loss was not altered by acivicin or AOAA, suggesting that weight loss and nephrotoxicity are via distinct mechanisms. These data support the hypothesis that the nephrotoxicity of cisplatin is due to the metabolism of a platinum-glutathione conjugate by GGT and cysteine S-conjugate beta-lyase to a potent nephrotoxin.
Topics: Aminooxyacetic Acid; Animals; Antimetabolites, Antineoplastic; Antineoplastic Agents; Blood Urea Nitrogen; Body Weight; Carbon-Sulfur Lyases; Cisplatin; Enzyme Inhibitors; Isoxazoles; Kidney; Kidney Diseases; Male; Mice; Mice, Inbred C57BL; Platinum; Survival Analysis; gamma-Glutamyltransferase
PubMed: 11752109
DOI: 10.1124/jpet.300.1.142 -
Anesthesiology Jun 1998The sevoflurane degradation product compound A is nephrotoxic in rats and undergoes metabolism to glutathione and cysteine S-conjugates, with further metabolism by renal...
BACKGROUND
The sevoflurane degradation product compound A is nephrotoxic in rats and undergoes metabolism to glutathione and cysteine S-conjugates, with further metabolism by renal cysteine conjugate beta-lyase to reactive intermediates. Evidence suggests that toxicity is mediated by renal uptake of compound A S-conjugates and metabolism by beta-lyase. Previously, inhibitors of the beta-lyase pathway (aminooxyacetic acid and probenecid) diminished the nephrotoxicity of intraperitoneal compound A. This investigation determined inhibitor effects on the toxicity of inhaled compound A.
METHODS
Fischer 344 rats underwent 3 h of nose-only exposure to compound A (0-220 ppm in initial dose-response experiments and 100-109 ppm in subsequent inhibitor experiments). The inhibitors (and targets) were probenecid (renal organic anion transport mediating S-conjugate uptake), acivicin (gamma-glutamyl transferase), aminooxyacetic acid (renal beta-lyase), and aminobenzotriazole (cytochrome P450). Urine was collected for 24 h, and the animals were killed. Nephrotoxicity was assessed by histology and biochemical markers (serum BUN and creatinine; urine volume; and excretion of protein, glucose, and alpha-glutathione-S-transferase, a predominantly proximal tubular cell protein).
RESULTS
Compound A caused dose-related proximal tubular cell necrosis, diuresis, proteinuria, glucosuria, and increased alpha-glutathione-S-transferase excretion. The threshold for toxicity was 98-109 ppm (294-327 ppm-h). Probenecid diminished (P < 0.05) compound A-induced glucosuria and excretion of alpha-glutathione-S-transferase and completely prevented necrosis. Aminooxyacetic acid diminished compound A-dependent proteinuria and glucosuria but did not decrease necrosis. Acivicin increased nephrotoxicity of compound A, and aminobenzotriazole had no consistent effect on nephrotoxicity of compound A.
CONCLUSIONS
Nephrotoxicity of inhaled compound A in rats was associated with renal uptake of compound A S-conjugates and cysteine conjugates metabolism by renal beta-lyase. Manipulation of the beta-lyase pathway elicited similar results, whether compound A was administered by inhalation or intraperitoneal injection. Route of administration does not apparently influence nephrotoxicity of compound A in rats.
Topics: Anesthetics, Inhalation; Animals; Biomarkers; Biotransformation; Carbon-Sulfur Lyases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Ethers; Hydrocarbons, Fluorinated; Kidney; Male; Rats; Rats, Inbred F344
PubMed: 9637657
DOI: 10.1097/00000542-199806000-00027 -
The Journal of Biological Chemistry Sep 2005Vitamin B6 is an essential nutrient in the human diet. It can act as a co-enzyme for numerous metabolic enzymes and has recently been shown to be a potent antioxidant....
Vitamin B6 is an essential nutrient in the human diet. It can act as a co-enzyme for numerous metabolic enzymes and has recently been shown to be a potent antioxidant. Plants and microorganisms have the ability to make the compound. Yet, studies of vitamin B6 biosynthesis have been mainly restricted to Escherichia coli, where the vitamin is synthesized from 1-deoxy-d -xylulose 5-phosphate and 4-phosphohydroxy-l-threonine. Recently, a novel pathway for its synthesis has been discovered, involving two genes (PDX1 and PDX2) neither of which is homologous to any of those participating in the E. coli pathway. In Bacillus subtilis, YaaD and YaaE represent the PDX1 and PDX2 homolog, respectively. The two proteins form a complex that functions as a glutamine amidotransferase, with YaaE as the glutaminase domain and YaaD as the acceptor and pyridoxal 5'-phosphate (PLP) synthesis domain. In this report we corroborate a recent report on the identification of the substrates of YaaD and provide unequivocal proof of the identity of the reaction product. We show that both the glutaminase and synthase reactions are dependent on the respective protein partner. The synthase reaction can also utilize an external ammonium source but, in contrast to other glutamine amidotransferases, is dependent on YaaE under certain conditions. Furthermore, we report on the detailed characterization of the inhibition of the glutaminase domain, and thus PLP synthesis, by the glutamine analog acivicin. Employing pull-out assays and native-PAGE, we provide evidence for the dissociation of the bi-enzyme complex under these conditions. The results are discussed in light of the nature of the interaction of the two components of the enzyme complex.
Topics: Antioxidants; Bacillus subtilis; Binding Sites; Carbon-Nitrogen Lyases; Chromatography; Chromatography, Gel; Chromatography, High Pressure Liquid; Cloning, Molecular; Cysteine; DNA; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli Proteins; Glutaminase; Glutamine; Immunochemistry; Isoxazoles; Kinetics; Mass Spectrometry; Models, Chemical; Organophosphates; Oxidoreductases; Pentosephosphates; Protein Structure, Tertiary; Pyridoxal Phosphate; Quaternary Ammonium Compounds; Spectrometry, Mass, Electrospray Ionization; Spectrophotometry; Threonine; Time Factors; Ultraviolet Rays; Vitamin B 6
PubMed: 16030023
DOI: 10.1074/jbc.M501356200 -
Frontiers in Plant Science 2019The vegetative phase change in flowering plants is controlled by microRNA156 (miR156) under transcriptional regulation. However, the developmental signals upstream of...
UNLABELLED
The vegetative phase change in flowering plants is controlled by microRNA156 (miR156) under transcriptional regulation. However, the developmental signals upstream of miR156 are not well understood. The glutathione/glutathione disulfide (GSH/GSSG) ratios and GSH levels decline significantly during phase change, which is consistent with miR156 expression in apple ( Borkh.). Here, we found that the content of protein conjugated glutathione was remarkably higher in chloroplasts and nuclei of adult than juvenile phase apple hybrids. The decrease in miR156 expression was most relevant to the activities of serine acetyltransferase (SAT) and soluble γ-glutamyl transpeptidase (GGT), and the expressions of or . Transgenic apples over-expressing or miR156-mimetic (MIM156) did not alter expression or the soluble GGT activity. Inhibition of GGT activity with serine-borate complex or acivicin led to significant reduction in GSH content, the GSH/GSSG ratio, and the expressions of , , and miR156. Depletion of GSH with diethyl maleate without altering GGT activity caused a dramatic decrease in the expression of , , and miR156. Manipulating GGT activity and GSH homeostasis by transgenic over-expressing or RNAi increased or decreased and levels, respectively. These data provided novel evidence that participates in transcriptional level of transcription regulation of miR156 precursors during ontogenesis.
HIGHLIGHTS
- MdGGT1 affects thiol redox status and indirectly participates in the regulation of miR156 expression during vegetative phase change.
PubMed: 31417600
DOI: 10.3389/fpls.2019.00994 -
ChemMedChem Sep 2012Acivicin analogues with an increased affinity for CTP synthetase (CTPS) were designed as potential new trypanocidal agents. The inhibitory activity against CTPS can be...
Acivicin analogues with an increased affinity for CTP synthetase (CTPS) were designed as potential new trypanocidal agents. The inhibitory activity against CTPS can be improved by increasing molecular complexity, by inserting groups able to establish additional interactions with the binding pocket of the enzyme. This strategy has been pursued with the synthesis of α-amino-substituted analogues of Acivicin and N1-substituted pyrazoline derivatives. In general, there is direct correlation between the enzymatic activity and the in vitro anti-trypanosomal efficacy of the derivatives studied here. However, this cannot be taken as a general rule, as other important factors may play a role, notably the ability of uptake/diffusion of the molecules into the trypanosomes.
Topics: Carbon-Nitrogen Ligases; Enzyme Inhibitors; HeLa Cells; Humans; Isoxazoles; Molecular Docking Simulation; Pyrazoles; Trypanocidal Agents; Trypanosoma brucei brucei; Trypanosomiasis, African
PubMed: 22865834
DOI: 10.1002/cmdc.201200304 -
Kidney International Jun 1999Current renal substitution therapy for acute or chronic renal failure with hemodialysis or hemofiltration is life sustaining, but continues to have unacceptably high...
BACKGROUND
Current renal substitution therapy for acute or chronic renal failure with hemodialysis or hemofiltration is life sustaining, but continues to have unacceptably high morbidity and mortality rates. This therapy is not complete renal replacement therapy because it does not provide active transport nor metabolic and endocrinologic functions of the kidney, which are located predominantly in the tubular elements of the kidney.
METHODS
To optimize renal substitution therapy, a bioartificial renal tubule assist device (RAD) was developed and tested in vitro for a variety of differentiated tubular functions. High-flux hollow-fiber hemofiltration cartridges with membrane surface areas of 97 cm2 or 0. 4 m2 were used as tubular scaffolds. Porcine renal proximal tubule cells were seeded into the intraluminal spaces of the hollow fibers, which were pretreated with a synthetic extracellular matrix protein. Attached cells were expanded in the cartridge as a bioreactor system to produce confluent monolayers containing up to 1.5 x 109 cells (3. 5 x 105 cells/cm2). Near confluency was achieved along the entire membrane surface, with recovery rates for perfused inulin exceeding 97 and 95% in the smaller and larger units, respectively, compared with less than 60% recovery in noncell units.
RESULTS
A single-pass perfusion system was used to assess transport characteristics of the RADs. Vectorial fluid transport from intraluminal space to antiluminal space was demonstrated and was significantly increased with the addition of albumin to the antiluminal side and inhibited by the addition of ouabain, a specific inhibitor of Na+,K+-ATPase. Other transport activities were also observed in these devices and included active bicarbonate transport, which was decreased with acetazolamide, a carbonic anhydrase inhibitor, active glucose transport, which was suppressed with phlorizin, a specific inhibitor of the sodium-dependent glucose transporters, and para-aminohippurate (PAH) secretion, which was diminished with the anion transport inhibitor probenecid. A variety of differentiated metabolic functions was also demonstrated in the RAD. Intraluminal glutathione breakdown and its constituent amino acid uptake were suppressed with the irreversible inhibitor of gamma-glutamyl transpeptidase acivicin; ammonia production was present and incremented with declines in perfusion pH. Finally, endocrinological activity with conversion of 25-hydroxy(OH)-vitamin D3 to 1,25-(OH)2 vitD3 was demonstrated in the RAD. This conversion activity was up-regulated with parathyroid hormone and down-regulated with increasing inorganic phosphate levels, which are well-defined physiological regulators of this process in vivo.
CONCLUSIONS
These results clearly demonstrate the successful tissue engineering of a bioartificial RAD that possesses critical differentiated transport, and improves metabolic and endocrinological functions of the kidney. This device, when placed in series with conventional hemofiltration therapy, may provide incremental renal replacement support and potentially may decrease the high morbidity and mortality rates observed in patients with renal failure.
Topics: Ammonia; Animals; Bicarbonates; Biological Transport, Active; Biomedical Engineering; Calcifediol; Calcitriol; Cells, Cultured; Diuresis; Equipment Design; Glucose; Glutathione; In Vitro Techniques; Kidney Tubules; Kidneys, Artificial; Microscopy, Electron; Swine; p-Aminohippuric Acid
PubMed: 10354300
DOI: 10.1046/j.1523-1755.1999.00486.x -
Proceedings of the National Academy of... Apr 2013The Large-neutral Amino Acid Transporter 1 (LAT-1)--a sodium-independent exchanger of amino acids, thyroid hormones, and prescription drugs--is highly expressed in the... (Comparative Study)
Comparative Study
The Large-neutral Amino Acid Transporter 1 (LAT-1)--a sodium-independent exchanger of amino acids, thyroid hormones, and prescription drugs--is highly expressed in the blood-brain barrier and various types of cancer. LAT-1 plays an important role in cancer development as well as in mediating drug and nutrient delivery across the blood-brain barrier, making it a key drug target. Here, we identify four LAT-1 ligands, including one chemically novel substrate, by comparative modeling, virtual screening, and experimental validation. These results may rationalize the enhanced brain permeability of two drugs, including the anticancer agent acivicin. Finally, two of our hits inhibited proliferation of a cancer cell line by distinct mechanisms, providing useful chemical tools to characterize the role of LAT-1 in cancer metabolism.
Topics: Amines; Analysis of Variance; Blood-Brain Barrier; Cell Line, Tumor; Crystallography, X-Ray; Cyclohexanecarboxylic Acids; Drug Delivery Systems; Flow Cytometry; Gabapentin; Glioblastoma; HEK293 Cells; Humans; Isoxazoles; Large Neutral Amino Acid-Transporter 1; Leucine; Ligands; Models, Molecular; Tritium; gamma-Aminobutyric Acid
PubMed: 23509259
DOI: 10.1073/pnas.1218165110 -
European Journal of Biochemistry Mar 1999Monoclonal antibodies (mAb) against the native form of rat kidney gamma-glutamyl transpeptidase (GGT) were isolated by screening hybridomas with rat kidney brush-border...
Monoclonal antibodies (mAb) against the native form of rat kidney gamma-glutamyl transpeptidase (GGT) were isolated by screening hybridomas with rat kidney brush-border membrane vesicles. They were directed against protein rather than sugar epitopes in that each recognized all GGT isoforms. All of them inhibited partially the enzyme activity of GGT. They were specific in that they inhibited the rat enzyme, but not the mouse or human enzyme. Kinetic analyses were carried out with free GGT and GGT-mAb complexes with d-gamma-glutamyl-p-nitroanilide in the presence or absence of maleate, or in the presence or absence of alanine, cysteine, cystine or glycylglycine as gamma-glutamyl acceptors. mAbs 2A10 and 2E9 inhibited the hydrolytic and glutaminase activities of GGT and had little effect on the transpeptidation activity of the enzyme, whereas mAbs 4D7 and 5F10 inhibited transpeptidation, but not hydrolytic or glutaminase activities. mAb 5F10 mimicked the effect of maleate on GGT, in that it inhibited transpeptidation, enhanced the glutaminase activity and increased the affinity of the donor site of GGT for acivicin. Such mAbs may be useful for long-term studies in tissue cultures and in vivo, and for the identification of GGT epitopes that are important for the hydrolytic and transpeptidase activities.
Topics: Animals; Antibodies, Monoclonal; Carbohydrates; Female; Glutaminase; Humans; Hydrolysis; Isoenzymes; Kidney; Mice; Mice, Inbred BALB C; Rats; Rats, Wistar; gamma-Glutamyltransferase
PubMed: 10103015
DOI: 10.1046/j.1432-1327.1999.00223.x