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Nico P. E. Vermeulen - One of the best experts on this subject based on the ideXlab platform.
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bioactivation of selenoCysteine se Conjugates by a highly purified rat renal Cysteine Conjugate beta lyase glutamine transaminase k
Journal of Pharmacology and Experimental Therapeutics, 2000Co-Authors: Jan N. M. Commandeur, Ioanna Andreadou, Martijn Rooseboom, M Out, L J De Leur, E J Groot, Nico P. E. VermeulenAbstract:SelenoCysteine Se-Conjugates have recently been proposed as potential prodrugs to target pharmacologically active selenol compounds to the kidney. Although rat renal cytosol displayed a high activity of beta-elimination activity toward these substrates, the enzymes involved in this activation pathway as yet have not been identified. In the present study, the possible involvement of Cysteine Conjugate beta-lyase/glutamine transaminase K (beta-lyase/GTK) in cytosolic activity was investigated. To this end, the enzyme kinetics of 15 differentially substituted selenoCysteine Se-Conjugates and 11 Cysteine S-Conjugates was determined using highly purified rat renal beta-lyase/GTK. The results demonstrate that most selenoCysteine Se-Conjugates are beta-eliminated at a very high activity by purified beta-lyase/GTK, implicating an important role of this protein in the previously reported beta-elimination reactions in rat renal cytosol. As indicated by the rapid consumption of alpha-keto-gamma-methiolbutyric acid, purified beta-lyase/GTK also catalyzed transamination reactions, which appeared to even exceed that of beta-elimination. The corresponding sulfur analogs also showed significant transamination but were beta-eliminated at an extremely low rate. Comparison of the obtained enzyme kinetic data of purified beta-lyase/GTK with previously obtained data from rat renal cytosol showed a poor correlation. By determining the activity profiles of cytosolic fractions applied to anion exchange fast protein liquid chromatography and gel filtration chromatography, the involvement of multiple enzymes in the beta-elimination of selenoCysteine Se-Conjugates in rat renal cytosol was demonstrated. The identity and characteristics of these alternative selenoCysteine Conjugate beta-lyases, however, remain to be established.
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Synthesis of novel Se-substituted selenoCysteine derivatives as potential kidney selective prodrugs of biologically active selenol compounds: evaluation of kinetics of beta-elimination reactions in rat renal cytosol.
Journal of Medicinal Chemistry, 1996Co-Authors: Ioanna Andreadou, Eduard A. Worthington, Wiro M.b.p. Menge, Jan N. M. Commandeur, Nico P. E. VermeulenAbstract:Eighteen Se-substituted selenoCysteine derivatives were synthesized as potential kidney selective prodrugs which can be activated by renal Cysteine Conjugate β-lyase to selenium-containing chemoprotectants or antitumor agents. SelenoCysteine derivatives with aliphatic and benzylic Se-substituents were synthesized by reducing selenocystine to selenoCysteine followed by a reaction with the corresponding alkyl and benzyl halogenides. SelenoCysteine derivatives with aromatic Se-substituents were synthesized by reaction of β-chloroalanine with substituted phenylselenol compounds, which were formed by reducing substituted diphenyl diselenides by NaBH4. The enzyme kinetic parameters (apparent Km and Vmax) of the β-elimination reaction of the selenoCysteine Conjugates were studied in rat renal cytosol. The results suggest that Se-substituted l-selenoCysteine Conjugates are extremely good substrates for renal Cysteine Conjugate β-lyases as indicated by low apparent Km and high Vmax values. The benzyl-substituted S...
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synthesis of novel se substituted selenoCysteine derivatives as potential kidney selective prodrugs of biologically active selenol compounds evaluation of kinetics of beta elimination reactions in rat renal cytosol
Journal of Medicinal Chemistry, 1996Co-Authors: Ioanna Andreadou, Eduard A. Worthington, Wiro M.b.p. Menge, Jan N. M. Commandeur, Nico P. E. VermeulenAbstract:Eighteen Se-substituted selenoCysteine derivatives were synthesized as potential kidney selective prodrugs which can be activated by renal Cysteine Conjugate beta-lyase to selenium-containing chemoprotectants or antitumor agents. SelenoCysteine derivatives with aliphatic and benzylic Se-substituents were synthesized by reducing selenocystine to selenoCysteine followed by a reaction with the corresponding alkyl and benzyl halogenides. SelenoCysteine derivatives with aromatic Se-substitutes were synthesized by reaction of beta-chloroalanine with substituted phenylselenol compounds, which were formed by reducing substituted diphenyl diselenides by NaBH4. The enzyme kinetic parameters (apparent Km and Vmax) of the beta-elimination reaction of the selenoCysteine Conjugates were studied in rat renal cytosol. The results suggest that Se-substituted L-selenoCysteine Conjugates are extremely good substrates for renal Cysteine Conjugate beta-lyases as indicated by low apparent Km and high Vmax values. The benzyl-substituted Se-Conjugates appeared to be better substrates than the phenyl- and alkyl-substituted Se-Conjugates. Corresponding L-Cysteine S-Conjugates were too poor substrates to obtain proper enzyme kinetics. Recently, local activation of Cysteine S-Conjugates by renal Cysteine Conjugate beta-lyases was proposed as a new strategy to target antitumor agents to the kidney. The present results show that Se-substituted selenoCysteine Conjugates may be more promising prodrugs because these compounds are much better substrates for beta-lyase.
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Purification of glutamine transaminase K/Cysteine Conjugate beta-lyase from rat renal cytosol based on hydrophobic interaction HPLC and gel permeation FPLC.
Protein expression and purification, 1993Co-Authors: A. Yamauchi, Jan N. M. Commandeur, G.j. Stijntjes, Nico P. E. VermeulenAbstract:Abstract Cysteine Conjugate β-lyase (β-lyase, EC 4.4.1.13) was purified to homogeneity from rat renal cytosol using a new and highly efficient method, based on C3-hydrophobic interaction (HI) high-performance liquid chromatography (HPLC) in combination with gel permeation fast protein liquid chromatography. The purity of the enzyme was judged from SDS-PAGE and C18-reversed-phase HPLC. The β-lyase was estimated to be a homodimer consisting of a 47,400-Da subunit with absorption maxima at 280 and 420-430 am. The specific activity of the purified β-lyase toward S-(1,2-dichloro-vinyl)-L-Cysteine (1,2-DCVC) in the presence of α-keto-γ-methiolbutyric acid (KMB) was 6.4 μmol/min/mg protein, which is by far the highest value so far reported. Kinetic analysis of 1,2-DCVC metabolism by the enzyme in the presence of KMB gave Km and Vmax of 0.33 mM and 8.4 μmol/min/mg protein, respectively. No significant activity of the purified enzyme was detectable with S-2-benzothiazolyl-L-Cysteine up to 2 mM. The purified enzyme also had glutamine transaminase K activity (EC 2.6.1.64) as assayed with phenylalanine and KMB as substrates. This specific activity was 16.0 μmol/min/mg. Amino acid analysis of the purified β-lyase was carried out and was found to be closely similar to the amino acid composition of five other pyridoxal phosphate (PLP)-containing amino acid aminotransferases. This suggests that glutamine transaminase K/Cysteine Conjugate β-lyase is a typical member of the PLP-containing aminotransferase group.
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High-performance liquid chromatography-fluorescence assay of pyruvic acid to determine Cysteine Conjugate β-lyase activity: Application to S-1,2-dichlorovinyl-l-Cysteine and S-2-benzothiazolyl-l-Cysteine
Analytical biochemistry, 1992Co-Authors: Gerard J. Stijntjes, Johan M. Te Koppele, Nico P. E. VermeulenAbstract:An HPLC-fluorescence assay has been developed for the determination of the activity of rat renal cytosolic Cysteine Conjugate beta-lyase. The method is based on isocratic HPLC separation and fluorescence detection of pyruvic acid, derivatized with o-phenylenediamine (OPD), and is shown to be rapid, specific, and very sensitive. The assay has been evaluated with two model substrates for rat renal cytosolic beta-lyase, notably S-1,2-dichorovinyl-L-Cysteine (DCVC) and S-2-benzothiazolyl-L-Cysteine (BTC). Equimolar formation of pyruvic acid and 2-mercaptobenzothiazole, a chromophoric thiol, indicated that pyruvic acid formation actually reflects the beta-elimination activity of beta-lyase during the beta-elimination of BTC. From this it follows that the pyruvic acid assay can be applied to the measurement of the beta-elimination activity of this enzyme, independent of the presence of chromophoric groups or radiolabels in substrates. Due to the large linear range and the very high sensitivity of the present HPLC-fluorescence assay (detection limit, 7.5 pmol of pyruvic acid), both good and poor substrates of beta-lyase can be measured. Enzyme kinetic data are presented for the model substrates BTC and DCVC and for four structurally related S-2,2-difluoroethyl-L-Cysteine Conjugates.
Evan D Kharasch - One of the best experts on this subject based on the ideXlab platform.
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Role of the Renal Cysteine Conjugate β-Lyase Pathway in Inhaled Compound A Nephrotoxicity in Rats
Anesthesiology, 1998Co-Authors: Evan D Kharasch, Gary M. Hoffman, David Thorning, Douglas C. Hankins, Cormac G. KiltyAbstract: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.
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evidence for metabolism of fluoromethyl 2 2 difluoro 1 trifluoromethyl vinyl ether compound a a sevoflurane degradation product by Cysteine Conjugate β lyase
Chemical Research in Toxicology, 1996Co-Authors: Douglas K Spracklin, Evan D KharaschAbstract:The volatile anesthetic sevoflurane is degraded to fluoromethyl 2,2-difluoro-1-(trifluoromethyl)vinyl ether (FDVE), a potent rat nephrotoxin. In rats in vivo, FDVE undergoes glutathione conjugation and metabolism to Cysteine Conjugates, whose bioactivation by renal Cysteine Conjugate β-lyase has been implicated by the protective effects of (aminooxy)acetic acid, an inhibitor of Cysteine Conjugate β-lyase. We specifically tested the hypothesis that FDVE is metabolized via the β-lyase pathway to yield 3,3,3-trifluoro-2-(fluoromethoxy)propanoic acid. Urine of rats administered FDVE (0.3 mmol/kg) was extracted and derivatized with diazomethane. Headspace GC/MS analysis demonstrated a peak whose retention time and mass spectrum were identical to those of synthetic methyl 3,3,3-trifluoro-2-(fluoromethoxy)propanoate. Pretreatment of rats with (aminooxy)acetic acid significantly decreased the amount of 3,3,3-trifluoro-2-(fluoromethoxy)propanoic acid detected in the urine of FDVE-treated animals. The 19F NMR spect...
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nephrotoxicity of sevoflurane compound a fluoromethyl 2 2 difluoro 1 trifluoromethyl vinyl ether in rats evidence for glutathione and Cysteine Conjugate formation and the role of renal Cysteine Conjugate β lyase
Biochemical and Biophysical Research Communications, 1995Co-Authors: Lixia Jin, Thomas A Baillie, Margaret R Davis, Evan D KharaschAbstract:Abstract Compound A, which is a breakdown product of the volatile anesthetic sevoflurane, is nephrotoxic in rats, although the mechanism of this toxicity is unknown. In the present investigation, the role of glutathione conjugation, glutathione Conjugate processing to Cysteine Conjugates, and renal Cysteine Conjugate β-lyase in the pathogenesis of Compound A nephrotoxicity was investigated in the rat. Following intraperitoneal administration of Compound A (1 mmol/kg), the presence in bile of two types of Compound A-glutathione Conjugates, and the urinary excretion of two types of Compound A-mercapturic acid Conjugates, was demonstrated by ionspray-tandem mass spectrometry. Aminooxyacetic acid, a competitive inhibitor of renal Cysteine Conjugate β-lyase, partially protected against Compound A-induced diuresis and proteinuria. These results suggest that glutathione Conjugate formation, subsequent processing to Cysteine Conjugates, and Cysteine Conjugate metabolism by renal β-lyase may be important factors in the pathogenesis of Compound A-mediated nephrotoxicity in rats.
Ioanna Andreadou - One of the best experts on this subject based on the ideXlab platform.
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bioactivation of selenoCysteine se Conjugates by a highly purified rat renal Cysteine Conjugate beta lyase glutamine transaminase k
Journal of Pharmacology and Experimental Therapeutics, 2000Co-Authors: Jan N. M. Commandeur, Ioanna Andreadou, Martijn Rooseboom, M Out, L J De Leur, E J Groot, Nico P. E. VermeulenAbstract:SelenoCysteine Se-Conjugates have recently been proposed as potential prodrugs to target pharmacologically active selenol compounds to the kidney. Although rat renal cytosol displayed a high activity of beta-elimination activity toward these substrates, the enzymes involved in this activation pathway as yet have not been identified. In the present study, the possible involvement of Cysteine Conjugate beta-lyase/glutamine transaminase K (beta-lyase/GTK) in cytosolic activity was investigated. To this end, the enzyme kinetics of 15 differentially substituted selenoCysteine Se-Conjugates and 11 Cysteine S-Conjugates was determined using highly purified rat renal beta-lyase/GTK. The results demonstrate that most selenoCysteine Se-Conjugates are beta-eliminated at a very high activity by purified beta-lyase/GTK, implicating an important role of this protein in the previously reported beta-elimination reactions in rat renal cytosol. As indicated by the rapid consumption of alpha-keto-gamma-methiolbutyric acid, purified beta-lyase/GTK also catalyzed transamination reactions, which appeared to even exceed that of beta-elimination. The corresponding sulfur analogs also showed significant transamination but were beta-eliminated at an extremely low rate. Comparison of the obtained enzyme kinetic data of purified beta-lyase/GTK with previously obtained data from rat renal cytosol showed a poor correlation. By determining the activity profiles of cytosolic fractions applied to anion exchange fast protein liquid chromatography and gel filtration chromatography, the involvement of multiple enzymes in the beta-elimination of selenoCysteine Se-Conjugates in rat renal cytosol was demonstrated. The identity and characteristics of these alternative selenoCysteine Conjugate beta-lyases, however, remain to be established.
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Synthesis of novel Se-substituted selenoCysteine derivatives as potential kidney selective prodrugs of biologically active selenol compounds: evaluation of kinetics of beta-elimination reactions in rat renal cytosol.
Journal of Medicinal Chemistry, 1996Co-Authors: Ioanna Andreadou, Eduard A. Worthington, Wiro M.b.p. Menge, Jan N. M. Commandeur, Nico P. E. VermeulenAbstract:Eighteen Se-substituted selenoCysteine derivatives were synthesized as potential kidney selective prodrugs which can be activated by renal Cysteine Conjugate β-lyase to selenium-containing chemoprotectants or antitumor agents. SelenoCysteine derivatives with aliphatic and benzylic Se-substituents were synthesized by reducing selenocystine to selenoCysteine followed by a reaction with the corresponding alkyl and benzyl halogenides. SelenoCysteine derivatives with aromatic Se-substituents were synthesized by reaction of β-chloroalanine with substituted phenylselenol compounds, which were formed by reducing substituted diphenyl diselenides by NaBH4. The enzyme kinetic parameters (apparent Km and Vmax) of the β-elimination reaction of the selenoCysteine Conjugates were studied in rat renal cytosol. The results suggest that Se-substituted l-selenoCysteine Conjugates are extremely good substrates for renal Cysteine Conjugate β-lyases as indicated by low apparent Km and high Vmax values. The benzyl-substituted S...
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synthesis of novel se substituted selenoCysteine derivatives as potential kidney selective prodrugs of biologically active selenol compounds evaluation of kinetics of beta elimination reactions in rat renal cytosol
Journal of Medicinal Chemistry, 1996Co-Authors: Ioanna Andreadou, Eduard A. Worthington, Wiro M.b.p. Menge, Jan N. M. Commandeur, Nico P. E. VermeulenAbstract:Eighteen Se-substituted selenoCysteine derivatives were synthesized as potential kidney selective prodrugs which can be activated by renal Cysteine Conjugate beta-lyase to selenium-containing chemoprotectants or antitumor agents. SelenoCysteine derivatives with aliphatic and benzylic Se-substituents were synthesized by reducing selenocystine to selenoCysteine followed by a reaction with the corresponding alkyl and benzyl halogenides. SelenoCysteine derivatives with aromatic Se-substitutes were synthesized by reaction of beta-chloroalanine with substituted phenylselenol compounds, which were formed by reducing substituted diphenyl diselenides by NaBH4. The enzyme kinetic parameters (apparent Km and Vmax) of the beta-elimination reaction of the selenoCysteine Conjugates were studied in rat renal cytosol. The results suggest that Se-substituted L-selenoCysteine Conjugates are extremely good substrates for renal Cysteine Conjugate beta-lyases as indicated by low apparent Km and high Vmax values. The benzyl-substituted Se-Conjugates appeared to be better substrates than the phenyl- and alkyl-substituted Se-Conjugates. Corresponding L-Cysteine S-Conjugates were too poor substrates to obtain proper enzyme kinetics. Recently, local activation of Cysteine S-Conjugates by renal Cysteine Conjugate beta-lyases was proposed as a new strategy to target antitumor agents to the kidney. The present results show that Se-substituted selenoCysteine Conjugates may be more promising prodrugs because these compounds are much better substrates for beta-lyase.
Jan N. M. Commandeur - One of the best experts on this subject based on the ideXlab platform.
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bioactivation of selenoCysteine se Conjugates by a highly purified rat renal Cysteine Conjugate beta lyase glutamine transaminase k
Journal of Pharmacology and Experimental Therapeutics, 2000Co-Authors: Jan N. M. Commandeur, Ioanna Andreadou, Martijn Rooseboom, M Out, L J De Leur, E J Groot, Nico P. E. VermeulenAbstract:SelenoCysteine Se-Conjugates have recently been proposed as potential prodrugs to target pharmacologically active selenol compounds to the kidney. Although rat renal cytosol displayed a high activity of beta-elimination activity toward these substrates, the enzymes involved in this activation pathway as yet have not been identified. In the present study, the possible involvement of Cysteine Conjugate beta-lyase/glutamine transaminase K (beta-lyase/GTK) in cytosolic activity was investigated. To this end, the enzyme kinetics of 15 differentially substituted selenoCysteine Se-Conjugates and 11 Cysteine S-Conjugates was determined using highly purified rat renal beta-lyase/GTK. The results demonstrate that most selenoCysteine Se-Conjugates are beta-eliminated at a very high activity by purified beta-lyase/GTK, implicating an important role of this protein in the previously reported beta-elimination reactions in rat renal cytosol. As indicated by the rapid consumption of alpha-keto-gamma-methiolbutyric acid, purified beta-lyase/GTK also catalyzed transamination reactions, which appeared to even exceed that of beta-elimination. The corresponding sulfur analogs also showed significant transamination but were beta-eliminated at an extremely low rate. Comparison of the obtained enzyme kinetic data of purified beta-lyase/GTK with previously obtained data from rat renal cytosol showed a poor correlation. By determining the activity profiles of cytosolic fractions applied to anion exchange fast protein liquid chromatography and gel filtration chromatography, the involvement of multiple enzymes in the beta-elimination of selenoCysteine Se-Conjugates in rat renal cytosol was demonstrated. The identity and characteristics of these alternative selenoCysteine Conjugate beta-lyases, however, remain to be established.
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Synthesis of novel Se-substituted selenoCysteine derivatives as potential kidney selective prodrugs of biologically active selenol compounds: evaluation of kinetics of beta-elimination reactions in rat renal cytosol.
Journal of Medicinal Chemistry, 1996Co-Authors: Ioanna Andreadou, Eduard A. Worthington, Wiro M.b.p. Menge, Jan N. M. Commandeur, Nico P. E. VermeulenAbstract:Eighteen Se-substituted selenoCysteine derivatives were synthesized as potential kidney selective prodrugs which can be activated by renal Cysteine Conjugate β-lyase to selenium-containing chemoprotectants or antitumor agents. SelenoCysteine derivatives with aliphatic and benzylic Se-substituents were synthesized by reducing selenocystine to selenoCysteine followed by a reaction with the corresponding alkyl and benzyl halogenides. SelenoCysteine derivatives with aromatic Se-substituents were synthesized by reaction of β-chloroalanine with substituted phenylselenol compounds, which were formed by reducing substituted diphenyl diselenides by NaBH4. The enzyme kinetic parameters (apparent Km and Vmax) of the β-elimination reaction of the selenoCysteine Conjugates were studied in rat renal cytosol. The results suggest that Se-substituted l-selenoCysteine Conjugates are extremely good substrates for renal Cysteine Conjugate β-lyases as indicated by low apparent Km and high Vmax values. The benzyl-substituted S...
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synthesis of novel se substituted selenoCysteine derivatives as potential kidney selective prodrugs of biologically active selenol compounds evaluation of kinetics of beta elimination reactions in rat renal cytosol
Journal of Medicinal Chemistry, 1996Co-Authors: Ioanna Andreadou, Eduard A. Worthington, Wiro M.b.p. Menge, Jan N. M. Commandeur, Nico P. E. VermeulenAbstract:Eighteen Se-substituted selenoCysteine derivatives were synthesized as potential kidney selective prodrugs which can be activated by renal Cysteine Conjugate beta-lyase to selenium-containing chemoprotectants or antitumor agents. SelenoCysteine derivatives with aliphatic and benzylic Se-substituents were synthesized by reducing selenocystine to selenoCysteine followed by a reaction with the corresponding alkyl and benzyl halogenides. SelenoCysteine derivatives with aromatic Se-substitutes were synthesized by reaction of beta-chloroalanine with substituted phenylselenol compounds, which were formed by reducing substituted diphenyl diselenides by NaBH4. The enzyme kinetic parameters (apparent Km and Vmax) of the beta-elimination reaction of the selenoCysteine Conjugates were studied in rat renal cytosol. The results suggest that Se-substituted L-selenoCysteine Conjugates are extremely good substrates for renal Cysteine Conjugate beta-lyases as indicated by low apparent Km and high Vmax values. The benzyl-substituted Se-Conjugates appeared to be better substrates than the phenyl- and alkyl-substituted Se-Conjugates. Corresponding L-Cysteine S-Conjugates were too poor substrates to obtain proper enzyme kinetics. Recently, local activation of Cysteine S-Conjugates by renal Cysteine Conjugate beta-lyases was proposed as a new strategy to target antitumor agents to the kidney. The present results show that Se-substituted selenoCysteine Conjugates may be more promising prodrugs because these compounds are much better substrates for beta-lyase.
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Purification of glutamine transaminase K/Cysteine Conjugate beta-lyase from rat renal cytosol based on hydrophobic interaction HPLC and gel permeation FPLC.
Protein expression and purification, 1993Co-Authors: A. Yamauchi, Jan N. M. Commandeur, G.j. Stijntjes, Nico P. E. VermeulenAbstract:Abstract Cysteine Conjugate β-lyase (β-lyase, EC 4.4.1.13) was purified to homogeneity from rat renal cytosol using a new and highly efficient method, based on C3-hydrophobic interaction (HI) high-performance liquid chromatography (HPLC) in combination with gel permeation fast protein liquid chromatography. The purity of the enzyme was judged from SDS-PAGE and C18-reversed-phase HPLC. The β-lyase was estimated to be a homodimer consisting of a 47,400-Da subunit with absorption maxima at 280 and 420-430 am. The specific activity of the purified β-lyase toward S-(1,2-dichloro-vinyl)-L-Cysteine (1,2-DCVC) in the presence of α-keto-γ-methiolbutyric acid (KMB) was 6.4 μmol/min/mg protein, which is by far the highest value so far reported. Kinetic analysis of 1,2-DCVC metabolism by the enzyme in the presence of KMB gave Km and Vmax of 0.33 mM and 8.4 μmol/min/mg protein, respectively. No significant activity of the purified enzyme was detectable with S-2-benzothiazolyl-L-Cysteine up to 2 mM. The purified enzyme also had glutamine transaminase K activity (EC 2.6.1.64) as assayed with phenylalanine and KMB as substrates. This specific activity was 16.0 μmol/min/mg. Amino acid analysis of the purified β-lyase was carried out and was found to be closely similar to the amino acid composition of five other pyridoxal phosphate (PLP)-containing amino acid aminotransferases. This suggests that glutamine transaminase K/Cysteine Conjugate β-lyase is a typical member of the PLP-containing aminotransferase group.
Denis Dubourdieu - One of the best experts on this subject based on the ideXlab platform.
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Measuring the aromatic potential of Vitis vinifera L. Cv. Sauvignon blanc grapes by assaying S-Cysteine Conjugates, precursors of the volatile thiols responsible for their varietal aroma.
Journal of agricultural and food chemistry, 2000Co-Authors: C. Peyrot Des Gachons, Takatoshi Tominaga, Denis DubourdieuAbstract:The method presented for measuring the aromatic potential of Sauvignon blanc must is based on an assay of the S-Cysteine Conjugate precursors of three volatile thiols involved in the characteristic aroma of wines made from this grape variety: 4-mercapto-4-methylpentan-2-one, 4-mercapto-4-methylpentan-2-ol, and 3-mercaptohexan-1-ol. These compounds were released enzymatically from their precursors by percolating the must through an immobilized tryptophanase column (EC 4.1.99.1), catalyzing an α,β-elimination reaction on the S-Cysteine Conjugate. The volatile thiols were analyzed by GC-MS, as were the deuterated analogues that had been released from synthesized deuterated precursors and were added as internal standards. The quantities of volatile thiols released under these conditions were proportional to the S-Cysteine Conjugate content of the must. Keywords: Aroma potential; S-Cysteine Conjugate; flavor precursor; Sauvignon blanc; stable isotope dilution assay; tryptophanase; Vitis vinifera
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a new type of flavor precursors in vitis vinifera l cv sauvignon blanc s Cysteine Conjugates
Journal of Agricultural and Food Chemistry, 1998Co-Authors: Takatoshi Tominaga, And Catherine Peyrot Des Gachons, Denis DubourdieuAbstract:Three flavor-active volatile thiols (4-mercapto-4-methylpentan-2-one, 4-mercapto-4-methylpentan-2-ol and 3-mercaptohexan-1-ol) involved in Vitis vinifera L. var. Sauvignon blanc wine aroma can be generated in vitro from nonvolatile must extracts by the enzyme action of a cell-free extract obtained from the gastrointestinal bacterium Eubacterium limosum. The specificity of a Cysteine Conjugate β-lyase (EC 4.4.1.13) which is contained in the cell-free extract, strongly suggested the existence of precursor for these volatile thiols having a structure of S-Cysteine Conjugate. Gas-phase chromatography analysis coupled with mass spectrometry of must extract in the form of trimethylsilylated derivatives, verified this hypothesis. The release of flavor-active volatile thiols during alcoholic fermentation of the must is shown to be due to the degradation, by yeast, of the corresponding S-Cysteine Conjugates. This mechanism explains the amplification of the typical Sauvignon blanc grape aroma during alcoholic ferme...
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A S-Cysteine Conjugate, precursor of aroma of White Sauvignon
OENO One, 1995Co-Authors: Takatoshi Tominaga, Isabelle Masneuf-pomarede, Denis DubourdieuAbstract:4-mercapto-4-methylpentan-2-one (4-MMP), a strongly odorant compound responsible for the « boxtree » or « broom plant » odour of the Sauvignon wines, can be enzymaticaly released in vitro from an odourless must extract. The enzyme source used is a cell-free extract of the gastrointestinal bacterium Eubacterium limosum. This crude preparation exhibits a Cysteine β-lyase activity which requires the presence of pyridoxal phosphate. The release of 4-MMP is inhibited when the substrate is previously treated with N-hydroxysuccimide acetate which reacts with a primary amine. The same bacterial extract is also able to release 4-MMP, pyruvic acid and ammonium, from S-(4-methylpentan-2-one)-L-Cysteine. On the other hand, the cleavage of S-(4-methylpentan-2-one)D,L-homoCysteine and S-(4-methylpentan-2-one)- glutathione is very limited. These results suggest that the precursor of 4-MMP in Sauvignon must is a S-Cysteine Conjugate. Such an aroma precursor in grapes or in other fruits has never been round berore.
