The Experts below are selected from a list of 225 Experts worldwide ranked by ideXlab platform
Ruy Pérez-montfort - One of the best experts on this subject based on the ideXlab platform.
-
Medical and Veterinary Importance of the Moonlighting Functions of Triosephosphate Isomerase.
Current protein & peptide science, 2019Co-Authors: Mónica Rodríguez-bolaños, Ruy Pérez-montfortAbstract:Triosephosphate Isomerase is the fifth enzyme in glycolysis and its canonical function is the reversible isomerization of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. Within the last decade multiple other functions, that may not necessarily always involve catalysis, have been described. These include variations in the degree of its expression in many types of cancer and participation in the regulation of the cell cycle. Triosephosphate Isomerase may function as an auto-antigen and in the evasion of the immune response, as a factor of virulence of some organisms, and also as an important allergen, mainly in a variety of seafoods. It is an important factor to consider in the cryopreservation of semen and seems to play a major role in some aspects of the development of Alzheimer's disease. It also seems to be responsible for neurodegenerative alterations in a few cases of human Triosephosphate Isomerase deficiency. Thus, Triosephosphate Isomerase is an excellent example of a moonlighting protein.
-
Sulfhydryl reagent susceptibility in proteins with high sequence similarity--Triosephosphate Isomerase from Trypanosoma brucei, Trypanosoma cruzi and Leishmania mexicana.
European journal of biochemistry, 1998Co-Authors: Georgina Garza-ramos, Pedro Ostoa-saloma, Marietta Tuena De Gómez-puyou, Ruy Pérez-montfort, Nallely Cabrera, Emma Saavedra-lira, Armando Gómez-puyouAbstract:The amino acid sequence of Triosephosphate Isomerase from Trypanosoma brucei, Trypanosoma cruzi, and Leishmania mexicana have an identity of 68 %. Using the numbering system for the T. brucei enzyme, in their aligned sequences, the T. cruzi and leishmanial enzymes have cysteine residues at positions 14, 40, 117 and 126. T. brucei Triosephosphate Isomerase has cysteine residues at positions 14, 40 and 126, and a valine residue at position 117. Dithionitrobenzoic acid and methylmethane thiosulfonate inhibited the three enzymes, but T. cruzi Triosephosphate Isomerase was more than 100-fold more sensitive. The sensitivity of wild type Triosephosphate Isomerase from T. cruzi and T. brucei to the reagents was equal to that of the Cys117Val and Val117Cys mutant enzymes, respectively. Triosephosphate Isomerases that have cysteine residues at positions 40 and 126, but lack a cysteine residue at position 14 are insensitive to methylmethane thiosulfonate. Thus, sulfhydryl reagents act on Cys14. At stoichiometric concentrations, the reagents inhibited the three enzymes as a consequence of structural alterations as measured by binding of 8-anilino-1-napthalenesulfonic acid to previously buried hydrophobic regions. However, the times for half-maximal alterations were 10 min, 15 hours and over 30 hours for T. cruzi, T. brucei and L. mexicana Triosephosphate Isomerase, respectively. The effect of pH on the action of the sulfhydryl reagents and molecular modeling showed no differences in the solvent accesibility of Cys14. As Cys14 forms part of the dimer interface, the data indicate that, in the three enzymes, barriers of different magnitude hinder the interaction between the sulfhydryl reagents and Cys14. The barrier is lower in T. cruzi Triosephosphate Isomerase which makes its dimer interface more susceptible for perturbation.
-
Cloning, Expression, Purification and Characterization Of Triosephosphate Isomerase from Trypanosoma Cruzi
European journal of biochemistry, 1997Co-Authors: Pedro Ostoa-saloma, Georgina Garza-ramos, Jorge Ramírez, Ingeborg Becker, Myriam Berzunza, Abraham Landa, Armando Gómez-puyou, Marietta Tuena De Gómez-puyou, Ruy Pérez-montfortAbstract:The gene that encodes for Triosephosphate Isomerase from Trypanosoma cruzi was cloned and sequenced. In T. cruzi, there is only one gene for Triosephosphate Isomerase. The enzyme has an identity of 72% and 68% with Triosephosphate Isomerase from Trypanosoma brucei and Leishmania mexicana, respectively. The active site residues are conserved: out of the 32 residues that conform the interface of dimeric Triosephosphate Isomerase from T. brucei, 29 are conserved in the T. cruzi enzyme. The enzyme was expressed in Escherichia coli and purified to homogeneity. Data from electrophoretic analysis under denaturing techniques and filtration techniques showed that Triosephosphate Isomerase from T. cruzi is a homodimer. Some of its structural and kinetic features were determined and compared to those of the purified enzymes from T. brucei and L mexicana. Its circular dichroism spectrum was almost identical to that of Triosephosphate Isomerase from T. brucei. Its kinetic properties and pH optima were similar to those of T. brucei and L. mexicana, although the latter exhibited a higher Vmax with glyceraldehyde 3-phosphate as substrate. The sensitivity of the three enzymes to the sulfhydryl reagent methylmethane thiosulfonate (MeSO2-SMe) was determined; the sensitivity of the T. cruzi enzyme was about 40 times and 200 times higher than that of the enzymes from T. brucei and L. mexicana, respectively. Triosephosphate Isomerase from T. cruzi and L. mexicana have the three cysteine residues that exist in the T. brucei enzyme (positions 14, 39, 126, using the numbering of the T. brucei enzyme); however, they also have an additional residue (position 117). These data suggest that regardless of the high identity of the three trypanosomatid enzymes, there are structural differences in the disposition of their cysteine residues that account for their different sensitivity to the sulfhydryl reagent. The disposition of the cysteine in Triosephosphate Isomerase from T. cruzi appears to make it unique for inhibition by modification of its cysteine.
-
Species-specific inhibition of homologous enzymes by modification of nonconserved amino acids residues The cysteine residues of Triosephosphate Isomerase
European journal of biochemistry, 1996Co-Authors: Georgina Garza-ramos, Marietta Tuena De Gómez-puyou, Ruy Pérez-montfort, Arturo Rojo-domínguez, Armando Gómez-puyouAbstract:The possibility of using non-conserved amino acid residues to produce selective inhibition of homologous enzymes from different species has been further explored with Triosephosphate Isomerase. S-phenylp-toluenethiosulfonate (MePhS0,-SPh), which produces phenyl disulfides with accessible Cys residues, inhibits the activity of rabbit Triosephosphate Isomerase. The inhibition is due to derivatization of one of the five Cys residues of rabbit Triosephosphate Isomerase. The effect of MePhS0,-SPh on Triosephosphate Isomerase from Saccharomyces cerevisiae, Escherichia coli, chicken and Schizosaccharomyces pombe was also determined. MePhS0,-SPh did not affect the activity of Triosephosphate Isomerase from S. cerevisiae and E. coli but it inhibited Triosephosphate Isomerase from chicken and S. pornhe. From an analysis of the Cys content of the various Triosephosphate Isomerases, it was evident that amongst the ones studied only those that have a Cys in position 217 (or in an equivalent position) were sensitive to MePhSOz-SPh. Methyl metanethiosulfonate (MeS0,-SMe), which produces methyl disulfides, had no effect on Triosephosphate Isomerases that lack Cys217 (S. cerevisiae and E. coli). In Triosephosphate Isomerases that have Cys217, MeS0,-SMe inhibited by 40-50% the activity of that from S. pornbe, 2025% that from rabbit but had no effect on the chicken enzyme. In the three latter Triosephosphate Isomerases, MeS0,-SMe protected against the strong inhibiting action of MePhS0,-SPh. The latter observations suggest that MeS0,-SMe and MePhS0,-SPh derivatize the same Cys and that significant inhibition of activity requires perturbation by the relatively large phenyl group. The intrinsic fluorescence of rabbit Triosephosphate Isomerase that had been derivatized to a phenyl disulfide was almost identical to that of the native enzyme. Thus, modification of Cys217 did not produce gross structural alterations, albeit it brought about important kinetic alterations, i.e. a nearly fivefold increase in the K,, for glyceraldehyde 3phosphate and a 65% decrease in V,,,,,. The effect of derivatizating Cys217 differs markedly from that produced by derivatization of Cysl4 (another non-conserved cysteine). The differences may be explained from their position in the three-dimensional structure of the enzyme.
-
Differential inactivation of rabbit and yeast Triosephosphate Isomerase: effect of oxidations produced by chloramine-T.
Archives of biochemistry and biophysics, 1994Co-Authors: Rafael A. Zubillaga, Ruy Pérez-montfort, Armando Gómez-puyouAbstract:Triosephosphate Isomerase from rabbit has 5 Cys and 2 Met, while Triosephosphate Isomerase from yeast has 2 Cys (present in the rabbit enzyme in equivalent positions) and no Met. Since chloramine-T oxidizes Cys and Met, we determined the effect it has on the activity and structure of both enzymes. The activity of Triosephosphate Isomerase from rabbit was more sensitive to chloramine-T than that of the yeast enzyme (under conditions where the rabbit Isomerase was completely inactive, the yeast enzyme exhibited approximately 50% activity). An initial effect of chloramine-T on Triosephosphate Isomerase was the oxidation of Cys and the formation of catalytically active acidic isoforms. For the yeast Isomerase, the two processes were slower. Our data suggest that oxidation of Cys 126, which is conserved in all of the studied species, does not abolish catalysis. Chloramine-T also oxidized the two Met of the rabbit enzyme. At ratios of 50 chloramine-T/monomer, circular dichroism studies showed that the rabbit enzyme, but not that from yeast, underwent extensive alterations of tertiary and secondary structures. This was accompanied by formation of stable dimers, whose cross-linking was not through disulfide bonds. Studies of dimer formation at various enzyme concentrations showed that cross-linking was between monomers of the same dimer. Under conditions that led to cross-linking, rabbit Triosephosphate Isomerase took up 2.7 mol of 3H from NaB3H4/mol dimer, and the yeast enzyme incorporated only 0.4 mol of 3H. Thus cross-linking was most likely via a Schiff base. The results revealed the points whose modification caused inactivation of the rabbit enzyme.
Armando Gómez-puyou - One of the best experts on this subject based on the ideXlab platform.
-
Sulfhydryl reagent susceptibility in proteins with high sequence similarity--Triosephosphate Isomerase from Trypanosoma brucei, Trypanosoma cruzi and Leishmania mexicana.
European journal of biochemistry, 1998Co-Authors: Georgina Garza-ramos, Pedro Ostoa-saloma, Marietta Tuena De Gómez-puyou, Ruy Pérez-montfort, Nallely Cabrera, Emma Saavedra-lira, Armando Gómez-puyouAbstract:The amino acid sequence of Triosephosphate Isomerase from Trypanosoma brucei, Trypanosoma cruzi, and Leishmania mexicana have an identity of 68 %. Using the numbering system for the T. brucei enzyme, in their aligned sequences, the T. cruzi and leishmanial enzymes have cysteine residues at positions 14, 40, 117 and 126. T. brucei Triosephosphate Isomerase has cysteine residues at positions 14, 40 and 126, and a valine residue at position 117. Dithionitrobenzoic acid and methylmethane thiosulfonate inhibited the three enzymes, but T. cruzi Triosephosphate Isomerase was more than 100-fold more sensitive. The sensitivity of wild type Triosephosphate Isomerase from T. cruzi and T. brucei to the reagents was equal to that of the Cys117Val and Val117Cys mutant enzymes, respectively. Triosephosphate Isomerases that have cysteine residues at positions 40 and 126, but lack a cysteine residue at position 14 are insensitive to methylmethane thiosulfonate. Thus, sulfhydryl reagents act on Cys14. At stoichiometric concentrations, the reagents inhibited the three enzymes as a consequence of structural alterations as measured by binding of 8-anilino-1-napthalenesulfonic acid to previously buried hydrophobic regions. However, the times for half-maximal alterations were 10 min, 15 hours and over 30 hours for T. cruzi, T. brucei and L. mexicana Triosephosphate Isomerase, respectively. The effect of pH on the action of the sulfhydryl reagents and molecular modeling showed no differences in the solvent accesibility of Cys14. As Cys14 forms part of the dimer interface, the data indicate that, in the three enzymes, barriers of different magnitude hinder the interaction between the sulfhydryl reagents and Cys14. The barrier is lower in T. cruzi Triosephosphate Isomerase which makes its dimer interface more susceptible for perturbation.
-
Cloning, Expression, Purification and Characterization Of Triosephosphate Isomerase from Trypanosoma Cruzi
European journal of biochemistry, 1997Co-Authors: Pedro Ostoa-saloma, Georgina Garza-ramos, Jorge Ramírez, Ingeborg Becker, Myriam Berzunza, Abraham Landa, Armando Gómez-puyou, Marietta Tuena De Gómez-puyou, Ruy Pérez-montfortAbstract:The gene that encodes for Triosephosphate Isomerase from Trypanosoma cruzi was cloned and sequenced. In T. cruzi, there is only one gene for Triosephosphate Isomerase. The enzyme has an identity of 72% and 68% with Triosephosphate Isomerase from Trypanosoma brucei and Leishmania mexicana, respectively. The active site residues are conserved: out of the 32 residues that conform the interface of dimeric Triosephosphate Isomerase from T. brucei, 29 are conserved in the T. cruzi enzyme. The enzyme was expressed in Escherichia coli and purified to homogeneity. Data from electrophoretic analysis under denaturing techniques and filtration techniques showed that Triosephosphate Isomerase from T. cruzi is a homodimer. Some of its structural and kinetic features were determined and compared to those of the purified enzymes from T. brucei and L mexicana. Its circular dichroism spectrum was almost identical to that of Triosephosphate Isomerase from T. brucei. Its kinetic properties and pH optima were similar to those of T. brucei and L. mexicana, although the latter exhibited a higher Vmax with glyceraldehyde 3-phosphate as substrate. The sensitivity of the three enzymes to the sulfhydryl reagent methylmethane thiosulfonate (MeSO2-SMe) was determined; the sensitivity of the T. cruzi enzyme was about 40 times and 200 times higher than that of the enzymes from T. brucei and L. mexicana, respectively. Triosephosphate Isomerase from T. cruzi and L. mexicana have the three cysteine residues that exist in the T. brucei enzyme (positions 14, 39, 126, using the numbering of the T. brucei enzyme); however, they also have an additional residue (position 117). These data suggest that regardless of the high identity of the three trypanosomatid enzymes, there are structural differences in the disposition of their cysteine residues that account for their different sensitivity to the sulfhydryl reagent. The disposition of the cysteine in Triosephosphate Isomerase from T. cruzi appears to make it unique for inhibition by modification of its cysteine.
-
Species-specific inhibition of homologous enzymes by modification of nonconserved amino acids residues The cysteine residues of Triosephosphate Isomerase
European journal of biochemistry, 1996Co-Authors: Georgina Garza-ramos, Marietta Tuena De Gómez-puyou, Ruy Pérez-montfort, Arturo Rojo-domínguez, Armando Gómez-puyouAbstract:The possibility of using non-conserved amino acid residues to produce selective inhibition of homologous enzymes from different species has been further explored with Triosephosphate Isomerase. S-phenylp-toluenethiosulfonate (MePhS0,-SPh), which produces phenyl disulfides with accessible Cys residues, inhibits the activity of rabbit Triosephosphate Isomerase. The inhibition is due to derivatization of one of the five Cys residues of rabbit Triosephosphate Isomerase. The effect of MePhS0,-SPh on Triosephosphate Isomerase from Saccharomyces cerevisiae, Escherichia coli, chicken and Schizosaccharomyces pombe was also determined. MePhS0,-SPh did not affect the activity of Triosephosphate Isomerase from S. cerevisiae and E. coli but it inhibited Triosephosphate Isomerase from chicken and S. pornhe. From an analysis of the Cys content of the various Triosephosphate Isomerases, it was evident that amongst the ones studied only those that have a Cys in position 217 (or in an equivalent position) were sensitive to MePhSOz-SPh. Methyl metanethiosulfonate (MeS0,-SMe), which produces methyl disulfides, had no effect on Triosephosphate Isomerases that lack Cys217 (S. cerevisiae and E. coli). In Triosephosphate Isomerases that have Cys217, MeS0,-SMe inhibited by 40-50% the activity of that from S. pornbe, 2025% that from rabbit but had no effect on the chicken enzyme. In the three latter Triosephosphate Isomerases, MeS0,-SMe protected against the strong inhibiting action of MePhS0,-SPh. The latter observations suggest that MeS0,-SMe and MePhS0,-SPh derivatize the same Cys and that significant inhibition of activity requires perturbation by the relatively large phenyl group. The intrinsic fluorescence of rabbit Triosephosphate Isomerase that had been derivatized to a phenyl disulfide was almost identical to that of the native enzyme. Thus, modification of Cys217 did not produce gross structural alterations, albeit it brought about important kinetic alterations, i.e. a nearly fivefold increase in the K,, for glyceraldehyde 3phosphate and a 65% decrease in V,,,,,. The effect of derivatizating Cys217 differs markedly from that produced by derivatization of Cysl4 (another non-conserved cysteine). The differences may be explained from their position in the three-dimensional structure of the enzyme.
-
Differential inactivation of rabbit and yeast Triosephosphate Isomerase: effect of oxidations produced by chloramine-T.
Archives of biochemistry and biophysics, 1994Co-Authors: Rafael A. Zubillaga, Ruy Pérez-montfort, Armando Gómez-puyouAbstract:Triosephosphate Isomerase from rabbit has 5 Cys and 2 Met, while Triosephosphate Isomerase from yeast has 2 Cys (present in the rabbit enzyme in equivalent positions) and no Met. Since chloramine-T oxidizes Cys and Met, we determined the effect it has on the activity and structure of both enzymes. The activity of Triosephosphate Isomerase from rabbit was more sensitive to chloramine-T than that of the yeast enzyme (under conditions where the rabbit Isomerase was completely inactive, the yeast enzyme exhibited approximately 50% activity). An initial effect of chloramine-T on Triosephosphate Isomerase was the oxidation of Cys and the formation of catalytically active acidic isoforms. For the yeast Isomerase, the two processes were slower. Our data suggest that oxidation of Cys 126, which is conserved in all of the studied species, does not abolish catalysis. Chloramine-T also oxidized the two Met of the rabbit enzyme. At ratios of 50 chloramine-T/monomer, circular dichroism studies showed that the rabbit enzyme, but not that from yeast, underwent extensive alterations of tertiary and secondary structures. This was accompanied by formation of stable dimers, whose cross-linking was not through disulfide bonds. Studies of dimer formation at various enzyme concentrations showed that cross-linking was between monomers of the same dimer. Under conditions that led to cross-linking, rabbit Triosephosphate Isomerase took up 2.7 mol of 3H from NaB3H4/mol dimer, and the yeast enzyme incorporated only 0.4 mol of 3H. Thus cross-linking was most likely via a Schiff base. The results revealed the points whose modification caused inactivation of the rabbit enzyme.
Zhuchu Chen - One of the best experts on this subject based on the ideXlab platform.
-
Triosephosphate Isomerase and peroxiredoxin 6 two novel serum markers for human lung squamous cell carcinoma
Cancer Science, 2009Co-Authors: Xiuzhi Zhang, Zhefeng Xiao, Danjuan Li, Maoyu Li, Zhiqiang Xiao, Cui Li, Feng Li, Fang Yang, Zhuchu ChenAbstract:There is currently substantial interest in the identification of human tumor antigens for the diagnosis and immunotherapy of cancer. In our previous study, secretion character and up-regulation of Triosephosphate Isomerase were observed in lung squamous cell carcinoma, and autoantibodies against Triosephosphate Isomerase and peroxiredoxin 6 were detected in the sera from over 25% of patients, but in none of the healthy controls. In this study, peroxiredoxin 6 was also found at higher levels in the sera of the patients. Up-regulated Triosephosphate Isomerase and peroxiredoxin 6 were further validated by enzyme-linked immunosorbent assay in an additional 61 lung squamous cell carcinoma patients, 23 lung adenocarcinoma patients, 56 other types of carcinoma patients, 12 benign lung disease patients, and 59 healthy controls. We found that both Triosephosphate Isomerase and peroxiredoxin 6 were specifically elevated in lung squamous cell carcinoma sera compared with other groups, with the exception of peroxiredoxin 6 in lung adenocarcinoma patients. Positive correlation between Triosephosphate Isomerase and distant metastasis was found. At the cut-off point 0.221 (optical density value) on the receiver operating characteristic curve, Triosephosphate Isomerase could comparatively discriminate lung squamous cell carcinoma from healthy controls with a sensitivity of 65.6%, specificity 84.7%, and total accuracy 75%. For peroxiredoxin 6, at the cut-off point 0.151, it could discriminate the two groups with a sensitivity of 70.5%, specificity 62.7%, and total accuracy 65.8%. With both Triosephosphate Isomerase and peroxiredoxin 6, discriminant analysis results showed that 68.9% of the lung squamous cell carcinoma and 83.1% of healthy controls were correctly classified. We concluded that Triosephosphate Isomerase and peroxiredoxin 6 could be markers for lung squamous cell carcinoma. (Cancer Sci 2009; 100: 2396–2401)
-
Triosephosphate Isomerase and peroxiredoxin 6, two novel serum markers for human lung squamous cell carcinoma.
Cancer science, 2009Co-Authors: Xiuzhi Zhang, Zhefeng Xiao, Zhiqiang Xiao, Fang Yang, Zhuchu ChenAbstract:There is currently substantial interest in the identification of human tumor antigens for the diagnosis and immunotherapy of cancer. In our previous study, secretion character and up-regulation of Triosephosphate Isomerase were observed in lung squamous cell carcinoma, and autoantibodies against Triosephosphate Isomerase and peroxiredoxin 6 were detected in the sera from over 25% of patients, but in none of the healthy controls. In this study, peroxiredoxin 6 was also found at higher levels in the sera of the patients. Up-regulated Triosephosphate Isomerase and peroxiredoxin 6 were further validated by enzyme-linked immunosorbent assay in an additional 61 lung squamous cell carcinoma patients, 23 lung adenocarcinoma patients, 56 other types of carcinoma patients, 12 benign lung disease patients, and 59 healthy controls. We found that both Triosephosphate Isomerase and peroxiredoxin 6 were specifically elevated in lung squamous cell carcinoma sera compared with other groups, with the exception of peroxiredoxin 6 in lung adenocarcinoma patients. Positive correlation between Triosephosphate Isomerase and distant metastasis was found. At the cut-off point 0.221 (optical density value) on the receiver operating characteristic curve, Triosephosphate Isomerase could comparatively discriminate lung squamous cell carcinoma from healthy controls with a sensitivity of 65.6%, specificity 84.7%, and total accuracy 75%. For peroxiredoxin 6, at the cut-off point 0.151, it could discriminate the two groups with a sensitivity of 70.5%, specificity 62.7%, and total accuracy 65.8%. With both Triosephosphate Isomerase and peroxiredoxin 6, discriminant analysis results showed that 68.9% of the lung squamous cell carcinoma and 83.1% of healthy controls were correctly classified. We concluded that Triosephosphate Isomerase and peroxiredoxin 6 could be markers for lung squamous cell carcinoma.
Georgina Garza-ramos - One of the best experts on this subject based on the ideXlab platform.
-
Sulfhydryl reagent susceptibility in proteins with high sequence similarity--Triosephosphate Isomerase from Trypanosoma brucei, Trypanosoma cruzi and Leishmania mexicana.
European journal of biochemistry, 1998Co-Authors: Georgina Garza-ramos, Pedro Ostoa-saloma, Marietta Tuena De Gómez-puyou, Ruy Pérez-montfort, Nallely Cabrera, Emma Saavedra-lira, Armando Gómez-puyouAbstract:The amino acid sequence of Triosephosphate Isomerase from Trypanosoma brucei, Trypanosoma cruzi, and Leishmania mexicana have an identity of 68 %. Using the numbering system for the T. brucei enzyme, in their aligned sequences, the T. cruzi and leishmanial enzymes have cysteine residues at positions 14, 40, 117 and 126. T. brucei Triosephosphate Isomerase has cysteine residues at positions 14, 40 and 126, and a valine residue at position 117. Dithionitrobenzoic acid and methylmethane thiosulfonate inhibited the three enzymes, but T. cruzi Triosephosphate Isomerase was more than 100-fold more sensitive. The sensitivity of wild type Triosephosphate Isomerase from T. cruzi and T. brucei to the reagents was equal to that of the Cys117Val and Val117Cys mutant enzymes, respectively. Triosephosphate Isomerases that have cysteine residues at positions 40 and 126, but lack a cysteine residue at position 14 are insensitive to methylmethane thiosulfonate. Thus, sulfhydryl reagents act on Cys14. At stoichiometric concentrations, the reagents inhibited the three enzymes as a consequence of structural alterations as measured by binding of 8-anilino-1-napthalenesulfonic acid to previously buried hydrophobic regions. However, the times for half-maximal alterations were 10 min, 15 hours and over 30 hours for T. cruzi, T. brucei and L. mexicana Triosephosphate Isomerase, respectively. The effect of pH on the action of the sulfhydryl reagents and molecular modeling showed no differences in the solvent accesibility of Cys14. As Cys14 forms part of the dimer interface, the data indicate that, in the three enzymes, barriers of different magnitude hinder the interaction between the sulfhydryl reagents and Cys14. The barrier is lower in T. cruzi Triosephosphate Isomerase which makes its dimer interface more susceptible for perturbation.
-
Cloning, Expression, Purification and Characterization Of Triosephosphate Isomerase from Trypanosoma Cruzi
European journal of biochemistry, 1997Co-Authors: Pedro Ostoa-saloma, Georgina Garza-ramos, Jorge Ramírez, Ingeborg Becker, Myriam Berzunza, Abraham Landa, Armando Gómez-puyou, Marietta Tuena De Gómez-puyou, Ruy Pérez-montfortAbstract:The gene that encodes for Triosephosphate Isomerase from Trypanosoma cruzi was cloned and sequenced. In T. cruzi, there is only one gene for Triosephosphate Isomerase. The enzyme has an identity of 72% and 68% with Triosephosphate Isomerase from Trypanosoma brucei and Leishmania mexicana, respectively. The active site residues are conserved: out of the 32 residues that conform the interface of dimeric Triosephosphate Isomerase from T. brucei, 29 are conserved in the T. cruzi enzyme. The enzyme was expressed in Escherichia coli and purified to homogeneity. Data from electrophoretic analysis under denaturing techniques and filtration techniques showed that Triosephosphate Isomerase from T. cruzi is a homodimer. Some of its structural and kinetic features were determined and compared to those of the purified enzymes from T. brucei and L mexicana. Its circular dichroism spectrum was almost identical to that of Triosephosphate Isomerase from T. brucei. Its kinetic properties and pH optima were similar to those of T. brucei and L. mexicana, although the latter exhibited a higher Vmax with glyceraldehyde 3-phosphate as substrate. The sensitivity of the three enzymes to the sulfhydryl reagent methylmethane thiosulfonate (MeSO2-SMe) was determined; the sensitivity of the T. cruzi enzyme was about 40 times and 200 times higher than that of the enzymes from T. brucei and L. mexicana, respectively. Triosephosphate Isomerase from T. cruzi and L. mexicana have the three cysteine residues that exist in the T. brucei enzyme (positions 14, 39, 126, using the numbering of the T. brucei enzyme); however, they also have an additional residue (position 117). These data suggest that regardless of the high identity of the three trypanosomatid enzymes, there are structural differences in the disposition of their cysteine residues that account for their different sensitivity to the sulfhydryl reagent. The disposition of the cysteine in Triosephosphate Isomerase from T. cruzi appears to make it unique for inhibition by modification of its cysteine.
-
Species-specific inhibition of homologous enzymes by modification of nonconserved amino acids residues The cysteine residues of Triosephosphate Isomerase
European journal of biochemistry, 1996Co-Authors: Georgina Garza-ramos, Marietta Tuena De Gómez-puyou, Ruy Pérez-montfort, Arturo Rojo-domínguez, Armando Gómez-puyouAbstract:The possibility of using non-conserved amino acid residues to produce selective inhibition of homologous enzymes from different species has been further explored with Triosephosphate Isomerase. S-phenylp-toluenethiosulfonate (MePhS0,-SPh), which produces phenyl disulfides with accessible Cys residues, inhibits the activity of rabbit Triosephosphate Isomerase. The inhibition is due to derivatization of one of the five Cys residues of rabbit Triosephosphate Isomerase. The effect of MePhS0,-SPh on Triosephosphate Isomerase from Saccharomyces cerevisiae, Escherichia coli, chicken and Schizosaccharomyces pombe was also determined. MePhS0,-SPh did not affect the activity of Triosephosphate Isomerase from S. cerevisiae and E. coli but it inhibited Triosephosphate Isomerase from chicken and S. pornhe. From an analysis of the Cys content of the various Triosephosphate Isomerases, it was evident that amongst the ones studied only those that have a Cys in position 217 (or in an equivalent position) were sensitive to MePhSOz-SPh. Methyl metanethiosulfonate (MeS0,-SMe), which produces methyl disulfides, had no effect on Triosephosphate Isomerases that lack Cys217 (S. cerevisiae and E. coli). In Triosephosphate Isomerases that have Cys217, MeS0,-SMe inhibited by 40-50% the activity of that from S. pornbe, 2025% that from rabbit but had no effect on the chicken enzyme. In the three latter Triosephosphate Isomerases, MeS0,-SMe protected against the strong inhibiting action of MePhS0,-SPh. The latter observations suggest that MeS0,-SMe and MePhS0,-SPh derivatize the same Cys and that significant inhibition of activity requires perturbation by the relatively large phenyl group. The intrinsic fluorescence of rabbit Triosephosphate Isomerase that had been derivatized to a phenyl disulfide was almost identical to that of the native enzyme. Thus, modification of Cys217 did not produce gross structural alterations, albeit it brought about important kinetic alterations, i.e. a nearly fivefold increase in the K,, for glyceraldehyde 3phosphate and a 65% decrease in V,,,,,. The effect of derivatizating Cys217 differs markedly from that produced by derivatization of Cysl4 (another non-conserved cysteine). The differences may be explained from their position in the three-dimensional structure of the enzyme.
Xiuzhi Zhang - One of the best experts on this subject based on the ideXlab platform.
-
Triosephosphate Isomerase and peroxiredoxin 6 two novel serum markers for human lung squamous cell carcinoma
Cancer Science, 2009Co-Authors: Xiuzhi Zhang, Zhefeng Xiao, Danjuan Li, Maoyu Li, Zhiqiang Xiao, Cui Li, Feng Li, Fang Yang, Zhuchu ChenAbstract:There is currently substantial interest in the identification of human tumor antigens for the diagnosis and immunotherapy of cancer. In our previous study, secretion character and up-regulation of Triosephosphate Isomerase were observed in lung squamous cell carcinoma, and autoantibodies against Triosephosphate Isomerase and peroxiredoxin 6 were detected in the sera from over 25% of patients, but in none of the healthy controls. In this study, peroxiredoxin 6 was also found at higher levels in the sera of the patients. Up-regulated Triosephosphate Isomerase and peroxiredoxin 6 were further validated by enzyme-linked immunosorbent assay in an additional 61 lung squamous cell carcinoma patients, 23 lung adenocarcinoma patients, 56 other types of carcinoma patients, 12 benign lung disease patients, and 59 healthy controls. We found that both Triosephosphate Isomerase and peroxiredoxin 6 were specifically elevated in lung squamous cell carcinoma sera compared with other groups, with the exception of peroxiredoxin 6 in lung adenocarcinoma patients. Positive correlation between Triosephosphate Isomerase and distant metastasis was found. At the cut-off point 0.221 (optical density value) on the receiver operating characteristic curve, Triosephosphate Isomerase could comparatively discriminate lung squamous cell carcinoma from healthy controls with a sensitivity of 65.6%, specificity 84.7%, and total accuracy 75%. For peroxiredoxin 6, at the cut-off point 0.151, it could discriminate the two groups with a sensitivity of 70.5%, specificity 62.7%, and total accuracy 65.8%. With both Triosephosphate Isomerase and peroxiredoxin 6, discriminant analysis results showed that 68.9% of the lung squamous cell carcinoma and 83.1% of healthy controls were correctly classified. We concluded that Triosephosphate Isomerase and peroxiredoxin 6 could be markers for lung squamous cell carcinoma. (Cancer Sci 2009; 100: 2396–2401)
-
Triosephosphate Isomerase and peroxiredoxin 6, two novel serum markers for human lung squamous cell carcinoma.
Cancer science, 2009Co-Authors: Xiuzhi Zhang, Zhefeng Xiao, Zhiqiang Xiao, Fang Yang, Zhuchu ChenAbstract:There is currently substantial interest in the identification of human tumor antigens for the diagnosis and immunotherapy of cancer. In our previous study, secretion character and up-regulation of Triosephosphate Isomerase were observed in lung squamous cell carcinoma, and autoantibodies against Triosephosphate Isomerase and peroxiredoxin 6 were detected in the sera from over 25% of patients, but in none of the healthy controls. In this study, peroxiredoxin 6 was also found at higher levels in the sera of the patients. Up-regulated Triosephosphate Isomerase and peroxiredoxin 6 were further validated by enzyme-linked immunosorbent assay in an additional 61 lung squamous cell carcinoma patients, 23 lung adenocarcinoma patients, 56 other types of carcinoma patients, 12 benign lung disease patients, and 59 healthy controls. We found that both Triosephosphate Isomerase and peroxiredoxin 6 were specifically elevated in lung squamous cell carcinoma sera compared with other groups, with the exception of peroxiredoxin 6 in lung adenocarcinoma patients. Positive correlation between Triosephosphate Isomerase and distant metastasis was found. At the cut-off point 0.221 (optical density value) on the receiver operating characteristic curve, Triosephosphate Isomerase could comparatively discriminate lung squamous cell carcinoma from healthy controls with a sensitivity of 65.6%, specificity 84.7%, and total accuracy 75%. For peroxiredoxin 6, at the cut-off point 0.151, it could discriminate the two groups with a sensitivity of 70.5%, specificity 62.7%, and total accuracy 65.8%. With both Triosephosphate Isomerase and peroxiredoxin 6, discriminant analysis results showed that 68.9% of the lung squamous cell carcinoma and 83.1% of healthy controls were correctly classified. We concluded that Triosephosphate Isomerase and peroxiredoxin 6 could be markers for lung squamous cell carcinoma.
