The Experts below are selected from a list of 141 Experts worldwide ranked by ideXlab platform
Lee Mcalister-henn - One of the best experts on this subject based on the ideXlab platform.
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Expression and gene disruption analysis of the Isocitrate Dehydrogenase family in yeast.
Biochemistry, 1996Co-Authors: Wen-ning Zhao, Lee Mcalister-hennAbstract:Mammalian and yeast cells contain three isozymes of Isocitrate Dehydrogenase: mitochondrial NAD- and NADP-specific enzymes and a cytosolic NADP-specific enzyme. Independent metabolic functions of ...
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Expression of pig heart mitochondrial NADP-dependent Isocitrate Dehydrogenase in Escherichia coli.
Protein expression and purification, 1996Co-Authors: Sambanthamurthy Soundar, Gary T. Jennings, Lee Mcalister-henn, Roberta F. ColmanAbstract:Abstract Pig heart mitochondrial NADP-specific Isocitrate Dehydrogenase is the most extensively studied among the mammalian Isocitrate Dehydrogenases. The 1.2-kbp cDNA encoding this porcine mitochondrial NADP-specific enzyme has now been inserted into an expression vector, pMAL-c2, to be expressed as a fusion protein with maltose binding protein. Initially, the vector was constructed with a cleavage site for protease Factor Xabetween the maltose binding protein and Isocitrate Dehydrogenase; however, since Factor Xawas also found to digest Isocitrate Dehydrogenase, a thrombin recognition site was substituted. The fusion protein was expressed inEscherichia coliby IPTG induction at 25°C, and was separated from the endogenousE. coliIsocitrate Dehydrogenase by affinity chromatography on an amylose resin which adsorbs maltose binding protein and its fusion products. Cleavage of the fusion protein with thrombin generated pig heart NADP-specific Isocitrate Dehydrogenase, which was purified to homogeneity by affinity chromatography on Matrex Gel Red-A resin and gel filtration by FPLC. A 41-fold increase in specific activity to 37 enzyme units/mg with an approximate yield of 34% for the expressed enzyme was achieved by this purification procedure. This enzyme exhibits a single band (Mr= 46,600) on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and, under standard assay conditions, has aKmfor DL -Isocitrate of 7.74 ± 0.18 μ M and aKmfor NADP+of 6.63 ± 1.34 μ M . These values are similar to theKms measured for the enzyme purified from pig heart. The amino-terminal sequence of the expressed enzyme is identical with that of authentic porcine enzyme and distinguishable from theE. colienzyme at 17 of the 18 residues determined. We conclude that this expression and purification system yields pure pig heart mitochondrial NADP-specific Isocitrate Dehydrogenase and should allow generation of wild-type and mutant enzymes in amounts suitable for their biochemical characterization and comparison.
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N-Ethylmaleimide Profiling of Yeast NADP-Dependent Isocitrate Dehydrogenase
Archives of biochemistry and biophysics, 1995Co-Authors: Yu-chu Huang, Lee Mcalister-henn, R. J. Haselbeck, Roberta F. ColmanAbstract:Abstract Yeast NADP-dependent Isocitrate Dehydrogenase is inactivated by N -ethyl-maleimide (NEM) at pH 7.7 and 30°C. Reaction with cysteine 382 occurs most rapidly and is accompanied by loss of about 50% of the enzymatic activity. A slower phase of inactivation ensues during which lysine 343 is the major target of NEM, while minor products result from reaction at cysteine 73 and cysteine 354 . Protection against the second phase of inactivation is provided by NADP, NADPH, or manganous-Isocitrate. Comparison of the time-dependence of inactivation and the products of reaction with N -ethyl-maleimide (NEM profiling) of the pig heart (G. E. Smyth and R. F. Colman, 1991, J. Biol. Chem. 266, 14918-14925) and yeast NADP-specific Isocitrate Dehydrogenases have been coupled with an examination of the crystal structure of the Escherichia coli Isocitrate Dehydrogenase. The following conclusions have been reached: while no cysteine is essential for activity, yeast Cys 382 /pig Cys 379 is close to the adenine portion of the NADP binding site, and pig Cys 269 is located in the region of the metal-Isocitrate binding site.
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Isolation and sequence of a cDNA encoding porcine mitochondrial NADP-specific Isocitrate Dehydrogenase
Biochemistry, 1992Co-Authors: Robert J. Haselbeck, Roberta F. Colman, Lee Mcalister-hennAbstract:The cDNA for porcine mitochondrial NADP-specific Isocitrate Dehydrogenase was isolated from a lambda gt11 library using polymerase chain reaction. Translation of the DNA sequence gave a 413-residue amino acid sequence and a calculated molecular weight of 46,600 for the mature polypeptide. Previously determined peptide sequences for the amino terminus and for internal tryptic peptides were all contained within the translated sequence. The porcine protein was found to share 63% residue identity with yeast mitochondrial NADP-specific Isocitrate Dehydrogenase and to be immunoreactive with an antiserum against the yeast protein. Highly conserved regions include residues which have been implicated in substrate and cofactor binding in previous studies of the porcine enzyme. The two eucaryotic enzymes exhibit only minimal homology with the NADP-dependent Isocitrate Dehydrogenase from Escherichia coli, with the exception of a striking conservation of residues implicated in formation of the metal-Isocitrate site of the procaryotic enzyme.
Roberta F. Colman - One of the best experts on this subject based on the ideXlab platform.
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Expression of pig heart mitochondrial NADP-dependent Isocitrate Dehydrogenase in Escherichia coli.
Protein expression and purification, 1996Co-Authors: Sambanthamurthy Soundar, Gary T. Jennings, Lee Mcalister-henn, Roberta F. ColmanAbstract:Abstract Pig heart mitochondrial NADP-specific Isocitrate Dehydrogenase is the most extensively studied among the mammalian Isocitrate Dehydrogenases. The 1.2-kbp cDNA encoding this porcine mitochondrial NADP-specific enzyme has now been inserted into an expression vector, pMAL-c2, to be expressed as a fusion protein with maltose binding protein. Initially, the vector was constructed with a cleavage site for protease Factor Xabetween the maltose binding protein and Isocitrate Dehydrogenase; however, since Factor Xawas also found to digest Isocitrate Dehydrogenase, a thrombin recognition site was substituted. The fusion protein was expressed inEscherichia coliby IPTG induction at 25°C, and was separated from the endogenousE. coliIsocitrate Dehydrogenase by affinity chromatography on an amylose resin which adsorbs maltose binding protein and its fusion products. Cleavage of the fusion protein with thrombin generated pig heart NADP-specific Isocitrate Dehydrogenase, which was purified to homogeneity by affinity chromatography on Matrex Gel Red-A resin and gel filtration by FPLC. A 41-fold increase in specific activity to 37 enzyme units/mg with an approximate yield of 34% for the expressed enzyme was achieved by this purification procedure. This enzyme exhibits a single band (Mr= 46,600) on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and, under standard assay conditions, has aKmfor DL -Isocitrate of 7.74 ± 0.18 μ M and aKmfor NADP+of 6.63 ± 1.34 μ M . These values are similar to theKms measured for the enzyme purified from pig heart. The amino-terminal sequence of the expressed enzyme is identical with that of authentic porcine enzyme and distinguishable from theE. colienzyme at 17 of the 18 residues determined. We conclude that this expression and purification system yields pure pig heart mitochondrial NADP-specific Isocitrate Dehydrogenase and should allow generation of wild-type and mutant enzymes in amounts suitable for their biochemical characterization and comparison.
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N-Ethylmaleimide Profiling of Yeast NADP-Dependent Isocitrate Dehydrogenase
Archives of biochemistry and biophysics, 1995Co-Authors: Yu-chu Huang, Lee Mcalister-henn, R. J. Haselbeck, Roberta F. ColmanAbstract:Abstract Yeast NADP-dependent Isocitrate Dehydrogenase is inactivated by N -ethyl-maleimide (NEM) at pH 7.7 and 30°C. Reaction with cysteine 382 occurs most rapidly and is accompanied by loss of about 50% of the enzymatic activity. A slower phase of inactivation ensues during which lysine 343 is the major target of NEM, while minor products result from reaction at cysteine 73 and cysteine 354 . Protection against the second phase of inactivation is provided by NADP, NADPH, or manganous-Isocitrate. Comparison of the time-dependence of inactivation and the products of reaction with N -ethyl-maleimide (NEM profiling) of the pig heart (G. E. Smyth and R. F. Colman, 1991, J. Biol. Chem. 266, 14918-14925) and yeast NADP-specific Isocitrate Dehydrogenases have been coupled with an examination of the crystal structure of the Escherichia coli Isocitrate Dehydrogenase. The following conclusions have been reached: while no cysteine is essential for activity, yeast Cys 382 /pig Cys 379 is close to the adenine portion of the NADP binding site, and pig Cys 269 is located in the region of the metal-Isocitrate binding site.
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Isolation and sequence of a cDNA encoding porcine mitochondrial NADP-specific Isocitrate Dehydrogenase
Biochemistry, 1992Co-Authors: Robert J. Haselbeck, Roberta F. Colman, Lee Mcalister-hennAbstract:The cDNA for porcine mitochondrial NADP-specific Isocitrate Dehydrogenase was isolated from a lambda gt11 library using polymerase chain reaction. Translation of the DNA sequence gave a 413-residue amino acid sequence and a calculated molecular weight of 46,600 for the mature polypeptide. Previously determined peptide sequences for the amino terminus and for internal tryptic peptides were all contained within the translated sequence. The porcine protein was found to share 63% residue identity with yeast mitochondrial NADP-specific Isocitrate Dehydrogenase and to be immunoreactive with an antiserum against the yeast protein. Highly conserved regions include residues which have been implicated in substrate and cofactor binding in previous studies of the porcine enzyme. The two eucaryotic enzymes exhibit only minimal homology with the NADP-dependent Isocitrate Dehydrogenase from Escherichia coli, with the exception of a striking conservation of residues implicated in formation of the metal-Isocitrate site of the procaryotic enzyme.
Keiko Murakami - One of the best experts on this subject based on the ideXlab platform.
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Role of phosphoenolpyruvate in the NADP-Isocitrate Dehydrogenase and Isocitrate lyase reaction in Escherichia coli.
Journal of bacteriology, 2006Co-Authors: Tadashi Ogawa, Keiko Murakami, Hirotada Mori, Nobuyoshi Ishii, Masaru Tomita, Masataka YoshinAbstract:Phosphoenolpyruvate inhibited Escherichia coli NADP-Isocitrate Dehydrogenase allosterically (Ki of 0.31 mM) and Isocitrate lyase uncompetitively (Ki′ of 0.893 mM). Phosphoenolpyruvate enhances the uncompetitive inhibition of Isocitrate lyase by increasing Isocitrate, which protects Isocitrate Dehydrogenase from the inhibition, and contributes to the control through the tricarboxylic acid cycle and glyoxylate shunt.
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Protective effect of NADP-Isocitrate Dehydrogenase on the paraquat-mediated oxidative inactivation of aconitase in heart mitochondria.
Environmental toxicology and pharmacology, 2006Co-Authors: Keiko Murakami, Miyako Haneda, T. Makino, Masataka YoshinoAbstract:Protective role of NADP-Isocitrate Dehydrogenase in the oxidative inactivation of mitochondrial enzymes was analyzed. Administration of paraquat to the rat inactivated liver mitochondrial enzymes: the aconitase activity decreased to one quarter, and citrate synthase and fumarase to half, whereas cytosolic enzymes were not affected. Activities of heart mitochondrial and cytosolic enzymes were not at all changed in the rat treated with paraquat, but paraquat directly inactivated aconitase in the heart mitochondria isolated from the non-treated rats. The paraquat-dependent inactivation of aconitase was prevented by activating NADP-Isocitrate Dehydrogenase in the presence of oxidized glutathione. NADP-Isocitrate Dehydrogenase could regenerate glutathione in isolated heart mitochondria, indicating that paraquat-mediated inactivation depends on the glutathione-regenerating activity by enhanced NADPH supply. Lower NADP-Isocitrate Dehydrogenase activity in liver mitochondria cannot regenerate reduced glutathione for scavenging reactive oxygen species, resulting in the paraquat-induced oxidative inactivation of mitochondrial enzymes. However, higher activity of NADP-Isocitrate Dehydrogenase participates in the regeneration of reduced glutathione causing stabilization of enzymes in heart mitochondria.
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Aluminum: a pH-dependent inhibitor of NADP-Isocitrate Dehydrogenase from porcine heart
Biometals, 1992Co-Authors: Masataka Yoshino, Keiko MurakamiAbstract:Aluminum showed a pH-dependent inhibitory effect on NADP-Isocitrate Dehydrogenase from porcine heart. Aluminum ions (Al^3+) acted as a partial competitive inhibitor of the enzyme with respect to the substrate threo -Ds-Isocitrate and inhibited the enzyme non-competitively with respect to NADP at pH 6.85. Fractional velocity plot analysis showed the K _i of the enzyme for aluminum ions to be 0.88μ m . When pH was elevated to 8.0, aluminum ions, which occur as a form of the Al(OH)_4 ^− anion, acted as partial uncompetitive and non-competitive inhibitors of the enzyme with respect to the substrates Isocitrate and NADP, respectively. The K′ _i of the enzyme was determined to be 5.64 μ m at pH 8.0 by fractional velocity plot analysis. The inhibition of NADP-Isocitrate Dehydrogenase by two forms of aluminum ions may explain aluminum toxicity in various tissues and organs.
Masataka Yoshino - One of the best experts on this subject based on the ideXlab platform.
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Protective effect of NADP-Isocitrate Dehydrogenase on the paraquat-mediated oxidative inactivation of aconitase in heart mitochondria.
Environmental toxicology and pharmacology, 2006Co-Authors: Keiko Murakami, Miyako Haneda, T. Makino, Masataka YoshinoAbstract:Protective role of NADP-Isocitrate Dehydrogenase in the oxidative inactivation of mitochondrial enzymes was analyzed. Administration of paraquat to the rat inactivated liver mitochondrial enzymes: the aconitase activity decreased to one quarter, and citrate synthase and fumarase to half, whereas cytosolic enzymes were not affected. Activities of heart mitochondrial and cytosolic enzymes were not at all changed in the rat treated with paraquat, but paraquat directly inactivated aconitase in the heart mitochondria isolated from the non-treated rats. The paraquat-dependent inactivation of aconitase was prevented by activating NADP-Isocitrate Dehydrogenase in the presence of oxidized glutathione. NADP-Isocitrate Dehydrogenase could regenerate glutathione in isolated heart mitochondria, indicating that paraquat-mediated inactivation depends on the glutathione-regenerating activity by enhanced NADPH supply. Lower NADP-Isocitrate Dehydrogenase activity in liver mitochondria cannot regenerate reduced glutathione for scavenging reactive oxygen species, resulting in the paraquat-induced oxidative inactivation of mitochondrial enzymes. However, higher activity of NADP-Isocitrate Dehydrogenase participates in the regeneration of reduced glutathione causing stabilization of enzymes in heart mitochondria.
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Aluminum: a pH-dependent inhibitor of NADP-Isocitrate Dehydrogenase from porcine heart
Biometals, 1992Co-Authors: Masataka Yoshino, Keiko MurakamiAbstract:Aluminum showed a pH-dependent inhibitory effect on NADP-Isocitrate Dehydrogenase from porcine heart. Aluminum ions (Al^3+) acted as a partial competitive inhibitor of the enzyme with respect to the substrate threo -Ds-Isocitrate and inhibited the enzyme non-competitively with respect to NADP at pH 6.85. Fractional velocity plot analysis showed the K _i of the enzyme for aluminum ions to be 0.88μ m . When pH was elevated to 8.0, aluminum ions, which occur as a form of the Al(OH)_4 ^− anion, acted as partial uncompetitive and non-competitive inhibitors of the enzyme with respect to the substrates Isocitrate and NADP, respectively. The K′ _i of the enzyme was determined to be 5.64 μ m at pH 8.0 by fractional velocity plot analysis. The inhibition of NADP-Isocitrate Dehydrogenase by two forms of aluminum ions may explain aluminum toxicity in various tissues and organs.
Yasuo Igarashi - One of the best experts on this subject based on the ideXlab platform.
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a novel biotin protein required for reductive carboxylation of 2 oxoglutarate by Isocitrate Dehydrogenase in hydrogenobacter thermophilus tk 6
Molecular Microbiology, 2003Co-Authors: Miho Aoshima, Masaharu Ishii, Yasuo IgarashiAbstract:Isocitrate Dehydrogenase was purified from Hydrogenobacter thermophilus, and the corresponding gene was cloned and sequenced. The enzyme had similar structural properties to the Isocitrate Dehydrogenase of Escherichia coli, but differed in its catalytic properties, such as coenzyme specificity, pH dependency and kinetic parameters. Notably, the enzyme catalysed the oxidative decarboxylation of Isocitrate, but not the reductive carboxylation of 2-oxoglutarate. The carboxylation reaction required the addition of cell extract and ATP-Mg, suggesting the existence of additional carboxylation factor(s). Further analysis of the carboxylation factor(s) resulted in the purification of two polypeptides. N-terminal amino acid sequencing revealed that the two polypeptides are homologues of pyruvate carboxylase with a biotinylated subunit, but do not catalyse pyruvate carboxylation. Pyruvate carboxylase was also purified, but was not active in stimulating Isocitrate Dehydrogenase. Isocitrate Dehydrogenase, the novel biotin protein, ATP-Mg and NADH were essential for the reductive carboxylation of 2-oxoglutarate. These observations indicate that the novel biotin protein is an ATP-dependent factor, which is involved in the reverse (carboxylating) reaction of Isocitrate Dehydrogenase.
