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Abstract:

An aromatic L-α-hydroxyacid dehydrogenase (AHADH) was purified to homogeneity from epimastigotes of Trypanosoma cruzi by a method involving chromatography on DEAE-cellulose, hydrophobic interaction chromatography on Phenyl-Sepharose and affinity chromatography on Affi-Gel Blue. The purified enzyme showed a single band in SDS-PAGE, with an apparent molecular mass of 36 kDa. Since the apparent molecular mass of the native enzyme, determined by gel filtration, is about 80 kDa, the native enzyme is a dimer of similar subunits. The amino acid composition was determined, as well as the sequences of 4 internal peptides obtained by CNBr cleavage at Met residues, and one peptide obtained after tryptic digestion. Three of the peptides presented considerable sequence similarity with the corresponding sequences of several malate dehydrogenases. The optimal pH for the enzyme reaction with p-hydroxyphenyl pyruvate and NADH as substrates was 7.5; that for the reverse reaction was 9.5. The apparent Km values for phenylpyruvate and p-hydroxyphenylpyruvate were 48 and 117μM, respectively; that for l-phenyllactate in the reverse reaction was 420μM. The enzyme was much less active with α-isocaproic acid as substrate, and other acids, including pyruvic and oxaloacetic, were not substrates at all. l-phenyllactic acid, but not the d-isomer, acted as substrate. The enzyme can therefore be considered as a general aromatic l-α-hydroxyacid dehydrogenase. The low apparent Km value for NADH (25 μM in the presence of phenylpyruvate) makes AHADH a candidate for the reoxidation of cytosolic NADH in T. cruzi. © 1994.

Registro:

Documento: Artículo
Título:Purification and partial structural and kinetic characterization of an aromatic L-α-hydroxy acid dehydrogenase from epimastigotes of Trypanosoma cruzi
Autor:Montemartini, M.; Santomé, JosA.; Cazzulo, J.J.; Nowicki, C.
Filiación:IQUIFIB (UBA-CONICET), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junin 956, 1113 Buenos Aires, Argentina
Instituto de Investigaciones Bioquímicas 'Luis F. Leloir', Fundación Campomar, CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, A. Machado 151, 1405 Buenos Aires Argentina
Palabras clave:Amino acid catabolism; Aromatic amino acid; Aromatic hydroxyacid; Dehydrogenase; Epimastigote; Trypanosoma cruzi; oxidoreductase; animal experiment; article; controlled study; enzyme analysis; enzyme purification; epimastigote; nonhuman; priority journal; protein degradation; trypanosoma cruzi; Alcohol Oxidoreductases; Amino Acid Sequence; Amino Acids; Animal; Comparative Study; Hydrogen-Ion Concentration; Kinetics; Molecular Sequence Data; Molecular Structure; Molecular Weight; Sequence Homology, Amino Acid; Substrate Specificity; Support, Non-U.S. Gov't; Trypanosoma cruzi; Animalia; Trypanosoma; Trypanosoma cruzi
Año:1994
Volumen:68
Número:1
Página de inicio:15
Página de fin:23
DOI: http://dx.doi.org/10.1016/0166-6851(94)00145-6
Título revista:Molecular and Biochemical Parasitology
Título revista abreviado:Mol. Biochem. Parasitol.
ISSN:01666851
CODEN:MBIPD
CAS:Alcohol Oxidoreductases, EC 1.1; Amino Acids; D-2-hydroxyacid dehydrogenase, EC 1.1.99.6; D-lactate dehydrogenase, EC 1.1.1.28
Registro:https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_01666851_v68_n1_p15_Montemartini

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Citas:

---------- APA ----------
Montemartini, M., Santomé, JosA., Cazzulo, J.J. & Nowicki, C. (1994) . Purification and partial structural and kinetic characterization of an aromatic L-α-hydroxy acid dehydrogenase from epimastigotes of Trypanosoma cruzi. Molecular and Biochemical Parasitology, 68(1), 15-23.
http://dx.doi.org/10.1016/0166-6851(94)00145-6
---------- CHICAGO ----------
Montemartini, M., Santomé, JosA., Cazzulo, J.J., Nowicki, C. "Purification and partial structural and kinetic characterization of an aromatic L-α-hydroxy acid dehydrogenase from epimastigotes of Trypanosoma cruzi" . Molecular and Biochemical Parasitology 68, no. 1 (1994) : 15-23.
http://dx.doi.org/10.1016/0166-6851(94)00145-6
---------- MLA ----------
Montemartini, M., Santomé, JosA., Cazzulo, J.J., Nowicki, C. "Purification and partial structural and kinetic characterization of an aromatic L-α-hydroxy acid dehydrogenase from epimastigotes of Trypanosoma cruzi" . Molecular and Biochemical Parasitology, vol. 68, no. 1, 1994, pp. 15-23.
http://dx.doi.org/10.1016/0166-6851(94)00145-6
---------- VANCOUVER ----------
Montemartini, M., Santomé, JosA., Cazzulo, J.J., Nowicki, C. Purification and partial structural and kinetic characterization of an aromatic L-α-hydroxy acid dehydrogenase from epimastigotes of Trypanosoma cruzi. Mol. Biochem. Parasitol. 1994;68(1):15-23.
http://dx.doi.org/10.1016/0166-6851(94)00145-6