RESUMEN
Trypanosoma brucei procyclic forms possess three different malate dehydrogenase isozymes that could be separated by hydrophobic interaction chromatography and were recognized as the mitochondrial, glycosomal and cytosolic malate dehydrogenase isozymes. The latter is the only malate dehydrogenase expressed in the bloodstream forms, thus confirming that the expression of malate dehydrogenase isozymes is regulated during the T. brucei life cycle. To achieve further biochemical characterization, the genes encoding mitochondrial and glycosomal malate dehydrogenase were cloned on the basis of previously reported nucleotide sequences and the recombinant enzymes were functionally expressed in Escherichia coli cultures. Mitochondrial malate dehydrogenase showed to be more active than glycosomal malate dehydrogenase in the reduction of oxaloacetate; nearly 80% of the total activity in procyclic crude extracts corresponds to the former isozyme which also catalyzes, although less efficiently, the reduction of p-hydroxyphenyl-pyruvate. The rabbit antisera raised against each of the recombinant isozymes showed that the three malate dehydrogenases do not cross-react immunologically. Immunofluorescence experiments using these antisera confirmed the glycosomal and mitochondrial localization of glycosomal and mitochondrial malate dehydrogenase, as well as a cytosolic localization for the third malate dehydrogenase isozyme. These results clearly distinguish Trypanosoma brucei from Trypanosoma cruzi, since in the latter parasite a cytosolic malate dehydrogenase is not present and mitochondrial malate dehydrogenase specifically reduces oxaloacetate.
Asunto(s)
Malato Deshidrogenasa/análisis , Trypanosoma brucei brucei/enzimología , Secuencia de Aminoácidos , Animales , Cromatografía en Agarosa/métodos , Reacciones Cruzadas/inmunología , Citosol/enzimología , Regulación del Desarrollo de la Expresión Génica/genética , Genes Protozoarios/genética , Isoenzimas/análisis , Isoenzimas/inmunología , Malato Deshidrogenasa/genética , Malato Deshidrogenasa/inmunología , Microcuerpos/enzimología , Microcuerpos/genética , Microcuerpos/inmunología , Mitocondrias/enzimología , Mitocondrias/genética , Mitocondrias/inmunología , Ácido Oxaloacético/metabolismo , Ácidos Fenilpirúvicos/metabolismo , Filogenia , Proteínas Protozoarias/metabolismo , Conejos , Proteínas Recombinantes/metabolismo , Alineación de Secuencia/métodos , Trypanosoma brucei brucei/inmunologíaRESUMEN
The nucleotide (ATP-ADP)/nucleoside (adenosine) ratio in the circulation can modulate the processes of vasoconstriction, vasodilatation and platelet aggregation. The main objective of the present study with rat blood serum was to evaluate the possibility of changes in nucleotide hydrolysis by phenylalanine (Phe) and phenylpyruvate (PP), the levels of which could increase in the circulation of individuals with phenylketonuria. Results demonstrated that Phe in the range 1.0-5.0 mM inhibited the ADP hydrolysis by rat serum. The effect of inhibition by Phe on ATP hydrolysis appeared only at a concentration of 5.0 mM. PP had no significant effect upon nucleotide hydrolysis. Kinetic analysis indicated that the inhibition of ADP and ATP hydrolysis by Phe in rat blood serum is uncompetitive. Conversely, Phe and PP did not affect the hydrolysis of p-nitrophenyl-5'-TMP by rat serum.
Asunto(s)
Adenosina Difosfato/sangre , Adenosina Trifosfato/sangre , Fenilalanina/metabolismo , Ácidos Fenilpirúvicos/metabolismo , Animales , Apirasa/antagonistas & inhibidores , Apirasa/metabolismo , Hidrólisis , Cinética , Masculino , Ratas , Ratas WistarRESUMEN
The main objective of the present study was to characterize the inhibition by phenylalanine and phenylpyruvate of ATP diphosphohydrolase activity in synaptosomes from the brain cortex of rats. This enzyme participates together with a 5'-nucleotidase in adenosine formation from the neurotransmitter, ATP, in the synaptic cleft. The inhibition of ATP diphosphohydrolase was competitive for nucleotide hydrolysis but 5'-nucleotidase was not affected by these metabolites. Furthermore, the two substances inhibited enzyme activity by acting at the same binding site. If the enzyme inhibition observed in vitro also occurs in the brain of PKU patients, it may promote an increase in ATP levels in the synaptic cleft. In this case, the neurotoxicity of ATP could possibly be one of the mechanisms leading to the characteristic brain damage of phenylketonuria.
Asunto(s)
Adenosina Trifosfato/metabolismo , Apirasa/metabolismo , Corteza Cerebral/enzimología , Fenilalanina/metabolismo , Fenilcetonurias/enzimología , Ácidos Fenilpirúvicos/metabolismo , Terminales Presinápticos/enzimología , 5'-Nucleotidasa/antagonistas & inhibidores , 5'-Nucleotidasa/metabolismo , Adenosina/biosíntesis , Adenosina Difosfato/metabolismo , Animales , Apirasa/antagonistas & inhibidores , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/fisiopatología , Hidrólisis/efectos de los fármacos , Cinética , Fenilalanina/farmacología , Fenilcetonurias/fisiopatología , Ácidos Fenilpirúvicos/farmacología , Terminales Presinápticos/efectos de los fármacos , Ratas , Ratas Wistar , SinaptosomasRESUMEN
Mn2+ catalyzes the aerobic oxidation of phenylpyruvic acid. Benzaldehyde and oxalate are the major products, the former being generated to some extent in an excited state, as indicated by chlorophyll-sensitized emission. Both oxygen consumption and emission are enhanced by addition of horseradish peroxidase. In the presence of the enzyme, halogen-containing xanthene dyes also sensitize the emission. From the values of the benzaldehyde/oxalate product ratio, it is inferred that two oxidation pathways are operative. One of these proceeds via a dioxetane intermediate and is responsible for the excited benzaldehyde; the other, which involves an alpha-keto-beta-peroxylactone, does not produce oxalate. The enzyme appears to favour the dioxetane route. The relative importance of these routes in biological systems is discussed.