The malate dehydrogenase isoforms from Trypanosoma brucei: subcellular localization and differential expression in bloodstream and procyclic forms.
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.
Aranda
Alejandro
A
Instituto de Química y Fisicoquímica Biológica IQUIFIB-CONICET, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, CP1113, Argentina.
Maugeri
Dante
D
Uttaro
Antonio D
AD
Opperdoes
Fred
F
Cazzulo
Juan J
JJ
Nowicki
C
C
D000595:Amino Acid Sequence; D000818:Animals; D002847:Chromatography, Agarose / Q000379:methods; D003429:Cross Reactions / Q000276:immunology; D003600:Cytosol / Q000201:enzymology; D018507:Gene Expression Regulation, Developmental / Q000235:genetics; D017125:Genes, Protozoan / Q000235:genetics; D007527:Isoenzymes / Q000032:analysis / Q000276:immunology; D008291:Malate Dehydrogenase / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D008830:Microbodies / Q000201:enzymology / Q000235:genetics / Q000276:immunology; D008928:Mitochondria / Q000201:enzymology / Q000235:genetics / Q000276:immunology; D062907:Oxaloacetic Acid / Q000378:metabolism; D010667:Phenylpyruvic Acids / Q000378:metabolism; D010802:Phylogeny; D015800:Protozoan Proteins / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment / Q000379:methods; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000276:immunology
D007527:Isoenzymes; D010667:Phenylpyruvic Acids; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C010590:4-hydroxyphenylpyruvic acid; D062907:Oxaloacetic Acid; D008291:Malate Dehydrogenase