title: Purification, cloning, and expression of the mitochondrial malate dehydrogenase (mMDH) from protoscolices of Echinococcus granulosus.
abstract: Protoscolices of the parasitic helminth Echinococcus granulosus contain two malate dehydrogenases (EC 1.1.1.37), one cytosolic and one mitochondrial. The latter has been separated from the other isoform and purified to protein homogeneity. Sequencing of tryptic peptides by Edman degradation allowed the design of oligonucleotide primers for PCR, leading to the cloning and sequencing of a full length cDNA. The encoding gene is present as a single copy per haploid genome and codes for a protein with high sequence identity (56-58%) with the similar enzymes from mammals, Caenorhabditis elegans and yeast. Active recombinant mitochondrial malate dehydrogenase was expressed in Escherichia coli, as protein fusions with glutathione S-transferase or a poly-His tail. The purified recombinant enzymes had a kinetic behaviour similar to that of the native enzyme, being inhibited by excess of the substrate oxaloacetate and unaffected by excess L-malate. The results indicate that E. granulosus contains two typical eukaryotic malate dehydrogenases, with relative levels quite different from those present in mammalian tissues like heart, in good agreement with the predominantly fermentative metabolism of the protoscolices.
authors.affiliation: Instituto de Investigaciones Biotecnológicas, Universidad Nacional de General San Martín, B 1650 KNA, San Martín, Argentina.
authors.forename: Fernán
authors.initials: F
authors.lastname: Agüero
authors.forename: Griselda
authors.initials: G
authors.lastname: Noé
authors.forename: Ulf
authors.initials: U
authors.lastname: Hellman
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Repetto
authors.forename: Arnaldo
authors.initials: A
authors.lastname: Zaha
authors.forename: Juan José
authors.initials: JJ
authors.lastname: Cazzulo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018509:DNA, Helminth / Q000235:genetics; D048209:Echinococcus granulosus / Q000201:enzymology* / Q000235:genetics*; D015870:Gene Expression; D017238:Genes, Helminth; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D018076:DNA, Complementary; D018509:DNA, Helminth; D011994:Recombinant Proteins; D008291:Malate Dehydrogenase