Purification, cloning, and expression of the mitochondrial malate dehydrogenase (mMDH) from protoscolices of Echinococcus granulosus.
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.
Agüero
Fernán
F
Instituto de Investigaciones Biotecnológicas, Universidad Nacional de General San Martín, B 1650 KNA, San Martín, Argentina.
Noé
Griselda
G
Hellman
Ulf
U
Repetto
Yolanda
Y
Zaha
Arnaldo
A
Cazzulo
Juan José
JJ
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
D018076:DNA, Complementary; D018509:DNA, Helminth; D011994:Recombinant Proteins; D008291:Malate Dehydrogenase