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Purification, cloning, and expression of the mitochondrial malate dehydrogenase (mMDH) from protoscolices of Echinococcus granulosus
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
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2004 (English)In: Molecular and biochemical parasitology (Print), ISSN 0166-6851, E-ISSN 1872-9428, Vol. 137, no 2, 207-214 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2004. Vol. 137, no 2, 207-214 p.
Keyword [en]
Amino Acid Sequence, Animals, Base Sequence, Cloning; Molecular, DNA; Complementary/genetics, DNA; Helminth/genetics, Echinococcus granulosus/*enzymology/*genetics, Gene Expression, Genes; Helminth, Kinetics, Malate Dehydrogenase/*genetics/*isolation & purification/metabolism, Mitochondria/enzymology, Molecular Sequence Data, Recombinant Proteins/genetics/metabolism, Research Support; Non-U.S. Gov't, Sequence Homology; Amino Acid
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Medical and Health Sciences Natural Sciences
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URN: urn:nbn:se:uu:diva-72966DOI: 10.1016/j.molbiopara.2004.06.003PubMedID: 15383291OAI: oai:DiVA.org:uu-72966DiVA: diva2:100877
Available from: 2005-05-31 Created: 2005-05-31 Last updated: 2017-12-14Bibliographically approved

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Hellman, UlfCazzulo, Juan José

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