Extracts of frozen rat liver were found to catalyse the formation of 3H2O from DL-2-hydroxy[2-3H]glutarate. Three peaks of enzyme activities were observed on separation by chromatography on DEAE-Sepharose. The first and second peaks corresponded to an enzyme acting on L-2-hydroxyglutarate and the third peak corresponded to an enzyme acting on D-2-hydroxyglutarate, as indicated by competitive inhibition of the detritiation of the racemic radioactive compound by the unlabelled L- and D-isomers respectively. The enzyme acting on the D-form was further characterized. It was independent of NAD or NADP and it converted D-2-hydroxyglutarate into α-ketoglutarate, transferring electrons to artificial electron acceptors. It also oxidized D-lactate, D-malate and meso-tartrate and was stimulated by Zn2+, Co2+ and Mn2+, but not by Mg2+ or Ca2+. Subcellular fractionation indicated that it was present in the mitochondrial fraction. The enzyme was further purified by chromatography on Blue Trisacryl and phenyl-Sepharose, up to a stage where only a few bands were still visible by SDS/ PAGE. Among the four candidate polypeptides that were identified by MS, one corresponded to a predicted mitochondrial protein homologous with FAD-dependent D-lactate dehydrogenase. The corresponding human protein was expressed in HEK-293 cells and it was shown to catalyse the detritiation of DL-2-hydroxy[2-3H]glutarate with similar properties as the purified rat enzyme.
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Research Article| June 22 2004
Identification of a dehydrogenase acting on D-2-hydroxyglutarate
Younes ACHOURI;
Younes ACHOURI
*Laboratory of Physiological Chemistry, Université catholique de Louvain and Christian de Duve Institute of Cellular Pathology (ICP), Avenue Hippocrate 75, B-1200 Brussels, Belgium
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Gaëtane NOËL;
Gaëtane NOËL
*Laboratory of Physiological Chemistry, Université catholique de Louvain and Christian de Duve Institute of Cellular Pathology (ICP), Avenue Hippocrate 75, B-1200 Brussels, Belgium
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Didier VERTOMMEN;
Didier VERTOMMEN
†Hormone and Metabolic Research Unit, Université catholique de Louvain and Christian de Duve Institute of Cellular Pathology (ICP), Avenue Hippocrate 75, B-1200 Brussels, Belgium
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Mark H. RIDER;
Mark H. RIDER
†Hormone and Metabolic Research Unit, Université catholique de Louvain and Christian de Duve Institute of Cellular Pathology (ICP), Avenue Hippocrate 75, B-1200 Brussels, Belgium
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Maria VEIGA-DA-CUNHA;
Maria VEIGA-DA-CUNHA
*Laboratory of Physiological Chemistry, Université catholique de Louvain and Christian de Duve Institute of Cellular Pathology (ICP), Avenue Hippocrate 75, B-1200 Brussels, Belgium
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Emile van SCHAFTINGEN
Emile van SCHAFTINGEN 1
*Laboratory of Physiological Chemistry, Université catholique de Louvain and Christian de Duve Institute of Cellular Pathology (ICP), Avenue Hippocrate 75, B-1200 Brussels, Belgium
1To whom correspondence should be addressed (e-mail vanschaftingen@bchm.ucl.ac.be).
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Publisher: Portland Press Ltd
Received: December 15 2003
Revision Received: March 16 2004
Accepted: April 07 2004
Accepted Manuscript online: April 07 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2004
Biochem J (2004) 381 (1): 35–42.
Article history
Received:
December 15 2003
Revision Received:
March 16 2004
Accepted:
April 07 2004
Accepted Manuscript online:
April 07 2004
Citation
Younes ACHOURI, Gaëtane NOËL, Didier VERTOMMEN, Mark H. RIDER, Maria VEIGA-DA-CUNHA, Emile van SCHAFTINGEN; Identification of a dehydrogenase acting on D-2-hydroxyglutarate. Biochem J 1 July 2004; 381 (1): 35–42. doi: https://doi.org/10.1042/BJ20031933
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