Rat liver d-3-phosphoglycerate dehydrogenase was purified to homogeneity and digested with trypsin, and the sequences of two peptides were determined. This sequence information was used to screen a rat hepatoma cDNA library. Among 11 positive clones, two covered the whole coding sequence. The deduced amino acid sequence (533 residues; Mr 56493) shared closer similarity with Bacillus subtilis 3-phosphoglycerate dehydrogenase than with the enzymes from Escherichia coli, Haemophilus influenzae and Saccharomyces cerevisiae. In all cases the similarity was most apparent in the substrate- and NAD+-binding domains, and low or insignificant in the C-terminal domain. A corresponding 2.1 kb mRNA was present in rat tissues including kidney, brain and testis, whatever the dietary status, and also in livers of animals fed a protein-free, carbohydrate-rich diet, but not in livers of control rats, suggesting transcriptional regulation. The full-length rat 3-phosphoglycerate dehydrogenase was expressed in E. coli and purified. The recombinant enzyme and the protein purified from liver displayed hyperbolic kinetics with respect to 3-phosphoglycerate, NAD+ and NADH, but substrate inhibition by 3-phosphohydroxypyruvate was observed; this inhibition was antagonized by salts. Similar properties were observed with a truncated form of 3-phosphoglycerate dehydrogenase lacking the C-terminal domain, indicating that the latter is not implicated in substrate inhibition or in salt effects. By contrast with the bacterial enzyme, rat 3-phosphoglycerate dehydrogenase did not catalyse the reduction of 2-oxoglutarate, indicating that this enzyme is not involved in human d- or l-hydroxyglutaric aciduria.
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April 1997
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Research Article|
April 15 1997
Cloning, sequencing and expression of rat liver 3-phosphoglycerate dehydrogenase
Younes ACHOURI;
Younes ACHOURI
*Laboratory of Physiological Chemistry, International Institute of Cellular and Molecular Pathology and Université Catholique de Louvain, B-1200 Brussels, Belgium
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Mark H. RIDER;
Mark H. RIDER
†Hormone and Metabolic Research Unit, International Institute of Cellular and Molecular Pathology and Université Catholique de Louvain, B-1200 Brussels, Belgium
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Emile VAN SCHAFTINGEN;
Emile VAN SCHAFTINGEN
*Laboratory of Physiological Chemistry, International Institute of Cellular and Molecular Pathology and Université Catholique de Louvain, B-1200 Brussels, Belgium
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Mariette ROBBI
Mariette ROBBI
‡
*Laboratory of Physiological Chemistry, International Institute of Cellular and Molecular Pathology and Université Catholique de Louvain, B-1200 Brussels, Belgium
‡To whom correspondence should be addressed.
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Publisher: Portland Press Ltd
Received:
October 02 1996
Accepted:
December 03 1996
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1997
1997
Biochem J (1997) 323 (2): 365–370.
Article history
Received:
October 02 1996
Accepted:
December 03 1996
Citation
Younes ACHOURI, Mark H. RIDER, Emile VAN SCHAFTINGEN, Mariette ROBBI; Cloning, sequencing and expression of rat liver 3-phosphoglycerate dehydrogenase. Biochem J 15 April 1997; 323 (2): 365–370. doi: https://doi.org/10.1042/bj3230365
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