Contribution of glutamate dehydrogenase to mitochondrial glutamate metabolism studied by (13)C and (31)P nuclear magnetic resonance.
The relative contribution of glutamate dehydrogenase (GDH) and the aminotransferase activity to mitochondrial glutamate metabolism was investigated in dilute suspensions of purified mitochondria from potato (Solanum tuberosum) tubers. Measurements of glutamate-dependent oxygen consumption by mitochondria in different metabolic states were complemented by novel in situ NMR assays of specific enzymes that metabolize glutamate. First, a new assay for aminotransferase activity, based on the exchange of deuterium between deuterated water and glutamate, provided a method for establishing the effectiveness of the aminotransferase inhibitor amino-oxyacetate in situ, and thus allowed the contribution of the aminotransferase activity to glutamate oxidation to be assessed unambiguously. Secondly, the activity of GDH in the mitochondria was monitored in a coupled assay in which glutamine synthetase was used to trap the ammonium released by the oxidative deamination of glutamate. Thirdly, the reversibility of the GDH reaction was investigated by monitoring the isotopic exchange between glutamate and [(15)N]ammonium. These novel approaches show that the oxidative deamination of glutamate can make a significant contribution to mitochondrial glutamate metabolism and that GDH can support the aminotransferases in funneling carbon from glutamate into the TCA cycle.
Aubert
S
S
Laboratoire de Physiologie Végétale, Département de Biologie Moléculaire et Structurale, CEA-Grenoble, 17 rue des Martyrs, F-38054 Grenoble Cédex 9, France.
Bligny
R
R
Douce
R
R
Gout
E
E
Ratcliffe
R G
RG
Roberts
J K
JK
D000625:Aminooxyacetic Acid / Q000494:pharmacology; D002247:Carbon Isotopes; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism*; D010759:Phosphorus Isotopes; D011198:Solanum tuberosum / Q000378:metabolism; D019802:Succinic Acid / Q000378:metabolism; D000637:Transaminases / Q000378:metabolism
D002247:Carbon Isotopes; D007656:Ketoglutaric Acids; D010759:Phosphorus Isotopes; D000625:Aminooxyacetic Acid; D018698:Glutamic Acid; D019802:Succinic Acid; D005969:Glutamate Dehydrogenase; D000637:Transaminases