Accumulation Of 2-oxoglutarate Research Articles

Characterization of Two Critical Residues in the Effector-Binding Domain of NtcA in the Cyanobacterium Anabaena sp. Strain PCC 7120

NtcA, highly conserved in cyanobacteria, regulates the expression of a large number of genes involved in nitrogen and carbon metabolisms, photosynthesis, and stress responses. In the filamentous diazotrophic cyanobacterium Anabaena PCC 7120, NtcA is also required for the initiation of heterocyst differentiation, triggered by the accumulation of 2-oxoglutarate (2-OG) following nitrogen starvation. Recent structural studies reveal the binding pocket of 2-OG on each of the two subunits of the NtcA homodimer, and indicate a route of signal transmission upon 2-OG binding. In this study, we studied the effect of mutations of two critical residues in the effector-binding domain of NtcA on heterocyst differentiation. Mutations of these residues could change strongly the ability of NtcA to sense the nitrogen-starvation signal in vivo. As a result of these mutations, the corresponding strains were unable to form any heterocysts, or form a few heterocysts at a very low frequency. Consistent with these phenotypes, these mutations were defective in initiating transcription by the RNA polymerase in the presence of 2-OG as determined by a reconstituted in vitro transcriptional assay. The different effects of the two mutations were consistent with the roles of the two corresponding residues in 2-OG binding highlighted by recent structural analysis of the NtcA-2-OG complex. These studies provided genetic evidence for the importance of the effector-binding domain in the regulatory function of NtcA.

Effect of free malonate on the utilization of glutamate by rat brain mitochondria.

Malonate is an effective inhibitor of succinate dehydrogenase in preparations from brain and other organs. This property was reexamined in isolated rat brain mitochondria during incubation with L-glutamate. The biosynthesis of aspartate was determined by a standard spectrofluorometric method and a radiometric technique. The latter was suitable for aspartate assay after very brief incubations of mitochondria with glutamate. At a concentration of 1 mM or higher, malonate totally inhibited aspartate biosynthesis. At 0.2 mM, the inhibitory effect was still present. It is thus possible that the natural concentration of free malonate in adult rat brain of 192 nmol/g wet weight exerts an effect on citric acid cycle reactions in vivo. The inhibition of glutamate utilization by malonate was readily overcome by the addition of malate which provided oxaloacetate for the transamination of glutamate. The reaction was accompanied by the accumulation of 2-oxoglutarate. The metabolism of glutamate was also blocked by inclusion of arsenite and gamma-vinyl-gamma-aminobutyric acid but again added malate allowed transamination to resume. When arsenite and gamma-vinyl-gamma-aminobutyric acid were present, the role of malonate as an inhibitor of malate entry into the mitochondrial interior could be determined without considering the inhibition of succinate dehydrogenase. The apparent Km and Vmax values for uninhibited malate entry were 0.01 mM and 100 nmol/mg protein/min, respectively. Malonate was a competitive inhibitor of malate transport (Ki = 0.75 mM).