Synthesis of glutamate from α-oxoglutarate and ammonia in rat-liver mitochondria: I. Comparison of different hydrogen donors

Abstract

1. 1. The synthesis of glutamate from α-oxoglutarate and ammonia in rat-liver mitochondria has been studied. Under conditions where oxaloacetate is formed, some of the glutamate synthesized transaminates with the oxaloacetate and gives rise to aspartate. 2. 2. In the presence of α-oxoglutarate, ammonia and oxygen, the synthesis of amino acids is stimulated by phosphate acceptor and by succinate. 3. 3. In the presence of α-oxoglutarate, ammonia, succinate and oxygen, and in the absence of phosphate acceptor, the synthesis of amino acids is stimulated by oligomycin, arsenite or dinitrophenol. When both arsenite and dinitrophenol are added, very little synthesis occurs. 4. 4. These results are interpreted in terms of three different mechanisms for the synthesis of amino acids from α-oxoglutarate and ammonia, in which reducing equivalents are supplied by α-oxoglutarate, succinate, and the products of oxidation of succinate, respectively. 5. 5. The synthesis of glutamate with α-oxoglutarate as hydrogen donor is stimulated by phosphate acceptor and is completely inhibited by arsenite. α-Oxoglutarate is a relatively inefficient hydrogen donor. 6. 6. With succinate as hydrogen donor, in the presence of arsenite, the amounts of glutamate and aspartate found represent the contribution of succinate and of malate (derived from succinate), respectively, as hydrogen donor. 7. 7. The synthesis of glutamate with succinate as hydrogen donor is greatly stimulated by oligomycin, and greatly inhibited by antimycin, malonate or Amytal. It is concluded that this synthesis represents an energy-linked reversal of the respiratory chain. 8. 8. The synthesis of glutamate + aspartate with malate as hydrogen donor, in the presence of arsenite and glutamate, is markedly inhibited by antimycin or Amytal, and stimulated by oligomycin. This shows that energy is also required for this reaction. 9. 9. When isocitrate is the hydrogen donor, the synthesis of glutamate is very little affected by antimycin, Amytal, oligomycin or dinitrophenol, indicating that no energy is required for this reaction. 10. 10. Inorganic phosphate or arsenate is necessary for maximum synthesis of amino acids from α-oxoglutarate and ammonia with succinate, malate or isocitrate as hydrogen donor. 11. 11. The possibility that glutamate dehydrogenase in the mitochondria reacts exclusively with NADP is discussed.