The role of glutamate transport in the regulation of the pathway of proline oxidation in rat liver mitochondria.

Mitochondrial proline oxidation was studied as a tool to investigate the regulation of the fate of intramitochondrially generated glutamate. Aspartate formation from proline was less than with glutamate as a substrate and was not appreciably stimulated by malate, indicating that the supply of oxalacetate was not limiting. A lag in aspartate synthesis from proline occurred which was relieved by external glutamate due to the involvement of the glutamate-aspartate antiporter. In the absence of added glutamate, the counterion for aspartate transport had to be supplied from internally generated glutamate by way of the glutamate uniporter. Addition of glutamate increased the activity of the glutamate-aspartate ranslocator, thereby stimulating aspartate formation but, in the absence of malate, it had little effect on proline oxidation or glutamate uniporter flux. A further stimulation of aspartate formation was obtained with added malate; under these conditions, increasing glutamate concentrations caused inhibition of proline oxidation. Ammonia was generated at a higher rate from proline than from added glutamate. This was not due to a difference in the oxidation-reduction state of NAD(P) with these substrates, although ammonia formation from either substrate was stimulated by agents that caused an oxidation of NAD(P). The higher rate of ammonia formation from proline than from added glutamate also appeared not to be due to a limiting capacity of the glutamate translocator. Addition of glutamate inhibited ammonia formation from proline, while it caused an accumulation of intramitochondrial glutamate so that a gradient of glutamate concentration was maintained equal to or in excess of the pH gradient across the mitochondrial membrane, allowing a continued efflux of intramitochondrial glutamate on the glutamate uniporter. The results illustrate the regulatory significance of the glutamate uniporter which maintains a relationship between the gradients of pH and glutamate concentration across the mitochondrial membrane, thereby integrating the flux of glutamate into its different metabolic pathways.