Effect of ATP translocation on citrulline and oxaloacetate synthesis by isolated rat liver mitochondria.

The possibility that the availability of ATP may affect the rate of synthesis of carbamoyl phosphate (measured as citrulline) by carbamoyl phosphate synthase (ammonia) was studied using respiring isolated rat liver mitochondria incubated with added ADP, with hexokinase, glucose, and ATP, or with atractylate, in order to enhance or prevent the efflux of mitochondrial ATP. The effects of these agents were compared with those on oxaloacetate synthesis from pyruvate. Addition of hexokinase, glucose, and ATP to isolated mitochondria resulted in an inhibition of citrulline synthesis which was proportional to the amounts of glucose 6-phosphate formed; under these conditions, matrix ATP and ATP/ADP tended to decrease. The addition of increasing amounts of ADP also resulted in proportional inhibition of citrulline synthesis, but in this case the matrix content of ATP and ADP increased, and ATP/ADP decreased very slightly. In the presence of atractylate, citrulline synthesis was maximal despite a 30% decrease in matrix ATP and ATP/ADP. These effects were observed whether pyruvate, succinate, glutamate, or β-OH-butyrate was used as the respiratory substrate. ADP, the hexokinase system, and atractylate had qualitatively similar but much less pronounced effects on oxaloacetate synthesis from pyruvate. Within the limits of variation observed in these experiments, the rate of synthesis of citrulline appears not to be affected by the matrix content of total ATP, total ADP, or by ATP/ADP. It is affected, however, by the velocity of translocation of ATP into the extramitochondrial medium. These findings suggest that carbamoyl phosphate synthase (ammonia) may be loosely associated with the mitochondrial inner membrane, and may compete for ATP with the ATP-ADP translocator to an extent determined by the extramitochondrial demands for ATP.     

Effect of ornithine concentration on citrulline synthesis in mouse liver mitochondria

The rate of citrulline synthesis in mitochondria from OTC-deficient spf-ash mice (15% of the normal activity) was found to be the same as that in mitochondria from control mice. The amount of NAG in their mitochondria varied markedly according to whether they had received a high- or low-protein diet, and the rate of citrulline synthesis was found to be affected by the level of NAG. These results indicate that the CPS stage, not the OTC stage, is rate-limiting in the citrulline synthesis process. Kinetic studies on the effect of ornithine concentration on citrulline synthesis in mitochondria showed that the Km for ornithine was very low in the mitochondria from the mice given a low-protein diet. Kinetic studies on the effect of ornithine concentration on mouse OTC at various concentrations of carbamylphosphate showed that OTC has a ping-pong mechanism, i.e., that the Km for ornithine and Vmax decrease with the reduction in carbamylphosphate concentration. This may explain the low Km value observed in citrulline synthesis in the mitochondria. We conclude that in mitochondrial citrulline synthesis the rate of carbamylphosphate synthesis by CPS in the presence of NAG plays a key role in determining the rate of citrulline synthesis and ornithine dependency.     

Uncoupler-inhibitor titrations of ATP-driven reverse electron transfer in isolated rat-liver mitochondria

Uncoupler-inhibitor titrations of ATP-driven reverse electron transfer across the first site of the respiratory chain were performed in isolated rat-liver mitochondria, and the experimental results were compared with the predictions of a simple delocalized chemiosmotic mechanism. The rates of ATP hydrolysis (Jp) and reverse electron transfer (-J0) were measured at different uncoupler (S-13) concentrations, either in the absence or in the presence of rotenone. When the rates -J0 and Jp measured at different uncoupler concentrations were expressed as percentages of the activity at zero uncoupler concentration, it was found that the efficiency of S-13 to uncouple the reverse electron transfer and to stimulate ATP hydrolysis was not significantly changed upon partial inhibition with rotenone. These results are in contrast with data from a study of uncoupler-inhibitor titrations in submitochondrial particles published previously, in which a higher effectiveness of several uncouplers to inhibit ATP-driven reverse electron transfer was observed in the presence of rotenone.     

Inhibition by salicylates of urea synthesis by isolated rat hepatocytes and citrulline synthesis by isolated rat mitochondria: an effect independent of uncoupling

1. Acetylsalicylate and salicylate inhibited urea synthesis by isolated rat hepatocytes and citrulline synthesis by isolated rat mitochondria. The effects were dose-dependent and occurred at drug concentrations seen in salicylate induced hepatoxicity. 2. Although ATP concentration was decreased in the hepatocytes the effect of the salicylates on citrulline synthesis remained after treatment with oligomycin and carbonyl cyanide m-chlorophenyl hydrazone. This suggests that the effect is independent of uncoupling of oxidative phosphorylation. 3. This in vitro inhibition of urea synthesis by salicylates is similar to that produced by valproate and endogenous organic acids, which are also associated with hyperammonaemic clinical toxicity, and is a possible mechanism for the action of salicylates in the hyperammonaemia of Reye's syndrome.