PubMed1981
The energetics of flux through carbamyl phosphate synthetase and of citrulline formation from added ammonia, bicarbonate, and ornithine have been investigated in liver mitochondria from rats fed a high protein diet. In the presence of an oxidizable substrate, but in the absence of ornithine, carbamyl phosphate accumulated as a function of the medium phosphate concentration (K'm approximately 1.5 mM) up to values of 30 nmol/mg of protein. Upon addition of ornithine, citrulline was produced at the rate of 70 nmol/mg/min, and the carbamyl phosphate content fell to below 1 nmol/mg. The intramitochondrial ATP/ADP ratio decreased after ornithine addition, indicating that release of inhibition of carbamyl phosphate synthetase by carbamyl phosphate predominated over the expected inhibition due to the fall of the ATP/ADP ratio. Under partially uncoupled conditions in the presence of ornithine, citrulline formation decreased linearly with a fall of the calculated intramitochondrial MgATP/MgADP ratio. Changes of the thermodynamic parameters of mitochondrial phosphorylation potential, delta Gp(m), proton electrochemical gradient, delta mu H+, and oxidation-reduction potential difference between NAD+ and cytochrome c, delta Eh, were measured under conditions of enhanced respiration induced by citrulline synthesis and compared with ADP-stimulated respiration. Under both conditions, delta Gp(m) decreased and delta Eh also decreased due to a net oxidation of NADH and reduction of cytochrome c. However, delta mu H+ showed no change after citrulline addition although it decreased during ADP-stimulated respiration. The average H+/2e stoichiometry over the first two phosphorylation sites calculated from the delta Eh/delta mu H+ ratio ranged from 3.0 to 3.5, while the H+/ATP stoichiometry calculated from the delta Gp(m)/delta mu H+ ratio ranged from 2.0 to 2.5. The calculated ratios of H+/2e and H+/ATP both increased as delta mu H+ was lowered by addition of an uncoupling agent. The overall data are apparently not in accordance with the commonly held view that delta mu H+ is an obligatory intermediate between the oxidation-reduction pumps of the respiratory chain and ATP synthase.