1. The ratio [ATP]/[ADP][P(i)], as measured by direct determination of the three components in rat liver, was found in various nutritional states to have approximately the same value as the ratio [ATP]/[ADP][P(i)] calculated from the concentrations of lactate, pyruvate, glyceraldehyde phosphate and 3-phosphoglycerate on the assumption that lactate dehydrogenase, glyceraldehyde phosphate dehydrogenase and 3-phosphoglycerate kinase are at near-equilibrium in the liver. This implies that the redox state of the NAD couple in the cytoplasm is linked to, and partially controlled by, the phosphorylation state of the adenine nucleotides. 2. The combined equilibrium constant of the glyceraldehyde 3-phosphate dehydrogenase and 3-phosphoglycerate kinase reactions at 38 degrees C and I0.25, was found to be 5.9x10(-6). 3. The fall of the [NAD(+)]/[NADH] ratio in starvation and other situations is taken to be the consequence of a primary fall of the [ATP]/[ADP][HPO(4) (2-)] ratio.

1. The equilibrium constant at 38 degrees and I 0.25 of the triose phosphate isomerase reaction was found to be 22.0 and that of the aldolase reaction, 0.99x10(-4)m. The [dihydroxyacetone phosphate]/[glyceraldehyde phosphate] ratio was found to be 9.3 in rat liver. The causes of the apparent deviation of the triose phosphate isomerase system from equilibrium in vivo have been investigated. 2. The equilibria of the triose phosphate isomerase and aldolase reactions were studied with relatively large concentrations of crystalline enzymes and small concentrations of substrates, approximating to those found in rat liver and muscle. There was significant binding of fructose diphosphate by aldolase under these conditions. There was no evidence that binding of glyceraldehyde phosphate by either enzyme affected the equilibria. 3. The deviation from equilibrium of the triose phosphate isomerase system in rat liver can be accounted for by the low activity of the enzyme, in relation to the flux, at low physiological concentrations of glyceraldehyde phosphate (about 3mum). It has been calculated that a flux of 1.8mumoles/min./g. wet weight of liver would be expected to cause the measured degree of disequilibrium found in vivo. 4. The conclusion that the triose phosphate isomerase is not at equilibrium is in accordance with the situation postulated by Rose, Kellermeyer, Stjernholm & Wood (1962) on the basis of isotope-distribution data. 5. The triose phosphate isomerase system is closer to equilibrium in resting muscle probably because of a very low flux and a high enzyme concentration. 6. The aldolase system deviated from equilibrium in rat liver by a factor of about 10 and by a much greater factor in resting muscle. 7. The measurement of total dihydroxyacetone phosphate and glyceraldehyde phosphate content indicates the concentrations of the free metabolites in the tissue. This may not hold for fructose diphosphate, a significant proportion of which may be bound to aldolase.

Experiments with carbamoyl phosphate synthetase (ammonia) in solution and in isolated mitochondria are reported which show the following. NH3 rather than NH4+ is the substrate of the enzyme. The apparent Km of NH3 for the purified enzyme is about 38 microM. The apparent Km for NH3 measured in intact isolated mitochondria is about 13 microM. This value was obtained for both coupled and uncoupled mitochondria and was unchanged when the rate of carbamoyl phosphate synthesis was increased 2-fold by incubating uncoupled mitochondria in the presence of 5 mM-N-acetylglutamate. According to the literature, the concentration of NH3 in liver is well below the measured apparent Km. On the basis of this and previous work we conclude that, quantitatively, changes in liver [NH3] and [ornithine] are likely to be the most important factors in the fast regulation of synthesis of carbamoyl phosphate and urea. This conclusion is consistent with all available evidence obtained with isolated mitochondria, isolated hepatocytes, perfused liver and whole animals.

Rat liver ornithine carbamoyltransferase appears to be located exclusively in the mitochondria; the activity that is found in the soluble fraction is indistinguishable from mitochondrial ornithine carbamoyltransferase by simple kinetic criteria, and seems to result from breakage of mitochondria during homogenization. Of several rat tissues studied, only the liver and the mucosa of small intestine contain significant amounts of ornithine carbamoyltransferase; the activity in intestinal mucosa is less than one thousandth of that in liver. Qualitatively, this distribution coincides with that of carbamoyl phosphate synthetase I and its cofactor, acetylglutamate. The rat liver contents of carbamoyl phosphate and ornithine were 0.1 and 0.15mumol/g wet wt. of tissue respectively. On the basis of these values, it is proposed that in vivo the ornithine carbamoyltransferase activity of liver may be much lower than its maximal activity in vitro might suggest.

Mitochondrial water spaces were determined by centrifugal filtration, by using 3H2O and [14C]-sucrose, -mannitol, -inulin and -dextran. The volume (in microliter/mg of mitochondrial protein) of each of the spaces was inversely proportional to the amount of mitochondria (mg of protein) centrifuged. The dextran space (representing extramitochondrial water carried down with the mitochondria) decreased the most, and accounted for most of the changes observed in the other spaces. However, the calculated matrix and intermembrane spaces also decreased when increasing amounts of mitochondria were centrifuged. For each space, the same value was obtained when centrifugal filtration was done at 8000 and at 15,600 g, and when the mitochondria were incubated with the markers for 15 s to 5 min, indicating that sucrose, mannitol and inulin do not penetrate the matrix, nor does dextran penetrate the intermembrane space, under the incubation and centrifugation conditions generally used to measure mitochondrial spaces.