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1. The digitonin fractionation procedure [Zuurendonk, P. F. and Tager, J. M. (1974) Biochim. Biophys. Acta, 333, 393 -3991 was used to determine the concentrations of adenine nucleotides and of inorganic phosphate in the mitochondrial and cytosolic compartments of hepatocytes isolated from the livers of starved rats.2. Using glycerol 3-phosphate as a cytosolic marker, it was concluded that leakage of cytosolic metabolites was complete after 15 s exposure of the hepatocytes to 2 mM digitonin. The recovery of the adenine nucleotides after digitonin fractionation was measured. The recovery of ATP was about 100% and that of ADP and AMP 88-109%. Radioactively labelled ATP or ADP were added to the fractionation medium in order to test whether redistribution or metabolism of adenine nucleotides occurs during the fractionation procedure. No significant redistribution of adenine nucleotides occurs during the fractionation. Although slight metabolic activity appears to occur in the cytosolic fraction this leads to a change of only 20% in ADP and no change in ATP. It is concluded that the digitonin fractionation procedure can be used to obtain an accurate estimation of the content of adenine nucleotides in the cytosolic and mitochondrial fractions of isolated rat-liver cells. 3. In isolated hepatocytes from fasted rats, 60% of total adenine nucleotides is found in the cytosolic fraction.4. When isolated hepatocytes from fasted rats are incubated with alanine, the ATPjADP ratio is 8.76 in the cytosolic fraction and 1.77 in the mitochondrial fraction. On addition of oleate there is no change in cytosolic ATP/ADP. 5. In hepatocytes incubated with alanine or alanine + oleate, the concentration of inorganic phosphate is 5-times higher in the mitochondrial fraction than in the cytosol.6. It can be calculated that in isolated hepatocytes incubated with alanine the phosphorylation state ([ATP]/[ADP] x [P,]) in the mitochondrial fraction is 105 M-' (AC, = 12.0 kJ/mol) (2.87 kcal/ mol) and that in the cytosolic fraction 2630 M-' (AG, = 20.3 kJ/mol) (4.85 kcal/mol). The addition of oleate did not alter the value of the cytosolic phosphorylation state whereas that in the mitochondria was increased by about 20%.7. The concentrations of the complexed and uncomplexed forms of the adenine nucleotides and inorganic phosphate in the cytosol were calculated assuming [K+Ifree = 100 mM, [M$+Ifree =0.4 mM, pH = 7.0 and 1=0.25 M. The cytosolic concentration of uncomplexed ATP was calculated to be 170 pM, and that of uncomplexed ADP 115 pM. The latter value is about 10-times greater than the K, for ADP that has been reported for the adenine nucleotide translocator at low temperatures.Adenine nucleotides play a central role in metabolism, acting as a link between energy-yielding and energy-utilizing processes in the cell. The rate of these processes can be controlled by the adenine nucleotides directly, since they are substrates and products of the reactions. Furthermore, studies with isolated enzymes have shown that adenine nucleotides can regula...

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1. Citrate, isocitrate and 2-oxoglutarate levels were determined in isolated rat hepatocytes and in particulate and soluble fractions thereof, obtained by the digitonin and silicone oil fractionation technique.2. Calculated from isocitrate/2-oxoglutarate ratios ("indicator metabolite method"), the redox potential of mitochondrial free NADPH is -402 mV, whereas that of the extramitochondrial (cytosolic) space is about 10 mV more positive, -392 mV.3. Addition of ammonia (either as ammonium chloride or from urea plus urease) to isolated hepatocytes causes preferential oxidation of mitochondrial NADPH, as demonstrated by spectrophotometry of the dihydro band and by the changes in the isocitrate/2-oxoglutarate ratios. The redox potential difference of free NADPH between mitochondria and cytosol is abolished or even reversed.4. It is concluded that during ureogenesis from ammonia mitochondrial isocitrate oxidation is shifted largely in favor of the NADP-linked as opposed to the NAD-linked enzyme; isocitrate concentration under these conditions is less than 10 pM, below the K, (isocitrate) of the NADlinked enzyme but in the range of that for the NADP-linked enzyme.5. Both in the absence and in the presence of ammonia there is a concentration gradient across the mitochondria1 inner membrane (from mitochondria to cytosol) for citrate, isocitrate, and also, to a smaller extent, for 2-oxoglutarate.6. These results and data in the literature on enzyme activity are in agreement with the assumption of near-equilibrium of NADP-dependent isocitrate dehydrogenases in the mitochondrial matrix and cytosolic spaces in the absence of ammonia; accordingly, during urea formation from added ammonia the redox potential of mitochondrial free NADPH is increased to -391 mV or possibly even higher if there exists an indicator error under this condition.Isocitrate dehydrogenases occur both intramitochondrially and extramitochondrially in rat liver [l] ; NADP-dependent enzymes are present in both spaces, whereas an NAD-dependent enzyme is present only intramitochondrially (see [2] for a review). The clear concept, put forward by Goebell and Klingenberg [3], of the dichotomy of mitochondrial isocitrate oxidation into a non-equilibrium, regulated (or regulatory) pathway catalyzed by the NAD-dependent enzyme and an equilibrium pathway catalyzed by the NADP-dependent enzyme implies that the compoEnzymes. Isocitrate dehydrogenase (NAD+) (EC 1.1.1.41); isocitrate dehydrogenase (NADP+) (EC 1.1.1.42).nents of the mitochondrial isocitrate dehydrogenase system may be utilized as 'indicator metabolites' for the mitochondrial NADP system, provided that the activity is high enough (see below). Regardins the extramitochondrial (cytosolic) space, there are no major complications to utilize the isocitrate dehydrogenase system as an indicator for the redox potential of cytosolic free NADPH. Veech etal. [4] have, in fact, utilized the isocitrate dehydrogenase system for this purpose ; however, their analytical data were the total tissue levels rather than the...

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