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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...