Intramitochondrial and Extramitochondrial Concentrations of Adenine Nucleotides and Inorganic Phosphate in Isolated Hepatocytes from Fasted Rats

Abstract

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...

Binding of ADP to rat liver cytosolic proteins and its influence on the ratio of free ATP/free ADP

“…If ADP-binding proteins have been lost owing to the chromatography on Ap5A-agarose, this ratio would be even higher. The ratio of 19 calculated on the basis of the present results is considerably higher than the cytosolic ratio of ATPtota1/ADPtotal of 2.6-10 found by various fractionation techniques (Siess & Wieland, 1976;Tischler et al, 1977;Akerboom et al, 1978;Soboll et al, 1978). However, the present results agree well with previous findings showing that a ratio of at least 20 is a prerequisite for obtaining physiological rates of gluconeogenesis in cell-free systems (Morikofer-Zwez et al, 1982;Stoecklin et al, 1986).…”

“…The total concentration of ATP in rat liver cytosol has been reported to be 2.8-3.4mm (Tischler et al, 1977;Akerboom et al, 1978;Siess et al, 1982). Since little, if any, ATP is bound to cytosolic proteins (Gankema et al, 1983), the concentration of free ATP in rat liver cytosol can be assumed to be about 3 mm.…”

“…In the present study a binding capacity of liver cytosol for ADP of 144 /LM was found. As the dissociation constants for the two binding sites are in the low-micromolar range, the observed cytosolic ADPbinding sites would lower the concentration of free cytosolic ADP to about 0.16 mM if a total cytosolic ADP concentration of 0.3 mm (Akerboom et al, 1978) is assumed. The value of 0.16 mm for free cytosolic ADP must, however, be regarded as an upper limit, since it cannot be excluded that ADP-binding proteins were lost during the chromatography necessary for the removal of adenylate kinase.…”

“…Physiological rates of hexose 6-phosphate formation from different substrates were obtained at an ATP/ADP ratio of at least 20. However, cytosolic ratios of ATP/ADP reported for rat liver and rat hepatocytes are not higher than 10 (Siess & Wieland, 1976;Tischler et al, 1977;Akerboom et al, 1978;Soboll et al, 1978). This apparent discrepancy may stem from the fact that in cell-free, highly diluted, systems the adenine nucleotides are essentially present as free compounds (Stoecklin et al, 1986), whereas in the cell they may be bound in part to cytosolic proteins.…”

“…Several attempts have been made to assess binding of adenine nucleotides to cytosolic proteins and to calculate the free concentration of ATP and ADP in liver. By using a metabolite indicator method, the concentration of free cytosolic ADP in freeze-clamped rat liver has been calculated to be 50 StM (Veech et al, 1979), suggesting that more than 80°/ % of the total cytosolic ADP (0.3 mM; Akerboom et al, 1978) is bound to proteins. This calculation has, however, been questioned by Groen et al (1982), who reported the metabolite-indicator method not to be valid under the conditions employed.…”