Adenine nucleotide pools

A quantitative description of oxidative phosphorylation within the cellular environment can be obtained on the basis of nonequilibrium thermodynamics. For this we consider the simple and purely phenomenological scheme depicted in Fig. 1. The input potential X0 applied to the converter is the redox potential of the respiratory substrates produced in intermediary metabolism. The input flow J0 conjugate to the input force X0 is the net rate of oxygen consumption. The input potential is converted into the output potential Xp which is the phosphate potential Xp = -[AG hoS -I- RT ln(ATP/ADP P,)]. The output flow Jp conjugate to the output force Xp is the net rate of ATP synthesis. The ATP produced by the converter is used to drive the ATP-utilizing reactions in the cell which are summarized by the load conductance L,. Since the net flows of ATP are large in comparison to the total adenine nucleotide pool to be turned over in the cell, the flow Jp is essentially conservative. 

Karl, D. M., and Bossard, P. (1985). Measurement and significance of ATP and adenine-nucleotide pool turnover in microbial-ceUs and environmental samples. J. Microbiol. Methods 3, 125—139. 

Bossard P. and Karl D. M. (1986) The direct measurement of ATP and adenine nucleotide pool turnover in microorganisms a new method for environmental assessment of metabolism, energy flux and phosphorus dynamics. J. Plankton Res. 8, 1-13. 

The other kinetic method [153], in which the rate constant of the first order approach to equilibrium is determined, provides ambiguous results because of uncertainty in the size of the exchangeable pool of nucleotides. The natural log plot of the percentage equilibrated is not linear regardless of the value chosen as 100% equilibrated. This is probably due to heterogeneity of the exchangeable in-tramitochondrial pool. The intramitochondrial adenine nucleotide pool is composed of ATP, ADP and AMP. AMP is not a substrate of the carrier, yet radioactive ATP... 

Knowles CJ. Microbial metabolic regulation by adenine nucleotide pools. In Microbial Energetics. In Haddock B, Hamilton WA, eds. Cambridge, Cambridge University Press, 1977 241-83. 

Giardia, Trichomonas and Entamoeba. These parasitic protozoans differ from the other protozoans discussed in this chapter in that they are all incapable of interconversion between their guanine and adenine nucleotide pools. They are dependent on their host environment to supply them with both guanine and adenine. With the exception of E. histolytica, these parasites lack ribonucleotide reductase. This requires that the host also supply purine and pyrimidine deoxynucleosides. 

Petruzzella, V., Baggetto, L.G., Penin, F., Cafagna, F., Ruggiero, F.M., Cantatore, P and Gadaleta, M.N. (1992) In vivo effect of acetyl-L-carnitine on succinate oxidation, adenine nucleotide pool and lipid composition of synaptic and non-synaptic mitochondria from cerebral hemispheres of senescent rats. A rc/i. Gerontol. Geriatr. 14 131-144... 

Two major messengers feed information on the rate of ATP utilization back to the TCA cycle (a) the phosphorylation state of ATP, as reflected in ATP and ADP levels, and (b) the reduction state of NAD, as reflected in the ratio of NADH/NAD. Within the cell, even within the mitochondrion, the total adenine nucleotide pool (AMP, ADP, plus ATP) and the total NAD pool (NAD plus NADH) are relatively constant. Thus, an increased rate of ATP utilization results in a small decrease of ATP concentration and an increase of ADP. Likewise, increased NADH oxidation to NAD by the electron transport chain increases the rate of pathways producing NADH. Under normal physiological conditions, the TCA cycle and other... 

The concentration of ATP in many types of healthy cells is 5 mM, while the concentration of ADP is much less at 0.2 mM AMP concentrations are typically 1 xM. The ratios of the concentrations of these adenine nucleotides are important in the control of metabolic processes. Except under extreme circumstances such as very fatigued muscle, the total concentration of the adenine nucleotide pool remains constant at 5 mM. The constancy of the total amount of recycled metabolites such as ATP is referred to as moiety conservation cells do this on time scales of hours to days. 

At very low values of EC, when AMP is elevated it is deaminated via AMP deaminase to inosine monophosphate (IMP). This further displaces the adenylate kinase reaction in the direction of ATP synthesis. The IMP is dephosphorylated by nucleotide phosphatase, and the inosine is phosphorylyzed via purine nucleotide phosphorylase, releasing hypoxanthine and ribose 1-phosphate. The latter is metabolized via the pentose phosphate pathway, and most of the carbon atoms enter glycolysis. Because this course of events depletes the overall adenine nucleotide pool, and hence the scope for ATP production in the longer term, it represents a metabolic last ditch stand by the cell to extract energy even from the energy currency itself ... 

According to Halestrap [98] activation of mitochondrial electron transport not only increases the proton-motive force but also the intramitochondrial ATP concentration which is important for intramitochondrial ATP utilising reactions like pyruvate carboxylation and citrulline synthesis, processes known to be activated by glucagon. Siess et al. [35], however, showed that in hepatocytes glucagon not only increased mitochondrial ATP, but also the sum of ATP, ADP and AMP at the expense of the cytosolic pool of adenine nucleotides, a phenomenon to which no attention has been paid in the literature. Exchange between ADP and ATP cannot increase the mitochondrial adenine nucleotide pool. Possibly net influx of adenine nucleotides can occur via exchange between ADP and mitochondrial phosphoenol-pyruvate (see [4] for literature). 

Changes in the intramitochondrial adenine nucleotide pool in rat liver are also found in the period around birth. The large increase in the rate of ADP translocation immediately after birth appears to be related to the intramitochondrial ATP content which increases about 4-fold in this period [99,100]. It is believed that at birth a short burst of hormones (adrenaline, glucagon) induces glycogenolysis, followed by enhanced glycolytic flux. This causes a spike in cytosolic ATP production which leads to an increased mitochondrial adenine nucleotide content and hence to increased rates of ADP transport so that the capacity of the mitochondria to produce ATP increases [101]. 

Theobald, U., Mailinger, W., Reuss, M., and Rizzi, M. (1993) In vivo analysis of glucose-induced fast changes in yeast adenine nucleotide pool applying a rapid sampling technique. Anal. Biochem., 214, 31-37. 

The radioactivity lost from the adenine nucleotide pool is recovered in various purine derivatives. There is a transient increase in radioactive inosine and hypoxanthine, and a nearly constant accumulation of radioactive allantoin (Fig. IB). There is also a transient accumulation of radioactive adenosine and IMP (data not shown). However, the extent of that accumulation depends upon the moment that the cells are exposed to glycerol. When glycerol is added at the beginning of the incubation, about 8% of the total radioactivity is found both in adenosine and in IMP. However, when glycerol is added after a 15 min incubation, 17% of the total radioactivity is found in IMP and only 3% in adenosine. All these changes are similar to those induced by fructose under identical conditions (data not shown). 

These data confirm that at this rate of ethanol administration, uric acid excretion did not decrease. The increased excretion of uric acid precursors suggests, in fact, that there is increased flux through the pathways of purine nucleotide degradation to uric acid. Excretion of labeled degradation products derived from the adenine nucleotide pool is significantly accelerated and suggests accelerated ATP or adenine nucleotide degradation. 

A decrease in the concentration of liver ATP has been repeatedly documented after chronic administration of ethanol to rats (Walker and Gordon, 1970 Bernstein et al., 1973 Gordon, 1977). Although several authors have not observed an acute effect of ethanol on hepatic ATP (Williamson et al., 1969 Veech et al., 1972) our results are in agreement with those of Soboll et al. (1978) who have shown an approx. 30 % decrease in the concentration of this nucleotide after the addition of ethanol to the perfused liver of fed rats. The finding (Fig. 2) that the effect of ethanol on the production of purine catabolites was still observed in the presence of coformycin at a concentration that inhibits selectively adenosine deaminase (Van den Berghe et al., 1980) indicates that the degradation of the adenine nucleotide pool, induced by ethanol, does not proceed by way of the dephosphorylation of AMP but involves AMP deaminase. 

The depletion of ATP was accompanied by an increase in the concentration of AMP, that proceeded more rapidly and reached higher levels in the hepatocytes from fasted animals In both conditions, a loss of the sum of the adenine nucleotides was recorded, that occured rapidly during the first 5 min of anoxia, but proceeded much more slowly thereafter These data indicate that potent control mechanisms restrict the further degradation of the hepatic adenine nucleotide pool in anoxic conditions. 

Further studies were aimed at the elucidation of the mechanisms whereby the hepatic adenine nucleotide pool is preserved in anoxic conditions, especially in the fasted state. In hepatocytes from fed rats, indeed, this protection is mainly due to a better better maintenance of the ATP concentration by anaerobic glycolysis. In hepatocytes from fasted animals, however, it is chiefly caused by a restriction of the degradation of AMP, as evidenced by the more marked accumulation of this nucleotide (Fig. 1) and the lower production of uric acid (Fig. 2) in comparison with the fed state. 

It can thus be concluded that the regulation of both AMP-degrading enzymes appears uniquely designed to preserve the adenine nucleotide pool when the liver is subjected to anoxic conditions. The importance of this preservation is evidenced by experiments that show that AMP, but not IMP, may be completely reconverted into ATP upon reoxygenation of the hepatocytes (Vincent et al., 1982). 

Addition of adenosine to isolated rat hepatocytes, as well as to other liver preparations, provokes marked increases in the intracellular concentration of ATP and total adenine nucleotides (Chagoya de Sanchez et al., 1972 Lund et al., 1975 Wilkening et al., 1975), that are explained by the utilisation of adenosine by adenosine kinase- The present work was initiated as a search for a mechanism whereby the rate of degradation of the adenine nucleotide pool would adapt to an increased rate of synthesis- It led to the unexpected ascertainment that, under normal conditions, there is a continuous foriaation of adenosine by the hepatocytes. This production does, however, not contribute to the formation of allantoin but is part of a futile cycle operating between AMP and adenosine. 

Fig. 1. depicts the sequence of events taking place after addition of 0.5 mM-adeno ne in hepatocyte suspensions that had been preincubated with [ C]adenine in order to label their adenine nucleotide pool (Van den Berghe et al., 1980). Concentrations of the adenine nucleotides in the hepatocytes and of adenosine and allantoin in the cell suspension are shown on the left, whereas the radioactivity in these compounds is displayed on the right. Cells incubated without adenosine are compared to cells incubated with 0.5 inM-adenosine, in the absence and in the presence... 

In control cells, the radioactivity in the adenine nucleotides decreased linearly with time, reflecting their rate of degradation. A more rapid loss of this radioactivity was recorded in the presence of adenosine, indicating a stimulation of the rate of degradation of the prelabelled adenine nucleotide pool. In the presence of coformycin, this stimulation was approx, halved. Since AMP deaminase is maximally inhibited at 50 M coformycin, this finding suggested that the degradation of the adenine nucleotides also proceeded by way of 5 -nucleotidase. This was confirmed by the determination of radioactivity in adenosine. Whereas no radioactive adenosine was detected in control conditions, the addition of unlabelled adenosine induced a prompt appearance of radioactivity in this nucleoside, which was transient in the absence of coformycin, but increased steadily in its presence. The formation of radioactive allantoin in the three experimental conditions was similar to that of the non-radioactive catabolite. 

These observations provide convincing evidence that adenosine is continuously formed by the hepatocytes, and has to be rephos-phorylated in order to maintain the adenine nucleotide pool. The results depicted in Fig. 2 were only slightly modified when a mixture of AOPCP and p-glycerophosphate was added to the cell suspensions in order to inhibit the membranous 5 -nucleotidase and aspe-cific phosphatases. This rules out a significant participation of the former enzyme, as well as of the extracellular catabolism of adenine nucleotides released by dammaged hepatocytes, in the formation of adenosine. The production of adenosine thus most likely results from dephosphorylation of AMP inside the cells, by the cytoplasmic 5 -nucleotidase. From the observation that this formation of adenosine does not contribute to the physiological production of allantoin, it can be concluded that both the formation and utilization of adenosine proceed at the same rate in control conditions, and thus constitute a "futile cycle. From... 

Rcssi, C., Alexandre, A., Carignani, G. Siliprandi, N. (1912) Adv. Enzyme Regul. 10,171-186. The role of mitochondrial adenine nucleotide pool on the regulation of fatty acid and -ketoglutarate oxidation. 

Regulation by variation of the adenine nucleotide pool or the NAD/ NADH ratio appears to be of particular physiologic import, since the adenylate nucleotides represent the principal storage form of chemical energy, while the pyridine nucleotides are the major source of reducing potential in the cell. With the two pools linked via electron transport and oxidative phosphorylation, any perturbation which affects either of the pools may have wide-ranging consequences on diverse reactions... 

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