The effect of nucleotides on glutamate dehydrogenase from the mealworm fat body

The effect of nucleotides: AMP, cAMP, ADP, ATP, GDP and GTP, on glutamate dehydrogenase (GDH) purified from the mealworm fat body was studied. Guanine nucleotides and ATP inhibited the enzyme strongly in both directions. GDH was partially protected from the inhibition by the addition of ADP to an assay medium. AMP and cAMP activated the enzyme slightly. The concerted effects of ADP and ATP indicate the importance of adenylate energy charge in the regulation of fat body GDH. It is suggested that GDH may play amphibolic role in the fat body and that the direction of GDH catalysed reaction is under strong influence of nucleotides. The enzyme may synthesize glutamate at high energy charge, but when the energy reserves are low, it oxidizes glutamate.     

Adenylate energy charge of rat and human cultured hepatocytes

Summary A simple and rapid method for the assay of adenine nucleotides (ATP, ADP, and AMP) was established to evaluate the adenylate energy charge (ATP+ADP/2)/(ATP+ADP+AMP) of cultured hepatocytes. The effects of inhibitors of glycolysis, fatty acid oxidation, or oxidative phosphorylation on the energy charge were examined. The energy charges of cultured hepatocytes in rats and human were almost identical and were maintained at a high level between 6 and 24 h after changing the media (rat: 0.908±0.008n=9, human: 0.918±0.014n=6, mean ± SD). Inhibition of glycolysis with sodium fluoride or oxidative phosphorylation with antimycin A irreversibly reduced both the adenine nucleotide contents and the energy charge. However, the inhibition of fatty acid oxidation with 2-tetradecylglycidic acid did not affect the nucleotide contents, and the energy charge only decreased transiently to recover within 8 h. When the inhibitor of oxidative phosphorylation was removed, the recovery in the energy charge preceded the recovery in the adenine nucleotide contents. These findings suggest that the adenylate energy charge is a more sensitive measure of the changes in energy metabolism than the adenine nucleotide contents. Furthermore, energy charge regulates adenine nucleotide contents in cultured hepatocytes. It is important to confirm that the high energy charge of the cultured hepatocytes is maintained when these cells are used for metabolic studies.     

Variations in the Adenylate Energy Charge During Phased Growth (Cell Cycle) of Candida utilis Under Energy Excess and Energy-Limiting Growth Conditions

The variations in the levels of adenine nucleotides during the phased growth (cell cycle) of the yeast Candida utilis growing under nitrogen, sulfate, or iron limitation with glycerol as carbon source have been determined. Synchronous cultures were obtained by the continuous phasing technique, and the results were compared with those of chemostat cultures growing at similar growth rates and under the same types of nutrient limitation. Whereas the chemostat experiments indicated only the average energy status of cultures growing at random, results from phased cultures showed that the adenylate energy charge, defined as (ATP + (1/2)ADP)/(ATP + ADP + AMP) (where ATP, ADP, and AMP signify adenosine 5'-triphosphate, -diphosphate, and -monophosphate, respectively), varied during the phased growth of the yeast. These variations were related to the stage of development of the cells and to the type of nutrient limitation. In every case the energy charge dropped to a low value during the first half of the phasing cycle (cell cycle). Whereas the energy charge was maintained at relatively high levels (ranging from 0.78 to 0.94), for sulfate- or nitrogen-limited cultures, it was very low when iron was the growth-limiting nutrient (0.44 to 0.78). In spite of the low energy charge, the yeast continued to grow under iron limitation. The main component of the adenylate pool of the iron-limited culture was ADP and not ATP as observed with other types of nutrient limitation. It is concluded that under iron limitation the growth of the organism is limited by energy and that under energy-limited growth the energy charge of a growing organism is maintained at low levels. The reason for maintaining a low energy charge in an energy-limited culture is discussed.     

Charges of nicotinamide adenine nucleotides and adenylate energy charge as regulatory parameters of the metabolism in Escherichia coli.

Methods for measurements of catabolic reduction charge (defined as NADH/(NADH+NAD+)) and anabolic reduction charge (defined as NADPH/(NADPH + NADP+)) are described using [14C]nicotinamide labeling of Escherichia coli cultures. Together with these parameters the adenylate energy charge (ATP + 1/2ADP)/(ATP + ADP + AMP) was measured using labeling with [2-3H]adenine. These three charges were found under different exponential growth conditions to have values independent of the growth conditions: catabolic reduction charge, 0.05; anabolic reduction charge, 0.45; and adenylate energy charge, 0.9. The charges were examined during interruption of growth primarily affecting catabolism, respiration, or anabolism, leading to changes of the charges. The changes of charges are evaluated as a possible regulation of the metabolic rates utilizing or producing the nucleotides by their respective charges.     

Effects of exogenous donor of nitric oxide-sodium nitroprusside on energy production of rat reticulocytes

The effects of the sodium nitroprusside (SNP), a nitric oxide (NO) donor clinically used in the treatment of hypertensive emergencies on the energy production of rat reticulocytes were investigated. Rat reticulocyte-rich red blood cell suspensions were aerobically incubated without (control) or in the presence of different concentrations of SNP (0.1, 0.25, 0.5, 1.0 mM). SNP decreased total and coupled, but increased uncoupled oxygen consumption. This was accompanied by the stimulation of glycolysis, as measured by increased glucose consumption and lactate accumulation. Levels of all glycolytic intermediates indicate stimulation of hexokinase-phosphofructo kinase (HK-PFK), glyceraldehyde 3-phosphate dehydrogenase (GAPD) and pyruvate kinase (PK) activities in the presence of SNP. Due to the decrease of coupled oxygen consumption in the presence of SNP, ATP production via oxidative phosphorylation was significantly diminished. Simultaneous increase of glycolytic ATP production was not enough to provide constant ATP production. In addition, SNP significantly decreased ATP level, which was accompanied with increased ADP and AMP levels. However, the level of total adenine nucleotides was significantly lower, which was the consequence of increased catabolism of adenine nucleotides (increased hypoxanthine level). ATP/ADP ratio and adenylate energy charge level were significantly decreased. In conclusion, SNP induced inhibition of oxidative phosphorylation, stimulation of glycolysis, but depletion of total energy production in rat reticulocytes. These alterations were accompanied with instability of energy status.     

Changes in adenosine phosphates and energy charge in chloroplastic and nonchloroplastic compartments of wheat leaves (author's transl)]

Changes in the adenine nucleotides and energy charge (= (ATP)+1/2(ADP)/(AMP)+(ADP)+(ATP)) levels were studied in chloroplastic and non-chloroplastic compartments using non-aqueously isolated wheat leaves chloroplasts. The two adenine nucleotides pools (of chloroplasts and non-chloroplastic part of the cell), though distinct, are linked. This linkage substantiates an energy-rich bond exchange between the two compartments. When both photphosphorylation and oxidative phosphorylation occur simultaneously, energy charge takes high values, generally higher than 0.80. When neither oxidative phosphorylation nor photophosphorylation occur, energy charge is very low and takes values generally lower than 0.45. When one compartment alone produces approximately P, energy charge in the two compartments takes intermediate values which remain relatively high. Dark-light transition in nitrogen resulted in changes of the AMP, ADP and ATP levels which quickly reach a steady state. Chloroplast energy charge shifts rapidly from 0.45 to 0.75 in 10 s; after 1 min it reaches 0.86, a value that corresponds to a steady level. In the cytoplasm, energy charge changes from 0.44 to 0.71 in 1 min. Energy charge increase in the non-chloroplastic compartment substantiates an energy transfer from chloroplasts to the cytoplasm. On nitrogen-air transition in the dark, the cytoplasm energy charge reaches a steady level in 30 s. In chloroplasts, it also increases but slowly. There is indeed a transfer of energy from cytoplasm to chloroplasts. Darkening of the leaves in air causes a drastic and lasting drop of energy charge in the chloroplasts where it has a low value after 5 min in the dark. Then it increases again but slowly and is still lower than 0.70 after 10 min in the dark. Meanwhile, energy charge in cytoplasm keeps values higher than 0.75. Metabolic regulation by energy charge and control of adenine nucleotides level by adenylate kinase (EC 2.7.4.3) are discussed.     

Adenine nucleotide content and energy charge of Bacillus megaterium during batch growth in low-phosphate medium

Abstract The effect of a low phosphate concentration on intracellular adenine nucleotides, oxygen consumption and poly-β-hydroxybutyric acid synthesis, was investigated with batch cultures of Bacillus megaterium . At low phosphate concentrations the cells contained much larger amounts of poly-β-hydroxybutyric acid, but displayed lower adenylate energy charge and oxygen uptake than did control cells. The ratio of ATP to ADP was much greater in the control cells. The levels of ATP and AMP were lower in low-phosphate cells.     

Postanoxic recovery of spontaneously beating isolated atria: pH related role of adenylate kinase

The functional activity, adenine nucleotides, and creatine phosphate content of spontaneously beating isolated rabbit atria were measured prior to anoxia, after 1 hr anoxia, and at the end of 1 hr reoxygenation at pH 6.7 and 7.2 During anoxia at pH 7.2 there was 13.3% loss of adenine nucleotides pool, 35.2% loss of ATP, 36.2% increase in ADP, 200% increase in AMP, and a decrease to 8.8% of CP assayed to the beating atria in oxygen. At pH 6.7 there was almost the same decrease in CP, about 10% decrease in ATP, no change in total adenine nucleotides, no change in AMP and a higher increase in ADP (88.7%). The postanoxic recovery was much more complete when the pH was 6.7 during anoxia, and the first 40 min of reoxygenation. The extent of recovery of functional activity correlated well with the level of ATP in all cases not CP. Since the adenylate kinase and ATPase activity both decrease at acidic pH, their combined diminution would tend to preserve the adenine nucleotide pool and thus the better recovery at pH 6.7, because of a decrease in energy demand and unavailability of AMP for the degradation process. This study also supports the notion of compartmented adenine nucleotides connected by the creatine phosphate-creatine energy shuttle.     

The ice worm, Mesenchytraeus solifugus, elevates adenylate levels at low physiological temperature

The ice worm, Mesenchytraeus solifugus, is among a few metazoan species that survive exclusively in glacier ice/snow. In this study, we demonstrate that ice worm adenylate levels [i.e. adenosine 5'-triphosphate (ATP), ADP and AMP] are maintained at levels well above their mesophilic counterparts, and that their response to temperature change is distinctly opposite, namely, ice worms increase energy levels as temperatures fall. Initially, this response is characterized by a sharp spike in [ATP] and the adenylate energy charge (even at sub-zero temperatures), which is followed by corresponding increases in [ADP] and [AMP] within a few days. These results suggest that ice worms have evolved a compensatory mechanism by which gains in adenylate nucleotides off-set, at least in part, the inherent lethargy and death usually associated with cold temperature.     

Energy aspects of the growth of Escherichia coli synchronized by starvation

The quantitative determination of adenyl nucleotides based on the separation of their dansyl derivatives by thin layer chromatography has made it possible to study the dynamics of changes in the pool of ATP, ADP and AMP in Escherichia coli K-12 during its synchronous growth after glucose starvation. The energy parameters (the adenylate pool, energy charge, teh ATP/ADP ratio, the rates of oxygen uptake and ATP generation, the economic coefficients of oxygen and ATP utilization) were compared with changes in the growth characteristics (the rate of growth and biomass concentration). This comparison allowed the authors to draw the conclusion about the uncoupled constructive and energy metabolism and about the possible regulatory role of energy parameters in the synchronised culture growth.     

In vitro adenine nucleotide catabolism in African catfish spermatozoa

It has been shown recently that African catfish (Clarias gariepinus) spermatozoa possess relatively low ATP content and low adenylate energy charge (AEC). One of the possible explanations for this phenomenon is that the spermatozoa actively catabolize adenine nucleotides. A relatively high rate of such catabolism could then contribute to the low ATP concentration and low adenylate energy charge observed in the spermatozoa in vitro. To check this hypothesis, we investigated ATP content and adenine nucleotide catabolism in African catfish spermatozoa stored at 4 °C in the presence of glycine as an energetic substrate. Our results indicate that the storage of African catfish sperm at 4 °C in the presence of glycine causes time-dependent ATP depletion. In contrast to ATP, the AMP content increases significantly during the same period of sperm storage, while the ADP increases only slightly. Moreover, a significant increase of inosine and hypoxanthine content was also found. Hypoxanthine was accumulated in the storage medium, but xanthine was found neither in spermatozoa nor in the storage medium. It indicates that hypoxanthine is not converted to xanthine, probably due to lack of xanthine oxidase activity in catfish spermatozoa. Present results suggest that adenine nucleotides may be converted to hypoxanthine according to the following pathway: ATP→ADP→AMP (adenosine/IMP)→inosine→hypoxanthine. Moreover, hypoxanthine seems to be the end product of adenine nucleotide catabolism in African catfish spermatozoa. In conclusion, our results suggest that a relatively high rate of adenine nucleotide catabolism contributes to the low ATP concentration and low adenylate energy charge observed in African catfish spermatozoa in vitro.     

Adenine nucleotides and the adenylate kinase equilibrium in livers of foetal and newborn rats

1. Measurements of ATP, ADP and AMP concentrations in livers of rats that had been delivered by Caesarian section indicate a rapid shift from a low to a high [ATP]/[AMP] ratio. This change is consistent with the cessation of glycolysis and the initiation of gluconeogenesis at birth. 2. When newborn animals are exposed to a 100% nitrogen atmosphere the hepatic ATP concentration falls and AMP increases. 3. Calculations of the [ATP][AMP]/[ADP](2) ratio give values that are close to the equilibrium constant of adenylate kinase except when the ATP concentration is high. 4. This difference cannot be accounted for by the preferential binding of available Mg(2+) to ATP(4-) rather than ADP(3-). It is concluded that the relative proportions of adenine nucleotides at any level of phosphorylation are only partly regulated by adenylate kinase.     

Preparation and isolation of dansyladenylates as fluorescent substrates in reactions of the energy metabolism

A method was developed for preparation of dansylated derivatives of adenine nucleotides characterized by fluorescence when being irradiated with UV-light. The involvement of dansylated ATP, ADP and AMP as substrate analogues in energy metabolism is demonstrated in the ATPase, hexokinase, pyruvate kinase and adenylate kinase reactions. The kinetics of the reactions is discussed.     

Energy status in the fat body of Trichoplusia ni parasitized by the insect parasite Hyposoter exiguae

The levels of adenylate nucleotides were examined in 4th-instar Trichoplusia ni larvae 3 days after parasitization by the insect parasite Hyposoter exiguae. In general, parasitization caused a decrease in the level of ATP and increased ADP and AMP levels. These changes resulted in alteration of the adenylate kinase mass-action ratio. The overall energy status of parasitized larvae, however, as indicated by energy ratios, including the “energy charge,” was affected only slightly. The result demonstrates that the host maintained an active and viable metabolic state despite extensive alterations in physiology which occur at this stage of the parasite-host association.     

Adenylate and nicotinamide nucleotides in developing soybean seeds during seed-fill

Profiles of adenylate and nicotinamide nucleotides in soybean seeds were determined during seed-fill. The ATP content per seed increased during the early seed-filling stages to a level of 10 to 12 micrograms per seed. Seed ATP decreased after 40 days of development and reached its lowest level of less than 1 microgram at maturity. The ATP:ADP ratios were relatively constant at all seed development stages. Sharp increases in AMP levels during the late seed-fill stages were paralleled with a disappearance of ATP and ADP pools resulting in a reduced seed energy charge. Energy charge varied from the highest value of 0.78 at mid-seed-fill to less than 0.10 at maturity.     

The effect of season and location on phosphoadenylate concentrations and adenylate energy charge in two species of freshwater clams

Summary Concentrations of phosphoadenylate nucleotides and the adenylate energy charge ((ATP+1/2ADP)/(ATP+ADP+AMP)) have been suggested as sensitive integrating measures of the energy state of organisms. This synoptic study investigated the seasonal and spatial variation of phosphoadenylate concentrations and AEC in two freshwater bivalve molluscs, the paper-shell clam, Anodonta imbecillis and the asian clam, Corbicula fluminea. Concentrations of all three adenylates, as well as the total adenylate concentration and adenylate energy charge of both species varied seasonally. These fluctuations were closely related to reproductive periods in both species. Total adenylate concentrations and ATP concentrations were slightly negatively correlated with shell length in A. imbecillis but the ADP and AMP concentrations and AEC were not significantly correlated with shell length. In C. fluminea the AEC was negatively correlated were positively correlated with shell length. Neither species exhibited significant differences in AEC between two collection locations. When C. fluminea collected from the Savannah River were acclimated and fed in the laboratory their AEC increased significantly.     

A kinetic study of a ternary cycle between adenine nucleotides

In the present paper, a kinetic study is made of the behavior of a moiety-conserved ternary cycle between the adenine nucleotides. The system contains the enzymes S-acetyl coenzyme A synthetase, adenylate kinase and pyruvate kinase, and converts ATP into AMP, then into ADP and finally back to ATP. L-Lactate dehydrogenase is added to the system to enable continuous monitoring of the progress of the reaction. The cycle cannot work when the only recycling substrate in the reaction medium is AMP. A mathematical model is proposed whose kinetic behavior has been analyzed both numerically by integration of the nonlinear differential equations describing the kinetics of the reactions involved, and analytically under steady-state conditions, with good agreement with the experimental results being obtained. The data obtained showed that there is a threshold value of the S-acetyl coenzyme A synthetase/adenylate kinase ratio, above which the cycle stops because all the recycling substrate has been accumulated as AMP, never reaching the steady state. In addition, the concept of adenylate energy charge has been applied to the system, obtaining the enabled values of the rate constants for a fixed adenylate energy charge value and vice versa.     

Net uptake of adenine nucleotides by newborn rat liver mitochondria

In newborn rat liver, the adenine nucleotide content (ATP + ADP + AMP) of mitochondria increases severalfold within 2 to 3 h of birth. The net increase in mitochondrial adenines suggests a novel mechanism by which mitochondria are able to accumulate adenine nucleotides from the cytosol (J. R. Aprille and G. K. Asimakis, 1980, Arch. Biochem. Biophys.201, 564.). This was investigated further in vitro. Isolated newborn liver mitochondria incubated with 1 mM ATP for 10 min at 30 °C doubled their adenine nucleotide content with effects on respiratory functions similar to those observed in vivo: State 3 respiration and adenine translocase activity increased, but uncoupled respiration was unchanged. The mechanism for net uptake of adenine nucleotides was found to be specific for ATP or ADP, but not AMP. Uptake was concentration dependent and saturable. The apparent K_m′s for ATP and ADP were 0.85 ± 0.27 mM and 0.41 ± 0.20 mM, respectively, measured by net uptake of [¹⁴C]ATP or [¹⁴C]ADP. The specific activities of net ATP and ADP uptake averaged 0.332 ± 0.062 and 0.103 ± 0.002 nmol/min/mg protein, respectively. ADP was a competitive inhibitor of net ATP uptake. If P_i was omitted from the incubations, net uptake of ATP or ADP was reduced by 51%. Either mersalyl or N-ethylmaleimide severely inhibited the accumulation of adenine nucleotides. Net ATP uptake was stoichiometrically dependent on MgCl₂, suggesting that Mg²⁺ is accumulated along with ATP (or ADP). Uptake was energy dependent as indicated by the following results: Net AdN uptake (especially ADP uptake) was stimulated by the addition of an oxidizable substrate (glutamate) and inhibited by FCCP (an uncoupler). Antimycin A had no effect on net ATP uptake but inhibited net ADP uptake, suggesting that ATP was able to serve as an energy source for its own accumulation. If carboxyatractyloside was added to inhibit the exchange translocase, thereby preventing rapid access of exogenous ATP to the matrix, net ATP uptake was inhibited; carboxyatractyloside had no effect on ADP uptake. It was concluded that the net uptake of adenine nucleotides from the extramitochondrial space occurs by a specific transport process distinct from the classic adenine nucleotide exchange translocase. The accumulation of adenine nucleotides may regulate matrix reactions which are allosterically affected by adenines or which require adenines as a substrate.     

Energy metabolism and blood perfusion in a mouse mammary adenocarcinoma during growth and following X irradiation

Biochemical and blood perfusion changes in a mouse tumor system (MDAH MCaIV) were studied relative to normal tissues under conditions of normal blood flow and clamped blood supply. Further studies were performed during tumor growth and after local X irradiation. The biochemical profiles of three untreated human soft tissue sarcomas were also investigated. Animal tumors were irradiated in situ with either a single or fractionated regime to total doses of 20 or 49 Gy. Assays of lactate, pyruvate, AMP, ADP, and ATP were made on freeze-clamped tissue following authentic or sham treatments. Blood perfusion to tumors treated in the same way was measured using iv injection of 201Tl. The human tumors were found to have a lower lactate to pyruvate ratio (L/P) than the MCaIV tumors; their ATP levels were also lower. L/P was much higher in the MCaIV tumors than in normal liver, kidney, and muscle in the mouse. Occlusion of the blood supplies of the normal kidney and the MCaIV tumor caused an increase in the lactate and L/P levels in both cases. However, whereas the ATP level in the kidney fell, the level in the tumor was maintained. There was some evidence that the adenine nucleotides were not in equilibrium via the adenyl kinase catalyzed reaction. In addition, tumors were found to contain the enzyme creatine kinase. These results suggest that energy charge calculations cannot be computed in a meaningful manner because the creatine kinase catalyzed phosphorylation of ADP would maintain a higher than normal ATP level. Lactate and L/P ratio was found to increase during tumor growth and decrease following X irradiation. The total adenine nucleotides (AMP + ADP + ATP) exhibited a trend toward lower values with increasing tumor size. There was no significant change in total adenine nucleotides after a single 20-Gy dose; however, fractionated radiation caused some fall in total nucleotides. It is concluded that, in this tumor system, lactate level is a sensitive index of radiation-induced biochemical changes which are likely to reflect changes in tumor oxygenation.     

In vivo effect of 2-deoxy-D-glucose on adenine nucleotide levels in the liver and skeletal muscle of rats

The present report indicates that 2-deoxy-D-glucose (2-DG) at a single dose causing reduction of Tre has no influence on liver and skeletal muscle content of ATP, ADP and AMP, the ATP/ADP ratio, energy charge potential (ECP) and total adenine nucleotides (TAN). After administration of 2-DG for 3) successive days, the level of ATP, ATP/ADP ratio, the values of ECP and TAN are decreased both in the liver and skeletal muscle. However, 72 hours after the last injection of 2-DG adenine nucleotide contents returned to the values observed in control group, indicating that the in vivo effect of this glucose analogue is fully reversible.