Effect of ethanol and ethylene glycol on the circadian rhythm of adenylic nucleotide levels in muscles]

The 24-hour's changes in ATP content in the gastrocnemiuses of intact rast are not significant. Both the ADP and AMP content is subjected to 24-hour's variations. It reaches its maximum within the time period from 12 to 15 o'clock p.m. for ADP and at 18 o'clock p.m. for AMP. Intragastric administration of ethanol and ethylene glycol to rats in a dose of 1/3 LD50 once per 24-hour period at 9 a. m. during a 7-days-long period dramatically changes the 24-hour's rhythm of adenylic nucleotide content in the rat gastrocnemius. It has been found that ethanol increases the average 24-hour's content of ATP, but decreases that of ADP. It increases the range of their 24-hour period variations and changes the acrophase. Ethylene glycol decreases the average 24-hour content of the both ATP and ADP, but it increases that of AMP. It changes their acrophases and increases the ranges of 24-hour-period variations of ATP.     

Changes in Acid-soluble Nucleotides in Cut-injured Sweet Potato Root Tissue

ATP and ADP increased in cut-injured sweet potato root tissue during the 3 to 6-hr incubation period, and showed the maximum for the 9 to 18-hr, and 6 to 9-hr incubation periods, respectively, then decreased. ATP was present in the highest amount among ATP, ADP and AMP throughout the 72-hr incubation period, while AMP was in the lowest. Total acid-soluble nucleotides increased gradually, and showed the peak content at the 12-hr incubation period, and decreased thereafter. Adenine mononucleotides such as ATP, ADP and AMP occupied about 40 to 65% of total acid-soluble nucleotides.     

The content of ATP,ADP, AMP,Pi, the activity of enzymes involved in the glycolytic pathway and some problems of its regulation,and energy balance in tobacco plants infected with potato virus Y

The content of ATP, ADP, AMP, P_i, the activity of the enzymes involved in the glycolytic pathway, some problems of their regulation by adenine nucleotides and some basic problems connected with tissue energy balance were studied in tobacco plants infected with the potato virus Y (PVY). The contents of ATP and ΣAdN were increased in virus-infected tissues when compared with healthy tissues and correlated with the PVY reproduction curve. ADP and AMP contents decreased just after the inoculation and increased at the end of the experimental period, P_i content was not influenced by the infection. The activities of the key enzymes of the glycolytic pathway (6-phosphofructokinase, hexosediphosphatase, and pyruvate kinase), determined both in crude homogenates and after partial purification, did not differ during the entire experimental period from the values found in healthy control tissues, similarly as the activities of glucosephosphate isomerase, glyceraldehydephosphate dehydrogenase, phosphoglyceromutase and enolase observed in crude homogenates. The unchanging AEC value in virus-infected tissues simultaneously indicated that no change in the rate of the glycolytic pathway occurred even under “invivo” conditions at the period of the acute stage of infection.     

Measurements of ATP in mammalian cells

Levels of phosphorylated adenosine nucleotides, including the universal energy carrier adenosine 5(')-triphosphate (ATP) and its metabolites adenosine 5(')-diphosphate (ADP) and adenosine 5(')-monophosphate (AMP), define the energy state in living cells and are dependent mainly on mitochondrial function. In this article, we describe a method based on the luciferase-luciferin system used to measure mitochondrial ATP synthesis continuously in permeabilized mammalian cells and mitochondria isolated from animal tissues. We also describe a technique that uses the expression of recombinant targeted luciferase to report ATP content in different cell compartments. Finally, we describe an HPLC-based method for accurate measurement of ATP, ADP, and AMP in cultured cells and animal tissues.     

ATP:AMP phosphotransferase activity, a new characteristic of Catharanthus roseus tonoplasts

The ATPase activity of Catharanthus roseus tonoplasts was examined using HPLC separation and quantification of adenine nucleotides. ATP seemed to be degraded into ADP and AMP by tonoplast vesicles. When ADP was the initial substrate, the appearance of AMP and concomitant ATP synthesis were observed; these reactions were inhibited by Ap⁵A. The apparent degradation of ATP into AMP was also inhibited by Ap⁵A. These results indicated that AMP arose from an ATP:AMP phosphotransferase activity and excluded the possibility of the hydrolysis of ADP into AMP by the tonoplast ATPase. AMP was degraded by the microsomal fraction from protoplasts or by the cytosol while the tonoplast vesicles did not hydrolyze it. This observation was used to assess the purity of tonoplasts.     

Content of Adenosine Phosphate Compounds in a Long-Day Duckweed, Lemna gibba G3, under Different Light and Nutritional Conditions

The levels of ATP ADP and AMP as well as the energy charge were examined in the long-day duckweed. Lemna gibba G3, under different light and nutritional conditions. ATP and ADP content, but not AMP, decreased slightly when the plant was cultured with a medium depleted of sucrose or was exposed to continuous darkness. The energy charge was not affected either by changes in the light conditions or by the depletion of sucrose from the medium, although increase of fresh weight changed drastically under these conditions. The levels of ADP and AMP and the value of the energy charge remained nearly constant throughout a 1-day period when the plant was exposed to short-day, continuous light and continuous dark. ATP content decreased gradually during the light period of the short day and thereafter remained constant during the dark period. When the plant was exposed to continuous light. ATP content decreased until the 8th hour after beginning of the continuous light, only to recover rapidly to its original level during the phase coinciding with the preceding dark period of the short day. Under continuous dark conditions the ATP content remained constant throughout the day. It was concluded that the diurnal rhythm of physiological activities previously reported cannot be related to the energy charge.     

Changes in the energy state of tissues in spontaneously hypertensive rats]

The contents of adenine nucleotides (ATP, ADP, AMP), phosphocreatine (PCr) and creatine (Cr) in the heart, skeletal muscle, liver and spleen in spontaneously hypertensive (SHR) and normotensive (WKY) rats. The ATP/ADP ratio in cardiac tissue was lower in SHR compared with WKY, while myocardial contents of adenine nucleotides, PCr and Cr did not differ significantly between the groups. A lower ATP/ADP ratio in the skeletal muscle SHR of was accompanied by a reduction of PCr content comparing with these indices in WKY rats. The liver and spleen of SHR exhibited lower ATP contents and higher ADP and AMP levels compared with those ones in WKY rats, despite of the close values of adenine nucleotide pools (sigma AN = ATP + ADP + AMP). This redistribution of tissue adenine nucleotides was corresponded to lower energy charges (EC = (ATP + 0.5 ADP)/sigma AN) and ATP/ADP ratios in SHR group. The reduction of the energy state of tissues in SHR rats increased in the following rank: heart > skeletal muscle > liver > spleen, thus, reflecting progressive decrease of intensity of oxidative metabolism. The results suggest changes in the balance of rates of ATP formation and hydrolysis occur at the system level in primary hypertension. Probably, consequences of such rearrangement in energy metabolism are functional disturbances of plasma membrane and sacroplasmic reticulum well-documented in a number of experimental and clinical studies.     

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.     

Cellular energy systems and reserpine ulcer in rats

Gastric ulcer was elicited in rats by reserpine (5 mg x kg-1 sc.) administration. Ulcer formation (number and severity) was measured 6, 12, 18 and 24 hr after reserpine administration. At the time of killing of the animals, tissue levels of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), cyclic adenosine monophosphate (cAMP) were measured enzymatically and by radioimmunoassay in the gastric fundal mucosa. The sum of ATP + ADP + AMP (adenylate pool) and the ratio of ATP x ADP-1 were calculated. It was found that (1) the tissue levels of ATP, AMP, cAMP, sum of ATP / ADP + AMP (adenylate pool) and ratio of ATP x ADP-1 increased significantly in the gastric fundal mucosa 6 hr after reserpine administration, thereafter these values decreased gradually and significantly; (2) the tissue level of ADP increased significantly in the gastric fundal mucosa 6 hr after reserpine administration, meanwhile its level increased significantly at 18 and 24 hr; (3) the value of energy charge (ATP + 0.5 ADP x ATP + ADP + AMP-1) remained unchanged; (4) the peaks of biochemical alterations in the gastric fundus mucosa preceded he appearance of ulcers. It was concluded that (1) reserpine ulcer appears after an active metabolic response in the rat gastric fundal mucosa; (2) hypoxaemic damage in the gastric fundal mucosa can be excluded as a possible underlying mechanism of ulcer formation produced by reserpine administration; (3) before the appearance of reserpine ulcer, significant changes in the feedback mechanism, system, i.e. between the ATP--membrane ATPase--ADP and the ATP--adenylate cyclase--cAMP energy systems, can be observed in the rat gastric fundal mucosa.     

Free pyrimidine nucleotide pool of Ehrlich ascites-tumour cells. Characteristics related to quantitative studies of RNA metabolism

The atractyloside-insensitive accumulation of adenine nucleotides by rat liver mitochondria (as opposed to the exchange-diffusion catalysed by the adenine nucleotide translocase) has been measured by using the luciferin/luciferase assay as well as by measuring [14C]ATP uptake. In foetal rat liver mitochondria ATP is accumulated more rapidly than ADP, whereas AMP is not taken up. The uptake of ATP occurs against a concentration gradient, and the rate of ATP uptake is greater in foetal than in adult rat liver mitochondria. The accumulated [14C]ATP is shown to be present within the mitochondrial matrix space and is freely available to the adenine nucleotide translocase for exchange with ATP present in the external medium. The uptake is specific for ATP and ADP and is not inhibited by adenosine 5'-[beta gamma-imido] triphosphate, GTP, CTP, cyclic AMP or Pi, whereas dATP and AMP do inhibit ATP accumulation. The ATP accumulation is also inhibited by carbonyl cyanide m-chlorophenylhydrazone, KCN and mersalyl but is insensitive to atractyloside. The ATP uptake is concentration-dependent and exhibits Michaelis-Menten kinetics. The divalent cations Mg2+ and Ca2+ greatly enhance ATP accumulation, and the presence of hexokinase inhibits the uptake of ATP by foetal rat liver mitochondria. These latter effects provide an explanation for the low adenine nucleotide content of foetal rat liver mitochondria and the rapid increase that occurs in the mitochondrial adenine nucleotide concentration in vivo immediately after birth.     

Adenylic nucleotides and energy charge during the embryonic development of Bufo arenarum

The contents of ATP, ADP and AMP were determined by HPLC and adenylic energy charge (AEC) was estimated during different stages of the embryonic development of Bufo arenarum up to the tailbud stage. All the developmental stages studied showed a high ATP content (about 1.04-1.48 nmol/emb.). The concentration of ADP was low (0.025-0.041 nmol/emb.) but rose slightly at the neural tube stage. AMP was undetectable before the tailbud stage. AEC values were almost constant (about 0.987-0.992) throughout the period studied. Only a fall at the tailbud stage could be detected which can be related to this more advanced cellular differentiation stage.     

Growth inhibition of transformed mouse fibroblasts by adenine nucleotides occurs via generation of extracellular adenosine

The growth of transformed mouse fibroblasts (3T6 cells) in medium containing 5% fetal bovine serum was inhibited after treatment with concentrations greater than 50 microM ATP, ADP, or AMP. Adenosine, the common catabolite of the nucleotides, had no effect on cell growth at concentrations below 1 mM. However, the following results indicate that the toxicity of ATP, ADP, and AMP is mediated by serum- and cell-associated hydrolysis of the nucleotides to adenosine. 1) ADP and AMP, but not ATP, were toxic to 3T6 cells grown in serum-free medium or medium in which phosphohydrolase activity of serum was inactivated. Under these conditions, the cells exhibited cell-associated ADPase and 5'-nucleotidase activity, but little ecto-ATPase activity. 2) Inhibition of adenosine transport in 3T6 cells by dipyridamole or S-(p-nitrobenzyl)-6-thioinosine prevented the toxicity of ATP in serum-containing medium and of ADP and AMP in serum-free medium. 3) A 16-24-h exposure to 125 microM AMP or ATP was needed to inhibit cell growth under conditions where serum- and cell-associated hydrolysis of the nucleotides generated adenosine in the medium continuously over the same time period. In contrast, 125 microM adenosine was completely degraded to inosine and hypoxanthine within 8-10 h. Furthermore, multiple doses of adenosine added to the cells at regular intervals over a 16-h period were significantly more toxic than an equivalent amount of adenosine added in one dose. Treatment of 3T6 cells with AMP elevated intracellular ATP and ADP levels and reduced intracellular UTP levels, effects which were inhibited by extracellular uridine. Uridine also prevented growth inhibition by ATP, ADP, and AMP. These and other results indicate that serum- and cell-associated hydrolysis of adenine nucleotides to adenosine suppresses growth by adenosine-dependent pyrimidine starvation.     

Metabolism of adenosine, AMP and ATP in rats

Metabolism of [14C]adenosine in a dose of 100 mg per 1 kg of mass and [14C]ATP in the equimolar quantity was studied in rats after intraperitoneal administration. Adenosine is shown to enter tissues of the liver, spleen, thymus, heart and erythrocytes where it phosphorylates into adenine nucleotides (mainly ATP) and deaminates into inosine. The content of adenosine increases for a short period in the above tissues, except for erythrocytes and plasma. The latter accumulates a considerable amount of inosine and hypoxanthine, but only traces of uric acid, xanthine and adenine nucleotides. ATP administered to rats catabolizes through the adenosine formation. The exogenic adenosine and ATP replace in tissues and erythrocytes only a slight part (1-12%) of their total adenine nucleotide pool. The content of these metabolites and ADP in the blood plasma does not change essentially under the effect of adenosine, ATP and AMP. It is shown on rats whose adenine nucleotide pool of cells is marked by the previous administration of [14C]adenine that injections of adenosine, ATP and inosine do not accelerate catabolism of adenine nucleotides in tissues and erythrocytes as well as do not increase the level of catabolism products in the blood plasma. Adenosine enhances and ATP lowers the content of cAMP in spleen and myocardium, respectively.     

Post-ischaemic synchronous purine nucleotide oscillations in perfused rat heart

Langendorff perfused rat hearts show synchronous, statistically significant, systematic variations in ATP and ADP. Here we show that AMP and IMP also vary in register with ATP and ADP and we suggest that the synchronizing trigger for these oscillations may be ischaemia. Oscillations in the ATP/ADP ratio were found to be significantly correlated with creatine phosphate content but by contrast these quantities vary quite differently from the GTP/GDP ratio. Cyclic GMP oscillations showed a significant negative correlation with variations in ADP. Epinephrine raised mean cyclic AMP content and stabilized cyclic GMP oscillations, but had little other effect on the purine nucleotide variations.     

Synthesis of bound adenosine triphosphate from bound adenosine diphosphate by the purified coupling factor 1 of chloroplasts. Evidence for direct involvement of the coupling factor in this "adenylate kinase-like" reaction.

Electrophoretically homogeneous coupling factor 1 from spinach chloroplasts binds ADP and converts the bound ADP to bound ATP and AMP. That this transphosphorylation of enzyme-bound ADP is catalyzed by the coupling factor itself, and not be a conventional adenylate kinase which might possibly contaminate preparations of the coupling factor, is supported by the following evidence. 1. The procedure for isolatio of the coupling factor is designed to separate this large (approximately 13 S) enzyme from the smaller (4.2 S) conventional adenylate kinase of spinach chloroplasts. The conventional adenylate kinase cannot be detected in purified preparations of the coupling factor by biochemical assay or by polyacrylamide gel electrophoresis. 2. The activity of spinach adenylate kinase is completely dependent upon magnesium ions. However, the production of bound ATP and AMP from bound ADP by the coupling factor can be assayed in the total absence of added magnesium ions or even in the presence of added EDTA. 3. Comparative studies with inhibitors show that the coupling factor can produce bound ATP from ADP under conditions where the activity of adenylate kinase is strongly inhibited. Conversely, the coupling factor is prevented from synthesizing bound ATP from ADP under other conditions where the conventional adenylate kinase has high levels of activity. 4. AMP, when added in solution to the coupling factor, does not bind to this enzyme, even in the presence of APT. Thus, it is unlikely that the appearance of AMP bound to the coupling factor after its incubation with ADP is due to the production of free AMP by contaminating adenylate kinase. These results demonstrate that the isolated, homogeneous coupling factor from spinach chloroplasts has the intrinsic capacity to perform a phosphoryl group transfer between two bound ADP molecules and thus to synthesize ATP. This reaction may have an important role in the photosynthetic production of ATP by the chloroplast, as is discussed in this communication.     

Regulation of glutamine metabolism in Chlorella pyrenoidosa. Regulation of glutamine synthetase activity by adenylic system components]

A decrease of glutamine synthetase (E. C. 6.3.1.2.) activity was observed under the assimilation of ammonium nitrogen in Chlorella. At the same time a decrease of ATP content in Chlorella cells took place. The ATP content was 7-fold decreased, while ADP and AMP contents were 4-fold and 3-fold increased respectively, after 15 min. of Chlorella incubation on "ammonium" medium. Further incubation for 45 min, resulted in gradual increase of ATP content and in decrease of ADP and AMP contents. The value of energy charge in ammonium assimilating Chlorella cells sharply decreased for first 15 min. of incubation and then it normalized gradually. The experiments with glutamine synthetase preparation, isolated from ammonium assimilating cells, have shown that ADP and AMP are strong inhibitors of the enzyme in the presence of Mg2+, and only ADP produces the inhibitory effect in the presence of Mn2+. No enzyme reactivation was observed after the transfer of ammonium assimilating cells into nitrogen-free medium or nitrate medium, the enzyme activity increasing at the expense of enzyme protein synthesis denovo.     

ADP is produced by firefly luciferase but its synthesis is independent of the light emitting properties

During experiments aimed at understanding the time course of appearance of reaction products in the Photinus pyralis luciferase system, an expected compound with a typical nucleotide UV spectrum was isolated. According to capillary electrophoresis (CE) analysis and 1H, 31P-NMR spectra, it was unambiguously found to be ADP, either with extracted or recombinant enzymes. The ADP synthesis was demonstrated by standard UV spectrophotometric methods and CE analysis. Also, the luciferase produced AMP and ATP from ADP. This reaction was completely inhibited by Ap(5)A at 250 nM and was independent of the light emitting properties of the enzyme. The only catalytic mechanism to explain the production of ADP is an intrinsic adenylate kinase activity of luciferase. The K(m) values of the AK activity are 0.3, 0.7, 0.06 mM for AMP, ADP, and ATP respectively. The multiple enzyme activities of luciferase may be partly responsible for the complex kinetics of light emission by changing the nucleotide concentrations.     

A rapid method for measuring ATP + ADP + AMP in marine sediment

In this report, I describe a method for rapid measurement of total adenylate (ATP + ADP + AMP) in marine sediment samples for estimating microbial biomass. A simple ‘boil and dilute’ method is described here, whereby adding boiled MilliQ water to sediments increases the detection limit for ATP + ADP + AMP up to 100-fold. The lowered detection limit of this method enabled the detection ATP + ADP + AMP in relatively low-biomass sub-seafloor sediment cores with 10⁴ 16S rRNA gene copies per gram. Concentrations of ATP + ADP + AMP correlated with 16S rRNA gene concentrations from bacteria and archaea across six different sites that range in water depth from 1 to 6000 m indicating that the ATP + ADP + AMP method can be used as an additional biomass proxy. In deep sea microbial communities, the ratio of ATP + ADP + AMP concentrations to 16S rRNA genes >1 m below seafloor was significantly lower compared to communities in the upper 30 cm of sediment, which may be due to reduced cell sizes and or lower ATP + ADP + AMP concentrations per cell in the deep sea sub-seafloor biosphere. The boil and dilute method for ATP + ADP + AMP is demonstrated here to have a detection limit sufficient for measuring low biomass communities from deep sea sub-seafloor cores. The method can be applied to frozen samples, enabling measurements of ATP + ADP + AMP from frozen sediment cores stored in core repositories from past and future international drilling campaigns.     

The development of gluconeogenesis in rat liver. Controlling factors in the newborn

1. Measurements in livers of rats delivered by Caesarian section show a rapid change in the relative proportion of adenine nucleotides. By 20min the ATP/ADP ratio had increased from 1.76 to 8.7 and the value of the relationship [ATP][AMP]/[ADP](2) increased from 1.0 to 4.4. These changes are dependent on the availability of oxygen to the animal. 2. The free [NAD(+)]/[NADH] ratio in the liver cytosol increases from 180 after delivery to reach a maximum of 1010 at 2h, before falling to 540 in the 24h-old animal. 3. The mitochondrial NAD redox potential also shows a sharp increase towards a more oxidized state in livers of delivered rats. 4. These results probably indicate that the foetal liver is hypoxic, with oxygenation occurring in the first hour after delivery. 5. Measurements in livers of naturally born rats 2min after birth also suggest that this tissue is hypoxic with an ATP/ADP ratio of 1.83 and a free [NAD(+)]/[NADH] ratio of 117. 6. Concentrations of intermediates in the gluconeogenic pathway have been determined in livers of foetal, 1h-old and 1-day-old rats. These experiments imply a facilitation of lactate dehydrogenase and glucose 6-phosphatase activities by 1h after birth, and a stimulation of phosphoenolpyruvate carboxykinase and glucose 6-phosphatase steps by 1 day after birth. 7. The appearance of gluconeogenesis in livers of newborn rats seems therefore to involve an oxygenation stage followed by an increase in phosphoenolpyruvate carboxykinase activity.     

Regulation of aflatoxin biosynthesis. III. Comparative study of adenine nucleotide pool in aflatoxigenic and non-aflatoxigenic strains.

The adenine nucleotide pools were studied in toxigenic (NRRL 3240) and non-toxigenic (NRRL 3537) strains of Aspergillus flavus group in relation to aflatoxin biosynthesis. The levels of ADP and AMP were comparatively low in the non-toxigenic strain throughout the growth period. In the toxigenic strain the levels of ADP and AMP increased significantly during the stationary phase. The ATP content of the toxigencic strain had higher ATP levels during the stationary phase compared to the toxigenic strain. The inorganic phosphate levels of the non-aflatoxigenic strain were higher when compared with the aflatoxigenic strain. High levels of inorganic phosphate have been reported to be inhibitory for secondary biosynthesis including aflatoxins. The energy charge values of the non-toxigenic strain, compared to the toxigenic strain, were lower during exponential phase but higher during stationary phase. The observed changes have been correlated with aflatoxin biosynthesis.