Measurements of the distribution of adenylate concentrations and adenylate energy charge across Pseudomonas aeruginosa biofilms.

Adenine nucleotide pools and adenylate energy charge distributions were determined by using a laboratory-generated quasi-steady-state Pseudomonas aeruginosa biofilm. The method used involved freezing and sectioning of the intact biofilm, followed by extraction and assay of the adenylates in the sectioned material. Results indicated an increase in adenylate energy charge of about 0.2 units from the bottom to the surface of the biofilm. However, energy charge values were generally low throughout the biofilm, reaching a maximum of only 0.6 units. Of the adenylates measured, AMP was the predominant nucleotide, especially in the deeper parts of the biofilm profile.     

Stabilization of the adenylate energy charge in erythrocytes of rats and humans at high altitude hypoxia.

1. Stabilization of adenylate energy charge and control of adenylate pool were analysed in the erythrocytes of the rat and the human exposed to highly hypoxic conditions. 2. Red cell energy charge was decreased in the rats exposed to a simulated altitude of 5000-8000 m, and then recovered to the normal value with the depletion of adenylate pool. 3. The energy charge and the adenylate pool size of the human erythrocytes did not show any change under highly hypoxic conditions. 4. Anaerobic incubation of rat erythrocytes caused a marked decrease in the energy charge, and its recovery was accompanied by the depletion of total adenylates. 5. The energy charge and total adenylates of human red cells did not change under the anaerobic incubation of erythrocytes. 6. These results suggest that the energy charge of rat erythrocytes can be controlled by depletion of the adenylate pool, but the adenylate degradation is not responsible for the stabilization of the energy charge in human erythrocytes.     

In situ studies on AMP deaminase as a control system of the adenylate energy charge in yeasts

The role of AMP deaminase reaction in the stabilization of the adenylate energy charge was investigated using permeabilized yeast cells. The addition of P_i or Zn²⁺, which inhibits AMP deaminase, remarkably retarded the depletion of total adenylate pool and the recovery of the adenylate energy charge. Polyamine, an activator of the enzyme, decreased total adenylates, resulting in the enhanced recovery of the energy charge in situ. AMP deaminase can act as a regulatory enzyme in the system that stabilizes the adenylate energy charge in yeast cells under the conditions of severe metabolic stress.     

Phosphoadenylate concentrations and adenylate energy charge of largemouth bass (Micropterus salmoides): relationship with condition factor and blood cortisol

1. Concentrations of phosphoadenylate nucleotides and adenylate energy charge in the dorsal muscle and blood of largemouth bass from a reservoir receiving heated effluent were investigated. These values were compared with the length, weight, body condition and blood cortisol concentrations. 2. Body condition of the largemouth bass ranged from 1043 to 2544, reflecting the range of condition due to starvation of some fish in the population. 3. Blood cortisol concentrations ranged from 4.0 to 23.1 micrograms/100 ml with a mean of 6.3 micrograms/100 ml. 4. The adenylate energy charge of muscle (MATP) and blood ranged from 0.37 to 0.98 and 0.74 to 0.99, respectively. 5. Blood cortisol concentration was positively correlated with body condition but not correlated with the adenylate energy charge of either blood or muscle. 6. Blood cortisol concentration was negatively correlated with concentration of adenylates in both muscle and blood.     

Response of nucleoside diphosphate kinase to the adenylate energy charge

The reaction catalyzed by nucleoside diphosphate kinase responds to the energy charge of the adenylate pool. The velocity is maximal at a charge of 1.0, and decreases sharply with a decrease in the charge. This response may control the flow of phosphate from ATP into the other nucleotide pools and thus participate in the regulation of macromolecular synthesis by the energy level of the cell, as reflected in the charge of the adenylate pool.     

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.     

Stabilization of adenylate energy charge and its relation to human sperm motility.

The adenylate energy charge of human ejaculated spermatozoa was studied when the sperm motility was perturbed by varying pH, prolonged incubation, and caffeine. Between pH 8 and 9, which was optimal for the sperm motility, the energy charge was in the physiological range of 0.8 to 0.9. Above pH 9, the mobility, ATP content, and adenine nucleotide pool declined rapidly but the energy charge was maintained slightly below 0.8. Below pH 8, the motility also dropped drastically, but the ATP, nucleotide pool, and energy charge fell only slightly. Prolonged incubations of the spermatozoa decreased the motility, ATP, and nucleotide pool. However, the energy charge would remain above 0.6. Caffeine stimulation of the motility caused a rapid fall of ATP and the reduction of the physiological energy charge by 0.2 unit, unless glucose was added. Imidazole which reduced the caffeine-stimulated motility did not alter the physiological energy charge of the spermatozoa. The study showed that the spermatozoa could maintain the energy charge above 0.6 under stress.     

Regulation of cytosol 5'-nucleotidase by adenylate energy charge

In the physiological range of the adenylate energy charge in liver (0.7-0.9), th rate of AMP-hydrolysis catalysed by rat liver cytosol 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) increased sharply with decreasing energy charge. In addition, a decrease in the concentration of Pi caused marked acceleration of the AMP-hydrolysing activity over the physiological range of adenylate energy charge. These responses seem to serve to protect the cells against a metabolic stress which could result from sudden utilization of ATP by removal of AMP. The AMP-hydrolysing activity of this enzyme decreased sharply as the size of the adenine nucleotide pool decreased in the physiological range. This effect may be a self-limiting response to prevent excess depletion of the pool. IMP-hydrolysing activity of this enzyme increased with increasing adenylate energy charge. But no marked response to its variation within the physiological range was observed. On the basis of the data obtained in this study, the IMP-hydrolysing activity of the cytosol 5'-nucleotidase in rat liver cells seems to be comparable to that of AMP deaminase reaction, but the AMP-hydrolysing activity was estimated to be less than 10% of AMP deaminase reaction at energy charge value of about 0.7. This strongly suggests that the AMP leads to IMP leads to inosine pathway is more significant that the AMP leads to adenosine leads to inosine pathway in rat liver.     

Regulation and biosynthesis of secondary metabolites

The relationship was studied between the energy metabolism of the actinomyceteStreptomyces aureofaciens and the biosynthesis of chlorotetracycline by this organism. The energy charge values in a culture of low-production strain were almost identical with those of a production variant but the total sum of adenylates was about 10 times higher. In the stationary growth phase both strains evinced a drop in energy charge values followed by a rise to the original level. An increase in the concentration of inorganic phosphate in fermentation medium caused a suppression of antibiotic formation in the lowproduction strain and further rise in the total adenylate level. The expression of the energy charge inStreptomyces aureofaciens acquires a complex character owing to the participation, apart from the adenylate system, of high-molecular polyphosphates as energy donors and the probable lack of a regulating mechanism such as the adenylate kinase reaction.     

Functions of chloroplastic adenylate kinases in Arabidopsis

Adenosine monophosphate kinase (AMK; adenylate kinase) catalyses the reversible formation of ADP by the transfer of one phosphate group from ATP to AMP, thus equilibrating adenylates. The Arabidopsis (Arabidopsis thaliana) genome contains 10 genes with an adenylate/cytidylate kinase signature; seven of these are identified as putative adenylate kinases. Encoded proteins of at least two members of this Arabidopsis adenylate kinase gene family are targeted to plastids. However, when the individual genes are disrupted, the phenotypes of both mutants are strikingly different. Although absence of AMK2 causes only 30% reduction of total adenylate kinase activity in leaves, there is loss of chloroplast integrity leading to small, pale-looking plantlets from embryo to seedling development. In contrast, no phenotype for disruption of the second plastid adenylate kinase was found. From this analysis, we conclude that AMK2 is the major activity for equilibration of adenylates and de novo synthesis of ADP in the plastid stroma.     

Influence of NO3 - and NH4 + nutrition on fermentation,nitrate reductase activity and adenylate energy charge of roots of Carex pseudocyperus L. and Carex sylvatica Huds. exposed to anaerobic nutrient solutions

The adenylate energy charge, production of ethanol and lactate, and nitrate reductase activity were determined in order to study the influence of different nitrogen sources on the metabolic responses of roots of Carex pseudocyperus L. and Carex sylvatica HUDS. exposed to anaerobic nutrient solutions. Determination of adenylates was carried out by means of a modified HPLC technique. Total quantity of adenylates was higher in Carex pseudocyperus than in Carex sylvatica under all conditions. In contrast, the adenylate energy charge was only slightly different between the species and decreased more or less in relation to the applied nitrogen source under oxygen deficiency. The adenylate energy charge in roots of plants under nitrate nutrition showed a smaller decrease under anaerobic environmental conditions than plants grown with ammonium or nitrate/ammonium. Roots of nitrate-fed plants showed a lower ethanol and lactate production than ammonium/nitrate- and ammonium-fed plants. Ethanol production was higher in C. pseudocyperus, formation of lactate was lower compared to that in Carex sylvatica. The activity of enzymes involved in fermentation processes (ADH, LDH and PDC) was enhanced significantly after 24 hours of exposure to anaerobic nutrient solutions in roots of both species. The induction of these enzymes was only slightly influenced by different nitrogen supply. In vivo nitrate reductase activity increased almost 3-fold compared to the aerobic treatment in both species and overcompensated loss of NADH reoxidation capacity caused by decrease of ethanol and lactate development. Induction of in vitro nitrate reductase activity was enhanced 313% in C. pseudocyperus and 349% in C. sylvatica under anaerobic environmental conditions and nitrate supply. These results indicate that nitrate may serve as an alternative electron acceptor in anaerobic plant root metabolism and that the nitrate-supported energy charge may be due to an accelerated glycolytic flux resulting from a more effective NADH reoxidation capacity by nitrate reduction plus fermentation than by fermentation alone.Abbreviations ADH alcohol dehydrogenase - AEC adenylate energy charge - DMSO dimethyl sulfoxide - EDTA ethylen diamine tetraacetic acid - HPLC high performance liquid chromatography - LDH lactate dehydrogenase - NRA nitrate reductase activity - PCA perchloric acid - PDC pyruvate decarboxylase - PVP polyvinylpyrrolidone - PVPP polyvinylpolypyrrolidone - TCA trichloroacetic acid, Tris-tris(hydroxymethyl)aminomethane     

Short term cadmium intoxication of the shrimp Palaemon serratus: Effect on adenylate metabolism

• 1.1. Shrimps were exposed for 96 hr to various concentrations of cadmium under laboratory conditions. The LC₅₀ was around 4 ppm Cd in water, which corresponded to 0.180 μg/g wet weight of cadmium in tail muscles.
• 2.2. The effect of various concentrations of cadmium on adenylates was analyzed in tail muscles: At subletal cadmium concentrations, no variation of ATP, ADP and of the adenylate sum occurred, while the AMP concentration began to decrease from 0.06 ppm.

At the LC₅₀, the ATP, ADP and AMP concentrations dropped acutely, the ATP/ADP ratio increased acutely.The apparent equilibrium constant of the adenylate kinase reaction increased significantly from 2 ppm Cd, indicating an impairment in energetic metabolism.Cadmium intoxication did not influence the value of the adenylate energy charge.     

Hypoxia and the energy charge of the cerebral adenylate pool

A brief period of anoxia in vivo causes a transitory decrease in the size of the adenylate pool in the rat brain. This is probably caused by feedback inhibition by AMP of adenine nucleotide synthesis. Exposing rats to various degrees of hypoxia suggests that the sensitivity of the brain to lack of O(2) results from the brain's limited ability to maintain an adequate energy charge in unfavourable circumstances.     

Stabilization of the adenylate energy charge by the depletion of adenylates without glycolytic stimulation

A relationship between the AMP deaminase reaction and the recovery of the energy charge was examined using permeabilized yeast cells. Citrate inhibited the glycolytic flux and the recovery of the energy charge. The addition of spermine enhanced the recovery of the energy charge without the reversal of the inhibition of glycolysis in the presence of excess citrate. The AMP deaminase reaction can participate in the stabilization of the energy charge only by the depletion of total adenylates, and not by the glycolytic stimulation under the conditions where citrate is highly increased during aerobic growth conditions.     

Informational aspects of Gibbs function output from nucleotide pools and of the adenylate kinase reaction

The physiological activity of living cells depends among other things on the available amounts of Gibbs energy stored in nucleotide pools and hence on the displacements of the triphosphate cleavage reactions from equilibrium. Since the chemical system in the cell is not ideal, concentrations of the nucleotides do not coincide with their activities. To ensure a relatively simple control of both the concentrations and the activities, the displacement from equilibrium must be encoded in terms of concentrations as exactly as possible.The adenylate kinase reaction provides a simple and sufficient means for establishing satisfactory coding. Since that coding is not quite exact, an information-theoretic error measure must be defined. The definition leads to a novel interpretation of energy charge as an information-theoretic quantity. The worst error values then select preferable values of the apparent equilibrium constant of the adenylate kinase reaction. These information-theoretic values are of the same order as reported experimental values, in spite of the simplification of the treatment by ignoring the influence of the ortho- and pyrophosphate.Supplying Gibbs energy to many processes in the cell the nucleotide pools can supply information to those of them which are properly designed. In exacting situations the displacement from equilibrium and energy charge must be sufficiently high.     

Close correlation between platelet responses and adenylate energy charge during transient substrate depletion

The relation between availability of metabolic energy and thrombin-induced platelet aggregation and secretion was investigated in a system of transient substrate depletion followed by restoration of ATP resynthesis. Substrate depletion induced a fall in the concentration of metabolic ATP and in the adenylate energy charge and a concurrent decline in aggregation and secretion of dense and α-granule contents. Restoration of energy generation completely restored the adenylate energy charge and restored aggregation and secretion, but led to incomplete recovery of the ATP concentration. A close correlation between the adenylate energy charge and aggregation and between the adenylate energy charge and the secretion of dense and α-granule contents could be demonstrated. No such correlation existed between these responses and the concentration of ATP. These results show that the adenylate energy charge monitors an energetic condition which is crucial for preservation of platelet aggregation and secretion of dense and α-granule contents.     

ATP content and adenylate energy charge ofBacillus stearothermophilus during growth

The ATP content and the adenylate energy charge of the thermophileBacillus stearothermophilus were determined during growth.Bacillus subtilis was used for comparison to determine whether there were differences in the ATP content and adenylate energy charge between a mesophile and a thermophile. Both the ATP content and the adenylate energy charge were lower in the thermophile than in the mesophile. These lower values may reflect a decreased activation energy required for the metabolic coupling when growth is occurring at the higher temperatures characteristic of the thermophile.     

Comparative analysis of mitochondrial and amyloplast adenylate translocators

Structurally intact and metabolically competent mitochondria isolated from liquid-culture cells of sycamore (Acer pseudoplatanus L.) were shown to incorporate ADPglucose. Employing the double silicone oil layer filtering centrifugation method, we examined the kinetic properties of the uptake of various adenylates as well as the inhibitory effects exerted by carboxyatractyloside, atractyloside and bongkrekic acid, known specific inhibitors of the mitochondrial adenylate translocator. Immunoblot patterns of peptides derived from the partial proteolytic digestion of the mitochondrial and plastid adenylate translocators were shown to be essentially the same. We conclude that the molecular entities engaged in the adenylate transport system operating in two different organelles, mitochondria and amyloplasts, are very similar.     

Delayed light studies on photosynthetic energy conversion. VIII. Evidence from millisecond emission of chloroplasts for two adenylate binding sites on membrane-bound coupling factor, CF1.

Effects of adenylates on chloroplast delayed light emission, at millisecond dark times, are inverse to the previously characterized effects of adenylates on electron transport rates. Either ADP alone or ATP alone increase intensity of delayed light, while ADP plus Pi decrease it. ADP alone requires the presence of an electron acceptor to have this effect on delayed light, but ATP does not. All three adenylate effects are abolished by uncoupling with gramicidin, by partial removal of photophosphorylation coupling factor (CF1) with EDTA, and by antibody to CF1. Readdition of CF1 re-established the adenylate effects in EDTA-stripped membranes. The three adenylate effects are differentially sensitive to pH, and pH differentially affected their abolition by antibody to CF1. The two adenylate effects shown in the absence of Pi are exhibited at lower adenylate concentrations than the ADP plus Pi effect, and are also less sensitive to phloridzin. These results are discussed in terms of probable adenylate effects on membrane-bound chloroplast coupling factor, CF1. At least two ADP binding sites would differ with respect to adenylate concentration for half maximal binding; pH of optimal binding capacity; phloridzin sensitivity; and functional regulation of electron transport, proton uptake, and energy storage within the membrane as measured by delayed light emission. It remains unclear whether the high affinity ADP binding site is identical to a high affinity ATP binding site on CF1.     

Intracellular ATP and total adenylate concentrations are critical predictors of reovirus productivity from Vero cells

The productivity of reovirus type-3 Dearing was studied in cultures of Vero cells in serum-free media. Viral productivity was dependent upon the metabolic state of the cells rather than the phase of growth at which the cells were infected. Cells at different energy states were established by 24-h incubation in nutrient-depleted media. This resulted in variable intracellular nucleotide concentrations but high cellular viability was maintained. Of the nucleotides analyzed at the time of infection only the intracellular [ATP] and total adenylate nucleotides were positively correlated with viral productivity. The correlated data followed a sigmoidal plot with an equation defined by polynomial regression analysis. Apparent threshold values of 3.2 fmol/cell and 3.3 fmol/cell were established for ATP and total adenylate, respectively, at which the viral production was 50% the maximal value. Cultures with lower ATP and total adenylate levels at the time of infection resulted in as much as a 95% reduction in overall viral titer compared to the control. The adenylate energy charge (AEC) showed a negative correlation with viral production with an AEC value >0.97 resulting in low virus productivity. Intracellular ATP or total adenylate concentration at the point of infection may be used as a predictor of viral yield in bioprocesses designed for virus/vaccine production.