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.     

The adenylate energy charge in marine phytoplantkon: The effect of temperature on the physiological state of Skeletonema costatum (Grev.) Cleve

The adenylate energy charge $\text{([ATP] +}\text{1}\text{2}\text{[ADP])}\text{[0ATP+ADP+AMP]}$ was measured in axenic batch cultures of Skeletonema costatum (Grev.) Cleve at 2°, 10°, 15°, 20°, 24° and 30°C. The results suggest that this eurythermal diatom is physiologically capable of adapting to the 28 °C range of temperature with little apparent difference in the potential energy available to the cell. In N-limited continuous cultures at 15 °C, the energy charge values were lower than those observed in batch culture by 0.2, implying nutrient stress may result in decreased intracellular chemical energy. The utilization of the adenylate energy charge as an indicator of physiological state is suggested.     

Interrelationships between the membrane-bound ATP-dependent energy systems of the gastric mucosa and the gastric cytoprotective effect of beta-carotene on the development of gastric mucosal damage produced by 0.6 M HCl in rats

The effects of different doses (0.01-0.1-1.0-10.0/mg/kg-1) of beta-carotene were studied on gastric secretory responses of 4 hr pylorus-ligated rats: development of gastric mucosal damage (as assessed by number and severity of lesions) produced by intragastric administration of 0.6 M HCl; tissue level of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenylate pool (ATP + ADP + AMP), ratio of ATP X ADP-1, "energy charge" (ATP + 0.5 ADP X X (ATP + ADP + AMP)-1) (during the development of gastric mucosal damage by 0.6 M HCl and of gastric cytoprotection by beta-carotene. It was found that beta-carotene did not decrease the gastric secretory responses of 4 hr pylorus-ligated rats; The development of gastric mucosal damage could be decreased dose-dependently by the administration of beta-carotene; the ATP transformation could be decreased by beta-carotene; the tissue levels of cAMP and AMP could be increased significantly and dose-dependently by beta-carotene; the ratio of ATP X ADP-1 could be increased significantly and dose-dependently by beta-carotene; the values of adenylate pool and "energy charge" remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Energy Metabolism of Rickettsia typhi: Pools of Adenine Nucleotides and Energy Charge in the Presence and Absence of Glutamate

The obligate intracellular bacterium Rickettsia typhi was examined for its ability to generate and maintain an adenylate energy charge in an extracellular environment. Freshly purified organisms were incubated, at 34 degrees C and pH 7.4, with or without glutamate and various other metabolites, and the levels of ATP, ADP, and AMP were determined. Of the metabolites tested, glutamate and glutamine were the most effective for the generation of ATP. In the presence of glutamate, there was a rapid increase in the level of ATP, followed by a moderate decrease during 150 min of incubation. The energy charge increased from a level of 0.2 to 0.5 to about 0.7 to 0.75, and then slowly declined to about 0.45 to 0.6. In the absence of glutamate, after an occasional initial surge in ATP level as the temperature was changed from 4 to 34 degrees C, there was a sharp decline in both ATP and energy charge (to 0.1 and sometimes to 0.01). The rickettsiae maintained their ability to regenerate their energy charge upon the addition of glutamate for about 30 min, but this ability declined with further incubation. In contrast to Escherichia coli, the decline in ATP in R. typhi was accompanied by a sharp increase in the level of AMP and the total adenylate pool. No adenine or adenosine was recovered from rickettsiae incubated with labeled AMP, ADP, or ATP. From these experiments and the demonstration reported elsewhere that rickettsiae transport the adenine nucleotides, it can be concluded that the adenylate energy charge in R. typhi is governed by the salvage of the adenine nucleotides rather than their unphosphorylated precursors. Thus, R. typhi undergoes greater shifts in energy charge than other bacteria, a phenomenon which may account for their instability in an extracellular environment. Under optimal conditions the adenylate energy charge of R. typhi approaches levels that border on those generally regarded as adequate for growth.     

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.     

ATP breakdown and resynthesis in the development of gastrointestinal mucosal damage and its prevention in animals and human (an overview of 25 years ulcer research studies).

The changes in membrane-bound ATP systems (breakdown and resynthesis) were analyzed in different experimental ulcer models (such as ETOH, HCl, NaOH, 25% NaCl-induced, pyloric ligated + epinephrine treated, stress, reserpine treated, indomethacin treated rat models) and chronic antral, duodenal and jejunal ulcers in patients. The energy system parameters (adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), cyclic AMP (cAMP), lactate) were measured from different sites of gastrointestinal mucosa, and values of ATP/ADP, adenylate pool (ATP + ADP + AMP) and energy charge ((ATP + 0.5 ADP)/(ATP + ADP + AMP)) were calculated. The biochemical measurements were done at different times during the development of gastrointestinal mucosal lesions, without and with application of different drugs (PGI2, atropine, cimetidine) and bilateral surgical vagotomy. The aims of our present paper were: 1.) To summarize the main directions of ATP breakdown during the development of gastrointestinal lesions or ulcers in different experimental models and human beings: 2.) To summarize the biochemical steps of defense of gastrointestinal mucosa against chemicals, drugs or unknown pathogenic factors; 3.) To analyze the importance of membrane-bound ATP-dependent energy systems in order to understand the mucosal lesions and their prevention; 4.) To evaluate the real values of changes in these parameters from the point of view of ulcerogenesis and its prevention; 5.) To find some correlation between the energy parameters during mucosal damage and its prevention: 6.) To understand better the types of tissue reactions (metabolic) due to development of mucosal lesions and prevention.(ABSTRACT TRUNCATED AT 400 WORDS)     

5''-ADENINE MONONUCLEOTIDES TN SYNAPTOSOMAL PREPARATIONS FROM GUINEA PIG NEOCORTEX: THEIR CHANGE ON INCUBATION, SUPERFUSION AND STIMULATION

–(l) The contents of potassium and of ATP, ADP and AMP of homogenates of guinea pig neocortex in 0.32 M-SUCROSE, and of synaptosomal preparations derived therefrom, were determined and effects of incubation, superfusion and stimulation of the preparation were examined. The synaptosome preparations in M-SUCTOSe carried a smaller content of 5′-nucleotide per unit protein than did the homogenate from which they were derived. However, the proportion of synaptosomal nucleotides present as ATP was markedly greater than in the homogenate as a whole, the adenylate energy-charge being 83% greater in the synaptosomes than in the homogenate. Dilution of the synaptosomal preparation from the 1 M-sucrose to isotonic sucrose, decreased the contcnt of ATP per unit protein, but did not change the sum ATP + ADP + AMP. (2) Examined as deposited beds during 5 to 20 min incubation in oxygenated glucose-bicarbonate salines, synaptosomal K content and adenylate energy charge increased. These changes were sustained during a subsequent 40 min of incubation and also during superfusion. During such continued superfusion, electrical stimulation caused diminution of the ATP and the adenylate energy charge of the beds, as also did superfusion with fluids of increased K-content. (3) Lactate formation by the superfused beds was relatively stable during 30 min incubation. By electrical stimulation, the rate of lactate formation was increased by up to 30%. Increase in the potassium content of superfusion fluids could however increase lactate production 2.4-fold. The basis for these actions is discussed.     

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.     

Effects of cimetidine administered in cytoprotective and antisecretory doses on the membrane-bound ATP-dependent energy systems in the gastric mucosal lesions induced by HCl in rats

CFY strain rats (both sexes, 180-210 g) were fasted for 24 hr. Different doses of cimetidine (2.5, 10 and 50 mg X kg-1 i.p.) were given 30 min prior to the gastric mucosal lesions induced by the intragastric application of 0.6 M HCl. Animals were sacrificed 1 hr after the administration of the necrotizing agent. The number of gastric lesions was determined and their severity scored. Samples from the gastric fundic mucosa were taken for biochemical analysis. The tissue levels of adenosine-5'-triphosphate (ATP), adenosine-5'-diphosphate (ADP), adenosine-5'-monophosphate (AMP) and L-(+)-lactate were determined enzymatically, while the tissue contents of cyclic 3',5' adenosine monophosphate were measured by radioimmunoassay. The values for adenylate pool (ATP + ADP + AMP)-1 were calculated. All biochemical results were computed for 1.0 mg mucosal protein. We found that (1) the levels of ADP and lactate rose significantly, while ATP, AMP, cAMP, ATP X ADP-1 and energy charge decreased during the development of gastric lesions induced by HCl: (2) cimetidine decreased dose-dependently the number and severity of lesions: (3) the levels of ATP, ADP X ADP-1, and energy charge were increased dose-dependently by cimetidine, while AMP and lactate were decreased: (4) the levels of ADP, adenylate pool and cAMP did not change significantly by cimetidine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coordination of adenylate energy charge and phosphorylation state during ischemia and under physiological conditions in rat liver and kidney

The relation of the adenylate energy charge $\text{(}\text{ATP}\text{+}\text{1}\text{2}\text{ADP/ATP}\text{+}\text{ADP}\text{+}\text{AMP}\text{)}$ to the phosphorylation state (ATP)/(ADP)(HPO₄^2?) in rat liver and kidney was analyzed. Under physiological conditions and in ischemia, the two regulatory parameters, calculated from reported values for adenine nucleotides and inorganic phosphate (P_i) and from new observations, were closely coordinated. Energy charge was an inverse linear function of P_i and -log (1 - energy charge) was a positive linear function of log phosphorylation state. To evaluate experimental data with known energy charge, but unknown P_i, and to determine the theoretical relation between energy charge and phosphorylation state, P_i was estimated from a) the regression equation: P_i, μmol/g wet wt tissue = 1.05 - energy charge/0.073 and b) the empirical relationship: (P_i/2P_a) + energy charge = k, where P_a = σAMP + 2ADP + 3ATP and k = 1. With both estimates, the relation between phosphorylation state and energy charge for the experimental data was, within error, the same as that observed with measured P_i and concordant with theoretical values. Over the physiological range of energy charge (～0.85 – 0.95, log phosphorylation state ～3.3 – 4.3), apparent ΔG_ATP (×2) was closer to the range of ΔG observed by Wilson $\text{et al}$ (Biochem. J. $\text{140}$:57, 1974) for transfer of two electrons from mitochondrial NAD to the cytochrome c couple than the ΔG_ATP (×2) they reported, supporting their conclusion that near-equilibrium exists between the mitochondrial respiratory chain and the cytoplasmic phosphorylation state under physiological conditions. From evidence presented, it is postulated that the phosphorylation state is regulated by the adenylate energy charge.     

Chronic environmental pollution alters adenylate levels in needles and fine roots of Scots pine

Contents of ATP, ADP, AMP, inorganic phosphate, and values of ATP/ADP ratio, adenylate energy charge (AEC), phosphorylation potential (PP) and adenylate kinase activity were analysed in needles and fine roots of Scots pine trees grown at the polluted and control (free of acute air pollution) site. Also chemical properties of the soil and mineral elements in needles from both sites were analysed. In comparison with the control, developing needles from the polluted site contained less ATP, the same amount of ADP and more AMP, and had lower values of ATP/ADP, AEC and PP. In one-year-old needles from the polluted site no change or a decrease in ATP was recorded, while ADP decreased, AMP increased, AEC did not change, and ATP/ADP ratio and PP were higher. In fine roots from the polluted site AMP level was higher, while ATP, ADP, ATP/ADP ratio, PP and AEC were lower than in the control.     

The relationship between nodule adenylates and the regulation of nitrogenase activity by O2 in soybean

Nodulated soybeans (Glycine max L. Merr, cv. Maple Arrow) were exposed to various physiological and environmental treatments to determine the relationship between nodule adenylate pools and the degree of O₂ limitation of nitrogenase. Adenylate energy charge (AEC = [ATP + 0.5 ADP]/[ATP + ADP + AMP]) and ATP/ADP ratios declined under conditions of decreased (10%) external pO₂ but increased in nodules exposed to elevated (30%) external pO₂. Nitrogenase activity was inhibited by both pO₂ treatments, but recovered towards initial levels within 45 min. AEC also returned to initial levels during this period. To account for these and related data in the literature, it was hypothesized that 1) legume nodules regulate infected cell O₂ concentration (Oi) to maintain adenylate pools at levels which limit respiratory metabolism: 2) treatments which decrease Oi alter the adenylate pools and further limit nodule metabolism; 3) treatments which increase Oi to levels in excess of a narrow range alter the adenylate pools and activate biochemical pathways which are not conducive to nitrogenase activity. In a preliminary test of these hypotheses, changes in AEC and ATP/ADP ratio were studied in nodules in which nitrogenase activity was inhibited by stem girdling, nitrate fertilization and exposure to an Ar:O₂ atmosphere. All three treatments caused an increased O₂ limitation of nodule respiration and nitrogenase activity. However, decreases in AEC were observed only in the stem girdling and nitrate fertilization treatment: Ar:O₂ exposure had no effect on whole nodule AEC. While this result challenged the hypotheses suggesting a central role for adenylates in the regulation of O₂-limited metabolism, it was noted that the Ar:O₂ treatment would differ from the other treatments in that it would have a specific effect on the ATP demands for NH₃ assimilation in the plant fraction. Since AEC and ATP/ADP ratio would be affected by both the rate of ATP synthesis (potentially an O₂-limited process) and the demand for ATP, changes in these parameters in the whole nodule may not be a reliable indicator of adenylate-mediated O₂ limitation. Futher studies are needed to examine in vivo changes in adenylate pools in the plant and bacteroid fractions in nodules which vary in their degree of O₂-limited metabolism.     

Substrate specificity and regulation of the maize (Zea mays) leaf ADP: protein phosphotransferase catalysing phosphorylation/inactivation of pyruvate, orthophosphate dikinase.

The protein substrate specificity of the maize (Zea mays) leaf ADP: protein phosphotransferase (regulatory protein, RP) was studied in terms of its relative ability to inactivate/phosphorylate pyruvate, orthophosphate dikinase from Zea mays and the non-sulphur purple photosynthetic bacterium Rhodospirillum rubrum. The dimeric bacterial dikinase was inactivated by the maize leaf RP via phosphorylation, with a stoichiometry of approximately 1 mol of phosphate incorporated/mol of 92.7-kDa protomer. Inactivation required both ADP and ATP, with ADP being the specific donor for regulatory phosphorylation. The requirements for inactivation/phosphorylation in this heterologous system were identical with those previously established for the tetrameric maize leaf dikinase. The ADP-dependent maize leaf RP did not phosphorylate alternative protein substrates such as casein or phosvitin, and its activity was not affected by cyclic nucleotides, Ca2+ or calmodulin. The regulation of the maize leaf ADP: protein phosphotransferase was studied in terms of changes in adenylate energy charge and pyruvate concentration. The change in adenylate energy charge necessary to substantially inhibit phosphorylation of maize leaf dikinase was not suggestive of it being a physiological modulator of phosphotransferase activity. Pyruvate was a potent competitive inhibitor of regulatory phosphorylation (Ki = 80 microM), consistent with its interaction with the catalytic phosphorylated intermediate of dikinase, the true protein substrate for ADP-dependent phosphorylation/inactivation.     

Levels of adenylic system components (ATP, ADP, AMP) and cAMP during development of Blastocladiella emersonii

Concentrations of ATP, ADP, AMP, cAMP as well as pyruvate and glucose-6-phosphate were measured in B. lastocladiella emersonii cells developing via RS morphogenetic pathway. They varied significantly in the course of development (1.3-14.8 mumole/g dry weight for the sum of ATP+ADP+AMP; 0.012-5.3 nmole for cAMP; 0.47-1.9 mumole for pyruvate; 0.36-4.78 mumole for glucose-6-phosphate). At the same time the adenylate energy charge remained essentially unchanged (about 0.8) from the middle of exponential growth till the end of the stationary phase. At the late stages of RS-sporangia formation the concentration of all the above compounds decreased by about 10 times, and the adenylate energy charge only by 30%. Positive correlation between the levels of ATP and cAMP in RS cells was demonstrated. The concentration of adenylic nucleotides and cAMP showed the most noticable changes at the end of exponential growth; transition of the point of no return was not accompanied by significant changes in the pools of adenylic system, cAMP or energy charge.     

Energy status of starving yeast cells immobilized by covalent linkage

Summary Changes in the adenylate pool size, energy charge values, ATP/ADP ratio, and the whole sum of nucleotide concentrations were studied during starvation of free and covalently immobilizedSaccharomyces cerevisiae cells. The results obtained indicate a better preservation of energy status of immobilized yeasts.     

Changes of gastric mucosal biochemistry in ethanol-treated rats with and without acute surgical vagotomy

The biochemical background of ethanol-(ETOH) induced gastric mucosal damage was studied in rats with intact vagus and after acute surgical vagotomy. Observations were carried out on Sprague-Dawley (CFY) strain rats of both sexes. Gastric mucosal lesions were produced by intragastric administration of 1 ml 96% ethanol. Bilateral truncal surgical vagotomy was carried out 30 min before ETOH administration. The number and severity of gastric mucosal lesions was noted 1 h after ETOH administration. Biochemical measurements (gastric mucosal level of ATP, ADP, AMP, cAMP and lactate) were carried out from the total homogenized gastric mucosa. The adenylate pool (ATP + ADP + AMP), energy charge ((ATP + 0.5 ADP)/(ATP + ADP + AMP)) and ratio of ATP/ADP were calculated. It was found that: 1) ATP transformation into ADP increased, while ATP transformation in cAMP decreased in ethanol-treated animals with intact vagus nerve, while these transformations were quite the opposite in vagotomized animals; 2) no significant changes were found in the tissue level of lactate: and 3) the extent of biochemical changes was significantly less after surgical vagotomy. It is concluded that an intact vagus is basically necessary for the metabolic adaptation of gastric mucosa.     

Adenine nucleotide extraction from multicellular organisms and beach sand: ATP recovery,energy charge ratios and determination of carbon/ATP ratios

Investigations were conducted comparing the efficiency of adenine nucleotide extraction from bacteria, unicellular algae, invertebrates (copepods, isopods and polychaetes), and beach sand using boiling buffers and cold acid extraction procedures. Cellular levels of ATP, ADP, and AMP obtained by these procedures were used to calculate the adenylate energy charge ratio ($\text{EC}{}_{\mathrm{<mtext>A</mtext>}}\text{= [}\text{ATP}\text{] +}\text{1}\text{2}\text{[}\text{ADP}\text{]/[}\text{ATP}\text{] + [}\text{ADP}\text{] + [}\text{AMP}\text{]}$). Although both extraction procedures efficiently extract ATP from unicellular micro-organisms, the results with multicells and beach sand indicate that the cold acid procedure preserves a greater percentage of the total adenine nucleotides ([A_T] = [ATP] + [ADP] + [AMP]) in the form of ATP, resulting in higher energy charge ratios. There were relatively large losses of ATP when multicellular organisms were extracted in boiling buffers. These data suggest that ATP hydrolysis may be important in certain fluid-solid mixtures, and also adds experimental support to the thermal gradient hypothesis.The C/ATP ratios calculated from these data indicate that multicellular organisms have C/ATP ratios < 100, as compared with the 250 ratio commonly found in micro-organisms. These results are discussed in terms of the proportion of structural (non-living) carbon vs protoplasm (living) carbon within each of these groups of organisms, as well as the relative intracellular levels of non-adenine nucleotide triphosphates. These differences in the C/ATP ratios must be considered whenever ATP measurements are used for biomass determinations.     

Composition and Distribution of Adenylates in Soybean (Glycine max L.) Nodule Tissue

Adenylates (ATP, ADP, and AMP) may play a central role in the regulation of the O2-limited C and N metabolism of soybean nodules. To be able to interpret measurements of adenylate levels in whole nodules and to appreciate the significance of observed changes in adenylates associated with changes in O2-limited metabolism, methods were developed for measuring in vivo levels of adenylate pools in the cortex, plant central zone, and bacteroid fractions of soybean (Glycine max L. Merr cv Maple Arrow x Bradyrhizobium japonicum strain USDA 16) nodules. Intact nodulated roots were either frozen in situ by flushing with prechilled Freon-113(-156[deg]C) or by rapidly (<1 s) uprooting plants and plunging them into liquid N2. The adenylate energy charge (AEC = [ATP + 0.5 x ADP]/[ATP + ADP + AMP]) of whole-nodule tissue (0.65 [plus or minus] 0.01, n = 4) was low compared to that of subtending roots (0.80 [plus or minus] 0.03, n = 4), a finding indicative of hypoxic metabolism in nodules. The cortex and central zone tissues were dissected apart in lyophilized nodules, and AEC values were 0.84 [plus or minus] 0.04 and 0.61 [plus or minus] 0.03, respectively. Although the total adenylate pool in the lyophilized nodules was only 41% of that measured in hydrated tissues, the AEC values were similar, and the lyophilized nodules were assumed to provide useful material for assessing adenylate distribution. The nodule cortex contained 4.4% of whole-nodule adenylates, with 95.6% being located in the central zone. Aqueous fractionation of bacteroids from the plant fraction of whole nodules and the use of marker enzymes or compounds to correct for recovery of bacteroids and cross-contamination of the bacteroid and plant fractions resulted in estimates that 36.2% of the total adenylate pool was in bacteroids, and 59.4% was in the plant fraction of the central zone. These are the first quantitative assessments of adenylate distribution in the plant and bacteroid fractions of legume nodules. These estimates were combined with theoretical calculations of rates of ATP consumption in the cortex (9.5 nmol g-1 fresh weight of nodule s-1), plant central zone (38 nmol g-1 fresh weight of nodule s-1), and bacteroids (62 nmol g-1 fresh weight of nodule s-1) of soybean nodules to estimate the time constants for turnover of the total adenylate pool and the ATP pool within each nodule fraction. The low values for time constant (1.6-5.8 s for total adenylate, 0.9-2.5 s for ATP only) in each fraction reflect the high metabolic activity of soybean nodules and provide a background for further studies of the role of adenylates in O2-limited nodule metabolism.