Adenine Nucleotide Levels, the Redox State of the NADP System, and Assimilatory Force in Nonaqueously Purified Mesophyll Chloroplasts from Maize Leaves under Different Light Intensities

Recently, a nonaqueous fractionation method of obtaining highly purified mesophyll chloroplasts from maize leaves was established. This method is now used to determine adenine nucleotide levels, the redox states of the NADP system, Pi levels and dihydroxyacetone phosphate/3-phosphoglycerate ratios in mesophyll chloroplasts of Zea mays L. leaves under different light intensities. The sum of the ATP, ADP, and AMP levels was estimated to be 1.4 millimolar and the ATP/ADP ratio was 1 in the dark and 2.5 to 4 in the light. The adenine nucleotides were equilibrated by adenylate kinase. The total concentration of NADP(H) in the chloroplasts was 0.3 millimolar in the dark and 0.48 millimolar in the light. The ratio of NADPH/NADP was 0.1 to 0.18 in the dark and 0.23 to 0.48 in the light. The Pi level was estimated to be 20 millimolar in the dark and 10 to 17 millimolar in the light. The 3-phosphoglycerate reducing system was under thermodynamic equilibrium in the light. The calculated assimilatory forces were 8 per molar and 40 to 170 per molar in the dark and the light, respectively. There was no relationship between the degree of activation of pyruvate, Pi dikinase, and adenylate energy charge, or ATP/ADP ratio or ADP level under various light intensities. Only a weak relationship was found between the degree of activation of NADP-malate dehydrogenase and the NADPH/NADP ratio or NADP(H) level with increasing light intensity. A possible regulatory mechanism which is responsible for the regulation of activation of pyruvate,Pi dikinase and NADP-malate dehydrogenase is discussed.     

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.     

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.     

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.     

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.     

Adenylate Levels, Energy Charge, and Phosphorylation Potential during Dark-Light and Light-Dark Transition in Chloroplasts, Mitochondria, and Cytosol of Mesophyll Protoplasts from Avena sativa L

The compartmentation of cellular energy relations during dark-light and light-dark transitions was studied by means of a newly developed technique to fractionate oat (Avena sativa L., var. Arnold) mesophyll protoplasts. Using an improved microgradient system with hydrophobic and hydrophilic layers of increasing density, a pure plastid pellet (up to 90% of total chloroplasts) could be separated from an interphase of only slightly contaminated mitochondria (70 to 80% of total mitochondria), and a cytoplasmic supernatant could be obtained within 60 seconds. Appropriate controls indicate that, under the conditions employed, metabolic interconversions of adenylates can be kept to a minimum and, thus, be determined and corrected for. Cross contamination of the fractions, as well as liberation of organelles to the supernatant, was assessed by specific markers, and the metabolite levels recorded were corrected accordingly. Using this technique, we found that, during dark-light transition, the chloroplastic and cytosolic ATP exhibits a rapid increase, while the mitochondrial ATP level decreases. In all compartments, ADP levels mirror alterations of the ATP pool in the opposite way, at least to some extent. To compensate fully for the rise in ATP, chloroplastic and mitochondrial AMP levels change accordingly, indicating that, due to the more or less unchanged level of total adenylates, there is no net flux of adenylates between the compartments. In contrast to the organelles, no AMP could be detected within the cytosol. When the light is turned off, a decrease of ATP coincides between chloroplast stroma and the cytosol for only about 30 seconds. Under prolonged dark treatment, cytosolic ATP rises again, while stroma ATP levels exhibit a further decrease. After about 60 seconds of darkness, the cytosolic ATP level is back to its initial value. This obviously is due to the immediate rise in mitochondrial ATP upon darkening, which cumulates after about 60 seconds; then, caused by an ATP/ADP exchange with the cytosol, it levels off again at the state before changing the conditions, as soon as the cytosolic ATP is also back to its original level. All of these events are closely mirrored by the change in the ATP/ADP ratio and the energy charge within the compartments. While the values for chloroplasts exhibit considerable differences between dark and light, those calculated for mitochondria and the cytosol exhibit only transient changes. These are limited to about 60 seconds of undershoot or overshoot, with respect to the cytosol, and then return to nearly the levels observed before changing the conditions. Adenylate kinase was found to be exclusively associated with chloroplasts (90% of total activity level) and mitochondria. Isotonic liberation of vacuoles did not point toward a significant association of adenylates with this compartment.     

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.     

Adenine nucleotide content and energy charge in dry cells and cysts of Azotobacter vinelandii

Abstract The levels of intracellular adenine nucleotides, energy charge, oxygen consumption and poly-β-hydroxybutyric acid stored, have been investigated in dry vegetative cells and cysts of Azotobacter vinelandii . The data show that under desiccation conditions the cysts retain viability at energy charge values of 0.20 and an ATP/ADP ratio of 0.24, whereas under the same desiccation conditions, vegetative cells die at energy charge values <0.5 and an ATP/ADP ratio of <0.6.     

Light-Induced Membrane Hyperpolarization and Adenine Nucleotide Levels in Perfused Characean Cells

This study examines the relationship between light-induced membranehyperpolarization and changes in adenine nucleotide levels intonoplast-free characean cells. When cells were perfused witha medium containing 1 mM ATP in the dark, the plasma membranedepolarized, the cytosolic ATP level decreased, and the ADPand AMP levels increased. Under light, the membrane hyperpolarized,the ATP level increased, and the ADP and AMP levels decreased.These changes in the adenine-nucleotide levels could partiallyexplain the membrane hyperpolarization. When cells were perfusedwith a medium containing an ATP-regenerating system consistingof phosphoenolpyruvate and pyruvate kinase, the membrane potentialremained in the hyperpolarized state, the ATP level remainedat a high level and no light-induced hyperpolarization was observed.The intracellular adenine nucleotide levels were also controlledby continuous perfusion. The membrane potential was determinedonly by the adenine nucleotide levels of perfusion media, irrespectiveof the light condition. Chloroplast-free Nitellopsis cells into which isolated Pisumchloroplasts were introduced also showed light-induced membranehyperpolarization. Pretreatment of chloroplasts with dicyclohexylcarbodiimide(DCCD) completely abolished the hyperpolarization with parallelinhibition of photophosphorylation. These results strongly suggestthat changes in adenine nucleotide levels caused by photophosphorylationare responsible for light-induced membrane hyperpolarizationin perfused cells. (Received August 17, 1985; Accepted December 13, 1985)     

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.     

Firefly assay of adenine nucleotides from algae: Comparison of extraction methods

Pools of ATP, ADP and AMP in algae were compared after extractionwith Trisbuffer, ethanol, chloroform, sulfuric acid, trichloroaceticacid and perchloric acid. Experimental organisms were Anacystisnidulans, Euglena gracilis, Chlorella kessleri and Scenedesmusobtusiusculus. Adenine nucleotides were determined by fireflyluciferase assay. Extraction of illuminated algae in situ with the different methodsresulted in great variations in the proportions between ATP,ADP and AMP. The sum of adenine nucleotides varied less. Extractionwith trichloroacetic acid or perchloric acid generally yieldedthe highest energy charge and ATP/ADP ratio. Experiments withilluminated Anacystis and Scenedesmus showed that extractionwith perchloric acid in situ gave higher yields of ATP in theextracts than if samples were withdrawn for extraction (timefor sample withdrawal {small tilde}1 sec). For Anacystis, itwas demonstrated that the extraction methods giving the highestproportions of ATP were suitable for studies on fluctuationsof the adenine nucleotide pools at transitions from dark tolight. ¹ Permanent address: National Defence Research Institute, Department4, S-172 04 Sundbyberg, Sweden. (Received September 4, 1978; )     

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.     

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.     

Stimulation of carbon dioxide fixation in isolated pea chloroplasts by catalytic amounts of adenine nucleotides

Carbon dioxide-dependent O(2) evolution by isolated pea (Pisum sativum var. Massey Gem) chloroplasts was increased two to 12 times by the addition of ATP. O(2) evolution was also stimulated by ADP and to a lesser extent by AMP. The ATP effects were not due to broken chloroplasts present in the preparations nor was ATP acting as a phosphate source. We concluded that the adenine nucleotides were acting catalytically. The concentration of ATP required for half-maximum rate of O(2) evolution was 16 to 25 mum. The degree to which ATP stimulated O(2) evolution depended on the age of pea plants from which the chloroplasts were isolated. Spinach (Spinacia oleracea var. True Hybrid 102) chloroplasts did not show a consistent stimulation of O(2) evolution by adenine nucleotides.The adenine nucleotide content of pea chloroplasts was not lower than that of spinach chloroplasts, but pea chloroplasts which showed a large stimulation of O(2) evolution by ATP contained an ATP-hydrolyzing reaction with rates of 10 to 50 mumol ATP hydrolyzed mg chlorophyll(-1) hour(-1). The rate of the ATP-consuming reaction was much lower in spinach chloroplasts and in chloroplasts from older pea plants which did not show large stimulation of O(2) evolution by ATP. We propose that the ATP-consuming reaction, with a high affinity for ATP, decreased the effective size of the ATP pool available for CO(2) fixation. Added adenine nucleotides could be transported into the chloroplasts increasing the concentration of internal nucleotides. Calculations showed that the adenine nucleotide transporter on the outer chloroplast membranes could operate at a sufficient rate to produce such an effect.     

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.     

A high-activity ATP translocator in mesophyll chloroplasts of Digitaria sanguinalis, a plant having the C-4 dicarboxylic acid pathway of photosynthesis

The effect of exogenous adenine nucleotides on CO₂ fixation and oxygen evolution was studied with mesophyll protoplast extracts of the C₄ plant Digitaria sanguinalis. Exogenous ATP was found to stimulate the rate of pyruvate and pyruvate + oxalacetate induced CO₂ fixation, as well as reverse the inhibition of CO₂ fixation by carbonyl cyanide m-chlorophenyl hydrazone and several electron transport inhibitors. The ATP-dependent stimulation of CO₂ fixation varied from 40 to 70 μmol CO₂ fixed/mg chlorophyll per h, suggesting that ATP was crossing the chloroplast membranes at rates of 80–140 μmol/mg chlorophyll per h, since 2 ATP are required for each CO₂ fixed. Fixation of CO₂ could also be induced in the dark by exogenous ATP, in which case ADP accumulated outside the chloroplasts. This suggests that external ATP is exchanging for internal ADP. In contrast, ADP and AMP were found not to traverse chloroplast membranes, on the basis that neither nucleotide inhibited CO₂ fixation or stimulated oxygen evolution that was limited by available ADP for phosphorylation. Further evidence that ATP can enter the chloroplasts was obtained by direct measurements of the increase in ATP in the chloroplasts due to addition of exogenous ATP in the dark. These studies yielded minimal rates of ATP uptake on the order of 30–40 μmol/mg chlorophyll per h. It is suggested that a membrane translocator exists that specifically transports ATP into the chloroplasts in exchange for ADP. The significance of these findings are considered with respect to the C₄ pathway of photosynthesis.     

Perinatal changes in adenine nucleotide content of developing rat kidney

Adenine nucleotides have been measured in fetal and newborn kidneys of rat using the luciferine-luciferase method. In fetuses, between days 18 and 21 of gestation there is a drop of the relative amount of ATP in the renal nucleotide pool. Consequently, the kidneys of 21 days-old fetuses have lowered ATP/ADP ratio (3.6) and energy charge (0.80) compared with values found on day 18 (6.9 and 0.91, respectively); this relative energy deficit is heightened in progesterone induced postmaturity. One hour after delivery whether the gestational stage is 21, 22 or 23 days, there is a rise in ATP and a decrease in AMP content which restore a high energy level in kidney of the newborn and a 30% increase in the total adenine nucleotide pool.     

Loose and tight binding of adenine nucleotides by membrane-associated chloroplast ATPase

Steady-state binding of adenine nucleotides by thylakoid membranes is measured by employing a centrifugation technique. By this method tightly bound nonexchangeable nucleotides can be discriminated from loosely bound, exchangeable nucleotides. Nucleotide binding requires membrane energization and is highly specific for medium ADP. In illuminated chloroplasts almost no exogenous AMP and only some ATP are incorporated, most being recovered as tightly bound nucleotides. In light-triggered chloroplasts, however, which are capable of hydrolyzing ATP, a high level of exchangeable nucleotides is found on the membranes. The sum of tightly bound and loosely bound nucleotides originating from medium ADP is about one per CF₁. The ratio between them decreases with increasing proton-motive force. Exchangeable nucleotides most probably represent the ligands involved in the catalytic process, as suggested from substrate specificity and the effect of a competitive inhibitor of photophosphorylation, naphthoyl ADP. This compound in a low concentration range supresses loose binding but not tight binding of medium ADP. Under phosphorylating conditions (presence of ADP, P_i and light), some of the tightly bound nucleotides exist as ATP even in the presence of a hexokinase system. The results are discussed in the context of the regulation of chloroplast ATPase by tight nucleotide binding.     

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.     

Stabilization of adenine nucleotide ratios at various values by an oxygen limitation of respiration in germinating lettuce (Lactuca sativa) seeds.

The concentrations of adenine nucleotides were determined in germinating lettuce (Lactuca sativa) seeds after transitions from air to hypoxic or anoxic atmospheres. The ratio ATP/ADP and the energy charge were rapidly lowered after the transitions and remained stable at low values for hours. The energy charge in anoxia stabilized at a value close to 0.3. After 24 h in anoxia the energy charge rose rapidly to high values (0.9) when N2 was replaced by air. The metabolic properties of lettuce seeds had then been conversed for hours at low energy charge. In hypoxia the O2 uptake was decreased and the energy charge was stabilized at values intermediate between that in air and that in anoxia. When the O2 partial pressures (pO2) were 5 and 2kPa, the values of O2 uptake were one-third and one-sixth of that in air, and the energy charges were 0.7 and 0.5. These results show that the energy charge is regulated over a wide range of values. The ratio ATP/ADP and the energy charge are indicators of the limitation of metabolic activity by hypoxia.