Transient Changes in the Energy State of Adenylates and the Redox States of Pyridine Nucleotides in Chlorella Cells Induced by Environmental Changes

The unicellular green algae Chlorella ellipsoidea was used tostudy transient changes in the energy state of adenylates andthe redox states of pyridine nucleotides induced by environmentalchanges. The transition from anaerobic to aerobic conditionsin the dark induced a sharp rise in the ATP ratio [ATP/(ATP+ADP+AMP)],a sudden decrease in the NADH ratio [NADH/(NAD⁺+NADPH)] anda transient drop in the NADPH ratio [NADPH/(NADP⁺+NADPH)]. Illuminationafter a dark period under anaerobic, CO₂-free conditions inducedsharp increases in the ATP and NADPH ratios and a slower decreasein the NADH ratio. Illumination under aerobic conditions, ineither the presence or absence of CO₂, caused a sharp increasein the NADPH ratio, a small increase in the ATP ratio and aslower increase in the NADH ratio. In the presence of CO₂, asubsequent large drop in the NADPH ratio occurred. Darkeningunder anaerobic, CO₂-free conditions induced a sudden decreasein the ATP ratio, a temporary fall in the NADPH ratio and aslow increase in the NADH ratio. Darkening under aerobic conditionsinduced transient drops in the ATP and NADPH ratios and a suddendrop in the NADH ratio. The addition of CO₂ to the atmospherewith illumination produced a decrease in all three parameters. These results are discussed in relation to current theoriesof the interaction between photosynthesis and respiration. Ourobservations indicate that the energy and reducing potentialsgenerated by photochemical processes are used for and controlother processes besides CO₂ fixation in photosynthetic cells. (Received December 3, 1981; Accepted May 4, 1982)     

Adenine and pyridine nucleotide levels during calcium-induced conidiation in Penicillium notatum

Concentrations of adenine and pyridine nucleotides and the associated charge values were examined in extracts of mycelium of Penicillium notatum during vegetative growth and reproductive development promoted by the addition of Ca²⁺ (10 mmol dm^-3). The significant increase in adenylate energy charge promoted by Ca²⁺ was due to a fall in intracellular AMP and a concomitant rise in ATP concentration. Intracellular concentrations of NADH and NAD fell within 1 h of the addition of Ca²⁺. The catabolic reduction charge was unchanged by Ca²⁺ whilst the anabolic reduction charge increased in Ca²⁺-induced mycelium due to lowered intracellular NADP concentration. Reduced concentration of NADPH in Ca²⁺-induced mycelium, relative to the vegetative controls, lowered the phosphorylated nucleotide fraction. The results are discussed in relation to metabolic economy during morphogenesis in P. notatum.     

Negative modulation of Escherichia coli NAD kinase by NADPH and NADH.

NAD kinase was purified 93-fold from Escherichia coli. The enzyme was found to have a pH optimum of 7.2 and an apparent Km for NAD+, ATP, and Mg2+ of 1.9, 2.1, and 4.1 mM, respectively. Several compounds including quinolinic acid, nicotinic acid, nicotinamide, nicotinamide mononucleotide, AMP, ADP, and NADP+ did not affect NAD kinase activity. The enzyme was not affected by changes in the adenylate energy charge. In contrast, both NADH and NADPH were potent negative modulators of the enzyme, since their presence at micromolar concentrations resulted in a pronounced sigmoidal NAD+ saturation curve. In addition, the presence of a range of concentrations of the reduced nucleotides resulted in an increase of the Hill slope (nH) to 1.7 to 2.0 with NADH and to 1.8 to 2.1 with NADPH, suggesting that NAD kinase is an allosteric enzyme. These results indicate that NAD kinase activity is regulated by the availability of ATP, NAD+, and Mg2+ and, more significantly, by changes in the NADP+/NADPH and NAD+/NADH ratios. Thus, NAD kinase probably plays a role in the regulation of NADP turnover and pool size in E. coli.     

Changes in the level of oxidized and reduced pyridine nucleotides during cold acclimation of winter rape plants

Prolonged cold (2°C) treatment of winter rape plants ( Brassica napus L. var. oleifera L. cv. Górczański) markedly modified the pattern of leaf growth and brought about changes in the level of pyridine nucleotides already during the first few days of treatment. The NAD⁺, NADP⁺ and NADPH levels markedly increased but there was practically no effect on the NADH level. Changes in the respective nucleotide levels were reflected by changes in anabolic and catabolic reduction charges. The former increased by 70%, whereas the latter decreased by 44%. Alterations in pyridine nucleotide levels and the reduction charges are discussed in terms of possible mechanisms involved, as well as in terms of their role in plant adaptation to cold.     

A relation between (NAD+)/(NADH) potentials and glucose utilization in rat brain slices.

Changes in several parameters involved in the control of metabolism were correlated with changes in glucose utilization in rat brain slices incubated under conditions which reduced glucose oxidation by 40 to 70%. The parameters included: the concentrations of ATP, ADP, AMP, and the adenylate energy charge; the cytoplasmic oxidation-reduction state ([NAD+]/[NADH]), determined from the [pyruvate]/[lactate] equilibrium; the mitochondrial oxidation-reduction state, determined from the [NH4+] ]2-oxoglutarate]/[glutamate] Equilibrium; the cytoplasmic and mitochondrial oxidation-reduction potentials (in volts), calculated from the respective [NAD+]/ [NADH] ratios using the Nernst equation; and the difference between the cytoplasmic and mitochondrial [NAD+]/[NADH] potentials. The conversion of [3, 4-14C] glucose to 14CO2 and of [U-14C] glucose to acetylcholine and to lipids, proteins, and nucleic acids by the brain slices were also determined. The values obtained by subtracting the mitochondrial from the cytoplasmic [NAD+1/[NADH] potentials correlated more closely with glucose utilization than did other parameters, under the conditions studied. For the synthesis of acetylcholine, the correlation coefficient was 0.96, and for the production of 14CO2 from [3, 4-14C] glucose it was 0.82.     

The stimulus--secretion coupling 4-methyl-2-oxopentanoate-induced insulin release.

1. Pancreatic islet insulin secretion and 45Ca uptake showed similar responses to variation in the extracellular concentration of 4-methyl-2-oxopentanoate with a threshold at 4 mM and a maximal response at a 25 mM concentration. 2. Islet respiration, acetoacetate production and rates of substrate utilization, oxidation and amination all changed as a simple hyperbolic function of 4-methyl-2-oxopentanoate concentration and exhibited a maximal response at 25 mM. 3. The responses of ATP content, [ATP]/[ADP] ratio, adenylate energy charge and [NADH]/[NAD+] ratio were also hyperbolic in nature but were maximally elevated at lower concentrations of the secretagogue. The islet [NADPH]/[NADP+] ratio, however, was tightly correlated with parameters of metabolic flux, 45Ca uptake and insulin release. 4. NH4+ and menadione, agents that promote a more oxidized state in islet NADP, did not affect islet ATP content or the rates of [U-14C]4-methyl-2-oxopentanoate oxidation or amination, but markedly inhibited islet 45Ca uptake and insulin release. 5. It is proposed that changes in the redox state of NADP and Ca transport may serve as mediators in the stimulus-secretion coupling mechanism of insulin release induced by 4-methyl-2-oxopentanoate.     

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.     

Effects of Temperature on Infected Cell O2 Concentration and Adenylate Levels in Attached Soybean Nodules

To assess the role of O2 in the regulation of nodule metabolism following a decrease or an increase in temperature, the fractional oxygenation of leghemoglobin (FOL) was measured in soybean (Glycine max L. Merr.) nodules during rapid and gradual changes in temperature from 20[deg]C to either 15 or 25[deg]C. The affinity of leghemoglobin for O2 was also measured at each temperature and the values were used to calculate the infected cell O2 concentration (Oi). After nodules were transferred to 15[deg]C, FOL and Oi increased and adenylate energy charge (AEC = [ATP + 0.5ADP]/[ATP + ADP + AMP]) increased from 0.70 to 0.78. The temperature increase was associated with a decrease in FOL and Oi. We concluded that changes in nodule temperature alter the respiratory demand of the nodules for O2, resulting in a change in Oi and a shift in the balance between ATP consumption and ATP production within the nodule tissue.     

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.     

Application of adenylate energy charge to problems of environmental impact assessment in aquatic organisms

Various physiological and biochemical methods have been proposed for assessing the effects of environmental perturbation on aquatic organisms. The success of these methods as diagnostic tools has, however, been limited. This paper proposes that adenylate energy charge overcomes some of these limitations. The adenylate energy charge (AEC) is calculated from concentrations of adenine nucleotides ([ATP+½ADP]/[ATP+ADP+AMP]), and is a reflection of metabolic potential available to an organism. Several features of this method are: correlation of specific values with physiological condition or growth state, a defined range of values, fast response times and high precision. Several examples from laboratory and field experiments are given to demonstrate these features. The test organisms used (mollusc species) were exposed to a variety of environmental perturbations, including salinity reduction, hydrocarbons and low doses of heavy metal. The studies performed indicate that the energy charge may be a useful measure in the assessment of environmental impact. Its use is restricted, however, as several limitations exist which need to be fully evaluated. Further work relating values to population characteristics of multicellular organisms needs to be completed before the method can become a predictive tool for management.     

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.     

Diurnal changes in adenylates and nicotinamide nucleotides in sugar beet leaves

Sugar beets (Beta vulgaris L. cv. F58-554H1) were cultured hydroponically in growth chambers at 25°C, with a photon flux density of 500 mol m^-2s^-1. Measurements were made of net CO₂ exchange, leaf adenylates (ATP, ADP and AMP), and leaf nicotinamide nucleotides (NAD⁺, NADP⁺, NADH, NADPH), over the diurnal period (16h light/8 h dark) and during photosynthetic induction. All the measurements were carried out on recently expanded leaves from 5-week-old plants. When the lights were switched on in the growth chamber, the rate of photosynthetic CO₂ uptake, and the levels of leaf ATP and NADPH increased to a maximum in 30 min and remained there throughout the light period. The increase in ATP over the first few minutes of illumination was associated with the phosphorylation of ADP to ATP and the increase in NADPH with the reduction of NADP⁺; subsequently, the increase in ATP was associated with an increase in total adenylates while the increase in NADPH was associated with an accumulation of NADP⁺ and NADPH due to the light-driven phosphorylation of NAD⁺ to NADP⁺. On return to darkness, ATP and NADPH values decreased much more slowly, requiring 2 to 4 hours to reach minimum values. From these results we suggest that (i) the total adenylate and NADPH and NADP⁺ (but not NAD⁺ and NADH) pools increase following exposure to light; (ii) the increase in pool size is not accompanied by any large change in the energy or redox states of the system; and (iii) the measured ratios of ATP/ADP and NADPH/NADP⁺ for intact leaves are low and constant during steady-state illumination.Abbreviations AEC adenylate energy charge - DHAP dihydroxyacetone phosphate - MTT 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide - PES phenazine ethosulfate - PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - PFD photon flux density - Ru5P ribulose-5-phosphate - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase     

Crabtree effect in tumoral pancreatic islet cells

The relationship between glycolysis and respiration was examined in a model of pancreatic B-cell dysfunction, namely in tumoral insulin-producing cells of the RINm5F line. A rise in D-glucose concentration from 2.8 to 16.7 mM increased the utilization of D-[5-3H]glucose and production of [14C]lactate from D-[U-14C]glucose, whereas decreasing the oxidation of either D-[U-14C]glucose or D-[6-14C]glucose. Whereas 2.8 mM D-glucose augmented O2 uptake above basal value, a further rise in D-glucose concentration to 16.7 mM decreased respiration, which remained higher, however, than basal value. Whether at low or high concentration, D-glucose exerted a pronounced sparing action upon the oxidation of endogenous nutrients in cells prelabeled with either L-[U-14C]glutamine or [14C]palmitate and, nevertheless, augmented above basal value the rate of lipogenesis, ATP/ADP content, adenylate charge, and cytosolic NADH/NAD+ and NADPH/NADP+ ratios. The generation of ATP resulting from the catabolism of either exogenous D-glucose or endogenous nutrients was not affected by the rise in hexose concentration from 2.8 to 16.7 mM. Thus, in sharp contrast with the situation found in normal islet cells, a rise in D-glucose concentration, instead of stimulating mitochondrial oxidative events, caused, through a Crabtree effect, inhibition of hexose oxidation and O2 consumption in tumoral islet cells.     

Changes of nicotinamide coenzymes and adenylate energy charge in leaves of hybrid and parental tomato forms in anin vitro culture

Interrelation between dry matter in leaves and catabolic and anabolic activity in parental forms and F₁-hybrids ofLycopersicon esculentum Mill was investigated underin vitro culture. Heterosis in dry matter was observed in hybrids and similar values of adenylate energy charge (AEC) and the redox charge (RC), displaying balance between the systems generating energy and the processes associated with its consumption, were revealed. A higher recovery degree of NADP-NADPH system in comparison with NAD-NADH one, showing inutilization of recovery equivalents in the NADPH form in biosynthetic processes, was detected in the initial cultivars. The content of adenylate nucleotides, nicotinamide coenzymes and their changes might play a role in regulatory mechanisms of biomass accumulation in F₁-tomato hybrids and their parents.     

Effects of adenosine phosphates and nicotinamide nucleotides on pyruvate carboxylase from baker''s yeast

1. Pyruvate carboxylase from baker's yeast is inhibited by ADP, AMP and adenosine at pH8.0 in the presence of magnesium chloride concentrations equal to or higher than the ATP concentration. The adenine moiety is essential for the inhibitory effect. 2. In the absence of acetyl-CoA (an allosteric activator) ADP, AMP and adenosine are competitive inhibitors with respect to ATP. In the presence of acetyl-CoA, besides the effect with respect to ATP, AMP competes with acetyl-CoA, whereas ADP and adenosine are non-competitive inhibitors with respect to the activator. 3. Pyruvate carboxylase is inhibited by NADH. The inhibition is competitive with respect to acetyl-CoA and specific with respect to NADH, since NAD(+), NADP(+) and NADPH do not affect the enzyme activity. In the absence of acetyl-CoA, NAD(+), NADH, NADP(+) and NADPH do not inhibit pyruvate carboxylase. 4. Pyruvate carboxylase is inhibited by ADP, AMP and NADH at pH6.5, in the presence of 12mm-Mg(2+), 0.75mm-Mn(2+) and 0.5mm-ATP, medium conditions similar to those existing inside the yeast cell. The ADP and NADH effects are consistent with a regulation of enzyme activity by the intracellular [ATP]/[ADP] ratio and secondarily by NADH concentration. These mechanisms would supplement the already known control of yeast pyruvate carboxylase by acetyl-CoA and l-aspartate. Inhibition by AMP is less marked and its physiological role is perhaps limited.     

Thermodynamics of oxidative phosphorylation in bovine heart submitochondrial particles.

The rates of both forward and reverse electron transfer in phosphorylating submitochondrial particles from bovine heart can be controlled by the thermodynamic phosphorylation potential (deltaGp) of the adenine nucleotide system. deltaGp is the Gibbs free energy of ATP synthesis and is defined by the relationship deltaGp = -deltaG'o + RTln([ATP]/[ADP][Pi]) where deltaG'o is the standard free energy of ATP hydrolysis. Studies of the effects of deltaGp on NADH respiration and the reduction of NAD+ by succinate show that increasing values of deltaGp cause an inhibition of forward electron transfer and a stimulation of reverse electron transfer. Between deltaGp values of 7.6 and 13.0 kcal/mol the rate of NADH respiration decreased 3-fold and the rate of NAD+ reduction by succinate increased 3-fold. Indirect phosphorylation potential titration experiments as well as direct chemical measurements indicate that steady state levels of ATP, ADP, and Pi are established during NADH respiration which correspond to a deltaGp equal to 10.7 to 11.4 kcal/mol.     

Oxygen deficiency and its effect on the adenylate system in Acetobacter in the submerse acetic fermentation

Summary It is well known that Acetobacter is extremely sensitive in high total concentrations (GK)¹ of ethanol and acetic acid. In the acetator, at a total concentration (GK) of 13%, ATP pool and growth show reverse behaviour. During the stationary, acidifying phase, the extracellular adenylate concentration amounts to 70% of the total edenylate pool (AN=ATP+ADP+AMP). In this range, the average value of the intracellular energy charge [EC=(ATP+1/2ADP)/(ATP+ADP+AMP)] is 0.82.After 45 s of interruption of aeration, the EC of the total culture dropped to a value of 0.58. After several weeks of storage, the EC of the inoculum amounted to 0.50.     

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.     

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.