Relationship between iron-limited growth and energy limitation during phased cultivation of Candida utilis

The yeast Candida utilis was continuously synchronized by the phasing technique (6 h doubling time) with either iron or nitrogen as the limiting nutrient. Iron limitations resulted in decreased molar growth yields with respect to the carbon substrates and ammonia and in increased specific rates of oxygen uptake. Relatively low energy-charge values were maintained by the iron-limited culture. All these taken together seemed to indicate that the growth of the yeast under iron limitation was also limited by metabolically available energy. Consideralbe amounts of ethyl acetate were produced by the yeast under phased cultivation when the growth was limited by iron but not by nitrogen. In vitro studies using cell-free extracts showed that the substrates for ethyl acetate synthesis were acetyl coenzyme A (acetyl CoA) and ethanol. Under iron-limited growth acetyl CoA seemed to be diverted to ethyl acetate formation rather than being oxidized through the tricarboxylic acid (TCA) cycle. The possibility of energy limitation under iron-limited growth being brought about by the reduced capacity of the yeast to oxidize acetyl CoA through the TCA cycle is considered.     

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.     

Intracellular adenylate pools and protein degradation during zoosporangial differentiation in Allomyces arbuscula

In Allomyces arbuscula Butl., strain Bali, the ratio of protein to dry weight remained constant in exponentially growing but decreased in differentiating cultures. The adenylate pools (ATP, ADP, AMP) and energy charge which integrates them, increased during zoospore germination and stabilized around 0.9 during differentiation. The level of ATP increased early during the induction of zoosporangia for up to 1 h and then declined. The ADP and AMP remained low for most of the time except for a transient increase in ADP (first 30 min induction). The energy charge was low in spores. The rate of turnover of proteins during growth and differentiation was more or less similar for up to 1.5 h after transfer. Subsequently very little turnover of proteins occurred in the growing plants. In differentiating plants, the rate of degradation was maintained and by the end of the 4 h experimental period 30% of the vegetative proteins were degraded. The intracellular ammonium showed a peak between 30 to 60 min of induction and was higher in the differentiating mycelia than in actively growing plants, while the glutamate pool remained around 1 μmol (mg protein)⁻¹ in both types of plants. The physiological role of these protein degradation products 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.     

Energy aspects of the growth of Escherichia coli synchronized by starvation

The quantitative determination of adenyl nucleotides based on the separation of their dansyl derivatives by thin layer chromatography has made it possible to study the dynamics of changes in the pool of ATP, ADP and AMP in Escherichia coli K-12 during its synchronous growth after glucose starvation. The energy parameters (the adenylate pool, energy charge, teh ATP/ADP ratio, the rates of oxygen uptake and ATP generation, the economic coefficients of oxygen and ATP utilization) were compared with changes in the growth characteristics (the rate of growth and biomass concentration). This comparison allowed the authors to draw the conclusion about the uncoupled constructive and energy metabolism and about the possible regulatory role of energy parameters in the synchronised culture growth.     

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.     

The effect of nucleotides on glutamate dehydrogenase from the mealworm fat body

The effect of nucleotides: AMP, cAMP, ADP, ATP, GDP and GTP, on glutamate dehydrogenase (GDH) purified from the mealworm fat body was studied. Guanine nucleotides and ATP inhibited the enzyme strongly in both directions. GDH was partially protected from the inhibition by the addition of ADP to an assay medium. AMP and cAMP activated the enzyme slightly. The concerted effects of ADP and ATP indicate the importance of adenylate energy charge in the regulation of fat body GDH. It is suggested that GDH may play amphibolic role in the fat body and that the direction of GDH catalysed reaction is under strong influence of nucleotides. The enzyme may synthesize glutamate at high energy charge, but when the energy reserves are low, it oxidizes glutamate.     

Cellular levels, excretion, and synthesis rates of cyclic AMP in Escherichia coli grown in continuous culture.

Changes in dilution rate did not elicit large and systematic changes in cellular cyclic AMP levels in Escherichia coli grown in a chemostat under carbon or phosphate limitation. However, the technical difficulties of measuring low levels of cellular cyclic AMP in the presence of a large background of extracellular cyclic AMP precluded firm conclusions in this point. The net rate of cyclic AMP synthesis increased exponentially with increasing dilution rate through either the entire range of dilution rates examined (phosphate limitation) or a substantial part of the range (lactose and glucose limitations). Thus, it is probable that growth rate regulates the synthesis of adenylate cyclase. The maximum rate of net cyclic AMP synthesis was greater under lactose than under glucose limitation, which is consistent with the notion that the uptake of phosphotransferase sugars is more inhibitory to adenylate cyclase than the uptake of other carbon substrates. Phosphate-limited cultures exhibited the lowest rate of net cyclic AMP synthesis, which could be due to the role of phosphorylated metabolites in the regulation of adenylate cyclase activity. Under all growth conditions examined, greater than 99.9% of the cyclic AMP synthesized was found in the culture medium. The function of this excretion, which consumed up to 9% of the total energy available to the cell and which evidently resulted from elaborate regulatory mechanisms, remains entirely unknown.     

Nucleotide Pools and Adenylate Energy Charge in Balanced and Unbalanced Growth of Chromatium

Adenine nucleotide pools and their energy charge were measured during balanced and unbalanced growth of photoheterotrophic Chromatium cultures. The methods used involved rapid sampling, accurate to within 1 s, from isotopically labeled cultures followed by chromatographic separation of individual nucleotides. During balanced growth, both energy charge and adenosine triphosphate (ATP) concentrations, whether expressed as a function of cell protein or intracellular water, were slightly higher in limiting light intensities than in cultures growing at their maximal rate in bright light. The ATP found corresponded to 4.67 +/- 0.08 nmol/mg of protein or 1.34 +/- 0.57 mM for low-light cells and to 4.41 +/- 0.58 mmol/mg of protein or 0.85 +/- 0.12 mM for high-light cells. Corresponding energy charges were 0.85 +/- 0.02 and 0.81 +/- 0.02. Illumination shifts caused differential synthesis of photosynthetic pigments lasting 2 to 3 h without corresponding perturbation of adenine nucleotide levels. Cultures in intermittent illumination were severely affected by some cycle durations; they had abnormal morphology and very high bacteriochlorophyll-to-protein ratios. In such cultures, energy charge and nucleotide concentrations were within normal limits and relaxed to the dark steady state during the dark periods. Arsenate at AsO(4) (3-) to PO(4) (3-) ratios of 10:1 in the medium retarded growth, but no abnormality of charge or quantity of phosphate-containing nucleotides was found. These experiments therefore suggest that, within experimental error, neither the size nor the charge of the adenylate pools governs growth rate in Chromatium. Moreover, these parameters do not appear to be concerned in regulating the synthesis of photosynthetic apparatus in this organism.     

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.     

Adenylate and nicotinamide nucleotides in developing soybean seeds during seed-fill

Profiles of adenylate and nicotinamide nucleotides in soybean seeds were determined during seed-fill. The ATP content per seed increased during the early seed-filling stages to a level of 10 to 12 micrograms per seed. Seed ATP decreased after 40 days of development and reached its lowest level of less than 1 microgram at maturity. The ATP:ADP ratios were relatively constant at all seed development stages. Sharp increases in AMP levels during the late seed-fill stages were paralleled with a disappearance of ATP and ADP pools resulting in a reduced seed energy charge. Energy charge varied from the highest value of 0.78 at mid-seed-fill to less than 0.10 at maturity.     

Energy state of spring and winter wheat during cold hardening. Soluble sugars and adenine nucleotides

Marked increases were found in the content of total soluble sugars, reducing sugars and ATP in winter wheat ( Triticum aestivum L. cv. Frederick) during cold hardening. The changes in soluble sugars and ATP of spring wheat ( T. aestivum L. cv. Glenlea) grown under similar conditions were less pronounced. The increase in ATP content during hardening of winter wheat was not associated with significant changes in the content of ADP or AMP. The adenylate energy charge did not change during hardening in either cultivar, but it was higher in the winter cultivar under both growth conditions. This difference could be related to the cold hardiness capacity of winter wheat.     

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.     

Several parameters of the growth of chemostatic culture of Candida utilis in the presence of optimal and submaximal temperatures

Candida utilis BKM Y-1668 was cultivated in the chemostat (limitation with glycerol) with the rate of flow D from 0.05 to 0.3 hr-1; the economic coefficient Y and Ks were constant at the optimum temperature of growth (30 degrees C). The maximum growth rate was 0.35 hr-1. The content of ATP in the cells and the energy charge of the cell decreased, and the content of ADP and AMP and the activity of phoshohydrolases increased in the cell, with an increase in D from 0.05 to 0.3 hr-1. Small amounts of glycerol and phosphorus were expended for maintaining life without multiplication (m) at 30 degrees C. At the submaximum temperature (40 degrees C), growth of the cells was inhibited, the rate of assimilation of glycerol and phosphorus, and m, increased. The content of ATP in the cells and their energy charge also increased.     

Effect of Temperature Stress on Adenylate Pools and Energy Charge in a Psychrophilic Bacterium, Vibrio sp. ABE-1

The adenylate energy charge in the psychrophilic bacterium Vibriosp. ABE-1 remained unchanged while the cells grew, althoughthe ATP pool varied in parallel with the growth rates underdifferent temperature conditions (0–20°C). However,at a nonpermissive temperature (25°C), the bacteria couldnot grow, the energy charge decreased due to temporary disappearanceof ATP, and before long, both the number of viable cells andthe energy charge decreased. Phosphoribosyl pyrophosphate synthetase, an ATP-utilizing enzyme,could be efficiently controlled by the energy charge at permissivetemperatures, but was regulated little or not at all at a nonpermissivetemperature (25°C). The regulation possibly arose from inhibitionof the enzyme activity by ADP or AMP, and especially by thereaction product AMP. (Received December 18, 1982; Accepted April 16, 1983)     

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.     

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 role of adenosine monophosphate nucleosidase in the regulation of adenine nucleotide levels in Azotobacter vinelandii during aerobic-anaerobic transitions

When cultures of Azotobacter vinelandii are made anaerobic the adenylate pool size remains constant or increases slightly while the adenylate energy charge decreases. Under these conditions, cell growth stops but the cells remain viable for at least 5 h with the decreased energy charge. The changes in the adenylate pool during the aerobic-anaerobic transition include: the formation of adenylates as a result of RNA degradation; the degradation of a portion of the excess AMP to form hypoxanthine by the sequential actions of AMP nucleosidase and adenine deaminase; an increase in the total adenylate pool which is stabilized at approximately 1.5 times the level in growing cells; and stabilization of the adenylate energy charge at a value near 0.3. The degradation of AMP is regulated by AMP nucleosidase, an allosteric enzyme which is activated by MgATP^2? and inhibited by P_i. The in vivo activity of AMP nucleosidase was estimated by measuring the rate of hypoxanthine formation in the culture or by measuring the activity of purified enzyme at the concentrations of AMP, ATP, and P_i found in the cells. The maximum estimated in vivo rate of AMP degradation was less than 3% of the catalytic capacity of AMP nucleosidase. Thus ample activity is present for rapid adjustments of the AMP levels in these cells. Expression of AMP nucleosidase catalytic activity is tightly controlled since high constant concentrations of intracellular AMP can be maintained for extended time periods at low adenylate energy charge values. Under these conditions controlled degradation of AMP can occur to maintain a constant AMP concentration.     

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)     

AMP deaminase from baker's yeast. Purification and some regulatory properties.

AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) was found in extract of baker's yeast (Saccharomyces cerevisiae), and was purified to electrophoretic homogeneity using phosphocellulose adsorption chromatography and affinity elution by ATP. The enzyme shows cooperative binding of AMP (Hill coefficient, nH, 1.7) with an s0.5 value of 2.6 mM in the absence or presence of alkali metals. ATP acts as a positive effector, lowering nH to 1.0 and s0.5 to 0.02 mM. P1 inhibits the enzyme in an allosteric manner: s0.5 and nH values increase with increase in Pi concentration. In the physiological range of adenylate energy charge in yeast cells (0.5 to 0.9), the AMP deaminase activity increases sharply with decreasing energy charge, and the decrease in the size of adenylate pool causes a marked decrease in the rate of the deaminase reaction. AMP deaminase may act as a part of the system that protects against wide excursions of energy charge and adenylate pool size in yeast cells. These suggestions, based on the properties of the enzyme observed in vitro, are consistent with the results of experiments on baker's yeast in vivo reported by other workers.