Pyridine Nucleotide Transhydrogenase from Azotobacter vinelandii

A method is described for the partial purification of pyridine nucleotide transhydrogenase from Azotobacter vinelandii (ATCC 9104) cells. The most highly purified preparation catalyzes the reduction of 300 mumoles of nicotinamide adenine dinucleotide (NAD(+)) per min per mg of protein under the assay conditions employed. The enzyme catalyzes the reduction of NAD(+), deamino-NAD(+), and thio-NAD(+) with reduced nicotinamide adenine dinucleotide phosphate (NADPH) as hydrogen donor, and the reduction of nicotinamide adenine dinucleotide phosphate (NADP(+)) and thio-NAD(+) with reduced NAD (NADH) as hydrogen donor. The reduction of acetylpyridine AD(+), pyridinealdehyde AD(+), acetylpyridine deamino AD(+), and pyridinealdehydedeamino AD(+) with NADPH as hydrogen donor was not catalyzed. The enzyme catalyzes the transfer of hydrogen more readily from NADPH than from NADH with different hydrogen acceptors. The transfer of hydrogen from NADH to NADP(+) and thio-NAD(+) was markedly stimulated by 2'-adenosine monophosphate (2'-AMP) and inhibited by adenosine diphosphate (ADP), adenosine triphosphate (ATP), and phosphate ions. The transfer of hydrogen from NADPH to NAD(+) was only slightly affected by phosphate ions and 2'-AMP, except at very high concentrations of the latter reagent. In addition, the transfer of hydrogen from NADPH to thio-NAD(+) was only slightly influenced by 2'-AMP, ADP, ATP, and other nucleotides. The kinetics of the transhydrogenase reactions which utilized thio-NAD(+) as hydrogen acceptor and NADH or NADPH as hydrogen donor were studied in some detail. The results suggest that there are distinct binding sites for NADH and NAD(+) and perhaps a third regulator site for NADP(+) or 2'-AMP. The heats of activation for the transhydrogenase reactions were determined. The properties of this enzyme are compared with those of other partially purified transhydrogenases with respect to the regulatory functions of 2'-AMP and other nucleotides on the direction of flow of hydrogen between NAD(+) and NADP(+).     

Redox involvement in acid secretion in the amphibian gastric mucosa

Summary Gastric fundic metabolism was studied by spectroscopic observation in frog mucosa during transitions of secretory status in vitro and by direct measurement of pyridine nucleotides and associated metabolites in biopsies of dog fundic mucosa also during secretory oxidation of the redox components from flavin adenine dinucleotide (FAD) to cytochromea ₃. Addition of histamine resulted in reduction of these components with onset of secretion by about 50%. In contrast, the effect of apparently, burimamide and subsequently histamine on the ratio of nicotinamide adenine dinucleotide to nicotinamide adenine dinucleotide, reduced (NAD⁺/NADH) was relatively slight. Further, the presence of burimamide substantially reduces the effect of amytal on the pyridine nucleotide spectrum and abolishes the effect of amytal on FAD and the cytochromes. Measurements of lactate, pyruvate, -ketoglutarate, NH₃ and glutamate in the dog showed that whereas the calculated NAD⁺/NADH ratio in the cytoplasm declined with onset of secretion, the calculated mitochondrial ratio rose. No change was noted in the nicotinamide adenine dinucleotide phosphate/nicotinamide adenine dinucleotide phosphate, reduced (NADP⁺/NADPH) ratio. It is concluded that (1) H₂ antagonists act by blocking substrate flow into the mitochondrial respiratory chain, (2) conversely, histamine stimulation acts at the level of substrate mobilization, and (3) there may be a cross-over in the mitochondrial chain between NAD⁺ and FAD.     

Further chemical characterization and biological activities of fluorescent I,N6-ethenoadenosine derivatives

The fluorescent 1,N⁶-ethenoadenosine derivatives of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, 3′:5′-cyclic adenosine monophosphate, adenosine and nicotinamide adenine dinucleotide have been prepared. Paper and thin layer chromatographic purification methods have been developed. Nuclear magnetic resonance and mass spectrum data indicate that only the purine ring has been modified.The 1,N⁶-ethenoadenosine triphosphate had about 70% of the activity of adenosine triphosphate as a substrate for total adenosine triphosphatase activity of hypophysectomized rat liver membranes. The 1,N⁶-ethenoadenosine diphosphate had about 86% of the activity of adenosine diphosphate as a substrate for adenosine diphosphatase of hypophysectomized rat liver membranes. The 1,N⁶-etheno derivative of nicotinamide adenine dinucleotide had about 8% of the activity of nicotinamide adenine dinucleotide as a substrate for nicotinamide adenine dinucleotide glycohydrolase and about 54% of the activity of nicotinamide adenine dinucleotide as a substrate for nicotinamide adenine dinucleotide pyrophosphatase of hypophysectomized rat liver membranes.K_m's for the ATPase, ADPase and yeast alcohol dehydrogenase using ε-ATP and ε-ADP and ε-NAD as substrates are presented.     

Fluorometric Determination of Adenosine Nucleotide Derivatives as Measures of the Microfouling, Detrital, and Sedimentary Microbial Biomass and Physiological Status

Adenosine, adenine, cyclic adenosine monophosphate (AMP), AMP, nicotinamide adenine dinucleotide, adenosine diphosphate, and adenosine triphosphate (ATP) were recovered quantitatively from aqueous portions of lipid extracts of microfouling, detrital, and sedimentary microbial communities. These could be detected quantitatively in the picomolar range by forming their 1-N⁶-etheno derivatives and analyzing by high-pressure liquid chromatography with fluorescence detection. Lipid extraction and subsequent analysis allowed the simultaneous measurement of the microbial community structure, total microbial biomass with the quantitative recovery of the adenine-containing cellular components, which were protected from enzymatic destruction. This extraction and fluorescent derivatization method showed equivalency with the luciferin-luciferase method for bacterial ATP measurements. Quick-freezing samples in the field with dry ice-acetone preserved the ATP and energy charge (a ratio of adenosine nucleotides) for analysis at remote laboratories. The metabolic lability of ATP in estuarine detrital and microfouling communities, as well as bacterial monocultures of constant biomass, showed ATP to be a precarious measure of biomass under some conditions. Combinations of adenosine and adenine nucleotides gave better correlations with microbial biomass measured as extractable lipid phosphate in the detrital and microfouling microbial communities than did ATP alone. Stresses such as anoxia or filtration are reflected in the rapid accumulation of intracellular adenosine and the excretion of adenosine and AMP into the surrounding milieu. Increases in AMP and adenosine may prove to be more sensitive indicators of metabolic status than the energy charge.     

Levels of oxidized and reduced pyridine nucleotides in dormant spores and during growth, sporulation, and spore germination of Bacillus megaterium.

Dormant spores of Bacillus megaterium contained no detectable reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) despite significant levels of the oxidized forms of these nucleotides (NAD and NADP). During the first minutes of spore germination there was rapid accumulation of NADH and NADPH. However, this accumulation followed the fall in optical density that is characteristic of the initiation of spore germination. Accumulation of NADH and NADPH early in germination was not blocked by fluoride or cyanide, and it occurred even when germination was carried out in the absence of an exogenous source of reducing power. In addition to pyridine nucleotide reduction, de novo synthesis also began early in germination as the pyridine nucleotide levels increased to those found in growing cells. Midlog-phase cells grown in several different media had 20 to 35 times as much total pyridine nucleotide as did dormant spores. However, as growth and sporulation proceeded, the NADH plus NAD level fell four- to fivefold whereas the NADPH plus NADP level fell by a lesser amount. From min 10 of spore germination until midway through sporulation the value for the ratio of NADH/NAD is about 0.1 (0.03 to 0.18) while the ratio of NADPH/ANDP is about 1.4 (0.3 to 2.4). Comparison of these ratios in log-phase versus stationary phase (sporulation) growth in all three growth media tested did not reveal any common pattern of changes.     

Effect of culture conditions on the NADH/NAD ratio and total amounts of NAD(H) in chemostat cultures of Enterococcus faecalis NCTC 775

Abstract Enterococcus faecalis was grown in chemostat culture on various energy sources at dilution rates ranging from 0.05 h⁻¹ to 0.5 h⁻¹, under both aerobic and anaerobic conditions. NADH/NAD ratios and total nicotinamide adenine dinucleotide pool size (NAD(H)) were determined. It was found that the NADH/NAD ratio was controlled by the steady state product concentrations rather than by the degree of reduction of the energy source. Highest ratios were observed when NADH was reoxidized via ethanol formation, whereas in aerobic cultures, in which predominantly acetate was produced and oxidation of NADH occurred via the NADH oxidase, ratios were lowest. Addition of ethanol to the medium resulted in an increase of the NADH/NAD ratio, both aerobically and anaerobically. The total amount of NAD(H) was found to be influenced by the culture conditions. Under anaerobic conditions, the NADH oxidation (NAD reduction) rate appeared to correlate with the total amount of nicotinamide nucleotides. In contrast, no effect of the culture conditions on the total amount of NAD(H) was observed in aerobically grown cells.     

Purine metabolites suppress proliferation of human NK cells through a lineage-specific purine receptor.

NK cell proliferation is suppressed in some patients with cancer by unknown mechanisms. Because purine metabolites released into the extracellular space during cell lysis may affect cell function, we hypothesized that these metabolites could serve as feedback regulators of NK cell proliferation. Sorted NK (CD56+/CD3-) cells were incubated with IL-2 (1000 U/ml) in a 4-day thymidine uptake assay with or without 10-10,000 microM of nucleotides. Adenine nucleotides inhibited NK cell proliferation, with ATP = ADP > 5'-adenylylimidodiphosphate > AMP = adenosine; ADP-ribose and nicotinamide adenine dinucleotide, but not nicotinamide or UTP, caused a dose-dependent suppression of thymidine uptake. A total of 100 microM ATP, a concentration that induced a maximal (80%) inhibition of thymidine uptake, did not inhibit cytotoxic activity against K562 targets. Because NK cells retained the ability to lyse K562 targets 4 days after exposure to 500 microM ATP or 1000 microM adenosine, inhibition of thymidine uptake was not due to cell death. Incubation of NK cells with dibutyryl cAMP and forskolin also suppressed thymidine uptake. Cholera toxin and pertussis toxin suppressed NK cell proliferation. Pertussis toxin did not block the adenine nucleotide effects. Further, ATP, but not adenosine or other nucleotides, markedly increased intracellular cAMP in a dose-dependent manner. The ATP-induced increase in cAMP was specific to cytolytic cells, because CD19+ B cells and CD4+ T cells did not increase their intracellular cAMP. These studies demonstrate that NK proliferation is regulated through purine receptors by adenine nucleotides, which may play a role in decreased NK cell activity. The response to adenine nucleotides is lineage-specific.     

Thiosulfate- and Sulfide-Dependent Pyridine Nucleotide Reduction and Gluconeogenesis in Intact Thiobacillus neapolitanus

Nicotinamide adenine dinucleotide phosphate (reduced form) is formed more rapidly after the addition of thiosulfate to suspensions of intact Thiobacillus neapolitanus in the absence of CO(2) than nicotinamide adenine dinucleotide (reduced form). Measurement of acid-stable metabolites shows this phenomenon to be the result of rapid reoxidation of nicotinamide adenine dinucleotide (reduced form) by 3-phosphoglyceric acid and other oxidized intermediates, which are converted to triose and hexose phosphates, and that, in reality, the rate of nicotinamide adenine dinucleotide (oxidized form) reduction exceeds that of nicotinamide adenine dinucleotide phosphate (oxidized form) by approximately 4.5-fold. The overall rate of pyridine nucleotide reduction by thiosulfate (264 nmol per min per mg of protein) is in excess of that rate needed to sustain growth. Pyridine nucleotide reduction, adenosine triphosphate synthesis, and carbohydrate synthesis are prevented by the uncoupler m-Cl-Carbonylcyanide phenylhydrazone. Sodium amytal inhibits pyridine nucleotide reduction and carbohydrate synthesis are prevented by the uncoupler m-Cl-carbonylcyanide observations are reproduced when sulfide serves as the substrate. The rate of pyridine nucleotide anaerobic reduction with endogenous substrates or thiosulfate is less than 1% of the aerobic rate with thiosulfate. We conclude that the principal, if not the only, pathway of pyridine nucleotide reduction proceeds through an energy-dependent and amytal-sensitive step when either thiosulfate or sulfide is used as the substrate.     

Nuclear magnetic resonance studies on pyridine dinucleotides. The pH dependence of the carbon-13 nuclear magnetic resonance of NAD+ analogs.

The pH dependence of the ¹³C chemical shifts for nicotinamide adenine dinucleotide (NAD⁺), thionicotinamide adenine dinucleotide (TNAD⁺), pyridine adenine dinucleotide (PyrAD⁺), N-methyl-nicotinamide adenine dinucleotide (N-Me-NAD⁺), acetylpyridine adenine dinucleotide (AcPyAD⁺), nicotinamide hypoxanthine dinucleotide (NHD⁺), and nicotinamide adenine dinucleotide phosphate (NADP⁺) are reported. In these analogs the ¹³C chemical shifts of the pyridinium moiety reflect the pK_a of the opposing purine base, while the ¹³C chemical shift dependence on pD for the pyridinium carbons of nicotinamide mononucleotide (NMN⁺) and adenosine monophosphate (AMP), 1,4-dihydronicotinamide adenine dinucleotide (NADH), 1,4-dihydronicotinamide adenine dinucleotide phosphate (NADPH), and nicotinic acid adenine dinucleotide (N(a)AD⁺) are not influenced by the adenine ring in the pD range tested. Through the use of ¹³C-labeled NAD⁺, the source of the pH dependence of the ¹³C chemical shifts was shown to be intramolecular in origin. However, serious doubt is cast on the utility of employing the pD dependence of chemical shift data to determine the nature of solution conformers or their relative populations.     

High-performance ion-exchange separation of oxidized and reduced nicotinamide adenine dinucleotides

High-performance anion-exchange chromatography of oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) on a Pharmacia Mono Q anion-exchange column is reported. Microgram quantities of all four nucleotides can be separated at pH 7.7 in approximately 20 min. For preparative purposes, greater than 7 mg of NADH can be purified in a single injection, and the peak fractions have an A260 of greater than 80 OD units with an A260/A340 ratio of 2.25.     

Determination of oxidized and reduced nicotinamide adenine dinucleotide in cell monolayers using a single extraction procedure and a spectrophotometric assay

While many investigations measuring oxidized nicotinamide adenine dinucleotide (NAD+) and reduced nicotinamide adenine dinucleotide (NADH) have been carried out on several mammalian tissues and blood cells, few reports have dealt with monolayers of cultured cells. Here we show a novel method to measure NAD+ and NADH in monolayers of a neuroblastoma cell line. The method was established by modifying a single extraction procedure originally developed for erythrocytes and an enzymatic cycling assay using a dye that absorbs in visible range. The following modifications were made. (i) Addition of 0.05% of a detergent, Triton X-100, to carbonate-bicarbonate extraction buffer enabled us to accurately measure cellular [NADH]/([NAD+]+[NADH]). (ii) Addition of N-ethyldibenzopyrazine ethyl sulfate salt (phenazine ethosulfate) immediately before the incubation suppressed the gradual decline of the sensitivity of the assay. The procedure presented here provides a simple and inexpensive measurement of NAD+ and NADH in cell monolayers.     

Oxidation of reduced nicotinamide adenine dinucleotide phosphate by isolated corn mitochondria

Isolated corn (Zea mays L.) mitochondria were found to oxidize reduced nicotinamide adenine dinucleotide phosphate in a KCl reaction medium. This oxidation was dependent on the presence of calcium or phosphate or both. Strontium and manganese substituted for calcium, but magnesium or barium did not. The oxidation of NADPH produced contraction of mitochondria swollen in KCl. Further evidence that the oxidation of NADPH was coupled was observed in respiratory control and adenosine diphosphate-oxygen ratios that were comparable to those reported for reduced nicotinamide adenine dinucleotide. The pathways of electron flow from NADH and NADPH were compared through the addition of electron transport inhibitors. The only difference between the two dinucleotides was that amytal was found to inhibit almost totally the state 3 oxidation of NADPH, but had little effect on the state 3 oxidation of NADH. The hypothetical pathways for electron flow from NADPH are discussed, as are the possible sites of calcium and phosphate stimulation.     

The levels of soluble nucleotides in wheat aleurone tissue

The content of soluble nucleotides in aleurone layers isolated from mature wheat (Triticum aestivum var. Olympic) grain was investigated. The most abundant nucleotides were adenosine triphosphate, uridine triphosphate, and uridine diphosphoglucose. Smaller amounts of guanosine triphosphate, cytidine triphosphate, adenosine diphosphate, and nicotinamide adenine dinucleotide were also identified. The levels of some of these nucleotides were increased after incubation of the tissue under certain conditions.     

A Metaphosphate-dependent Nicotinamide Adenine Dinucleotide Kinase from Brevibacterium ammoniagenes

The enzyme utilizing metaphosphate for nicotinamide adenine dinucleotide phosphorylation was purified 500-fold from B. ammoniagenes and its properties were studied. The isolated enzyme appeared homogeneous on disc gel electrophoresis; its molecular weight was determined to be 9.0 × 10⁴ by gel filtration. This enzyme specifically phosphorylated nicotinamide adenine dinucleotide at the optimum pH at 6.0. Of phosphoryl donors tested, metaphosphate was most effective for the reaction, and adenosine-5′-triphosphate was less effective. The activity was inhibited by adenosine-5′-monophosphate, adenosine-5′-diphosphate or reduced pyridine nucleotides. The enzyme did not exhibit catalytic activity in the absence of a divalent cation. We concluded that the enzyme phosphorylating nicotinamide adenine dinucleotide in the presence of metaphosphate is distinct from adenosine-5′-triphosphate-dependent nicotinamide adenine dinucleotide kinase, and tentatively designated it metaphosphate-dependent nicotinamide adenine dinucleotide kinase.     

Purification and Characterization of the Two 6-Phosphogluconate Dehydrogenase Species from Pseudomonas multivorans

The two species of 6-phosphogluconate dehydrogenase (EC 1.1.1.43) from Pseudomonas multivorans were resolved from extracts of gluconate-grown bacteria and purified to homogeneity. Each enzyme comprised between 0.1 and 0.2% of the total cellular protein. Separation of the two enzymes, one which is specific for nicotinamide adenine dinucleotide phosphate and the other which is active with nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate was facilitated by the marked difference in their respective isoelectric points, which were at pH 5.0 and 6.9. Comparison of the subunit compositions of the two enzymes indicated that they do not share common peptide chains. The enzyme active with nicotinamide adenine dinucleotide was composed of two subunits of about 40,000 molecular weight, and the nicotinamide adenine dinucleotide phosphate-specific enzyme was composed of two subunits of about 60,000 molecular weight. Immunological studies indicated that the two enzymes do not share common antigenic determinants. Reduced nicotinamide adenine dinucleotide phosphate strongly inhibited the 6-phosphogluconate dehydrogenase active with nicotinamide adenine dinucleotide by decreasing its affinity for 6-phosphogluconate. Guanosine-5'-triphosphate had a similar influence on the nicotinamide adenine dinucleotide phosphate-specific 6-phosphogluconate dehydrogenase. These results in conjunction with other data indicating that reduced nicotinamide adenine dinucleotide phosphate stimulates the conversion of 6-phosphogluconate to pyruvate by crude bacterial extracts suggest that in P. multivorans, the relative distribution of 6-phosphogluconate into the pentose phosphate and Entner-Doudoroff pathways might be determined by the intracellular concentrations of reduced nicotinamide adenine dinucleotide phosphate and purine nucleotides.     

A new ternary complex between horse liver alcohol dehydrogenase, NAD+, and acetone

Acetone was found to form a dead-end ternary complex with horse liver alcohol dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD⁺) when the reactants were incubated for a long time at relatively high concentrations. The complex formation was demonstrated by measuring the increase in absorbance at 320 nm, the quenching of protein fluorescence, and the loss of enzyme activity. Since acetone is a substrate of liver alcohol dehydrogenase, and the presence of acetaldehyde or pyrazole prevents acetone from forming the dead-end complex with liver alcohol dehydrogenase and NAD⁺, the acetone molecule in the complex may be bound to the substrate binding site of liver alcohol dehydrogenase. The dissociation of the complex was demonstrated by prolonged dialysis or by addition of reduced nicotinamide adenine dinucleotide (NADH) and iso-butyramide. A modified nicotinamide adenine dinucleotide was obtained as a main product from the dead-end complex after dissociation of the complex or denaturation of the apoenzyme. The modified nicotinamide adenine dinucleotide was found to exhibit an absorption spectrum similar to that of NADH; however, it was not oxidizable by liver alcohol dehydrogenase in the presence of acetaldehyde and exhibited no fluorescence.     

The Role of Phosphate Anions in Autoinduced and Photoinduced Oxidation of NADH

Biophysics - The effects of mono- and disubstituted phosphates on the photolysis reaction of reduced nicotinamide adenine dinucleotide (NADH) in a frozen (77 K) aqueous solution were studied by...     

Glutamate dehydrogenase from Mycoplasma laidlawii

Mycoplasma laidlawii possesses a single glutamate dehydrogenase (GDH) with dual coenzyme specificity [specificity for nicotinamide adenine dinucleotide (H) and nicotinamide adenine dinucleotide phosphate (H)]. A purification procedure is reported which results in an enzyme preparation with a specific activity of 79.5 units/mg and which displays only one significant protein band after gel electrophoresis. This one band was determined, by activity staining, to have all of the GDH nucleotide specificities. The molecular weight of the enzyme is 250,000 +/- 10%, and it has a subunit size of about 48,000. The enzyme exhibits measurable activity with aspartate and pyruvate but is inactive with eight other possible substrates. Purine nucleotides do not affect the activity. The K(m) for reduced nicotinamide adenine dinucleotide was 1.8 x 10(-4)m. The optimal substrate concentrations and pH optimum for each of the respective GDH activities are also reported.     

Regulation of Autotrophic and Heterotrophic Carbon Dioxide Fixation in Hydrogenomonas facilis

After growth on various carbon sources, sonic extracts of Hydrogenomonas facilis contained ribulosediphosphate (RuDP) carboxylase and phosphoribulokinase (Ru5-P kinase). After very short sonic treatment, a reductive adenosine triphosphate (ATP)-dependent incorporation of (14)CO(2) was also detectable. Reduced nicotinamide adenine dinucleotide (NADH(2)) served as reductant 30-fold more effectively than reduced nicotinamide adenine dinucleotide phosphate (NADPH(2)). Adenosine 5'-phosphate (AMP) and adenosine 5'-pyrophosphate (ADP) inhibited Ru5-P kinase and NADH(2)-, ATP-dependent CO(2) fixation. The levels and duration of CO(2) fixation suggested that it is a cyclic process. The requirement of reduced pyridine nucleotide and ATP and the sensitivity of fixation to AMP and ADP support the conjecture that it occurs via the Calvin cycle. After thorough study of variables affecting catalysis, specific activities (millimicromoles of substrate disappearing per milligram of protein) at 30 C were determined for RuDP carboxylase (C), Ru5-P kinase (K) and ATP-, NADH(2)- dependent CO(2) fixation (CO(2) F) after growth autotrophically on fructose, glucose, ribose, glutamate, lactate, succinate, and acetate. Values for these growth modes were, respectively-for C: 67.3, 51.1, 51.4, 24.6, 2.05, 10.2, 2.25, 1.4; for K: 24.7, 24.0, 23.2, 14.2, 12.8, 12.9, 13.4, 2.8; and for CO(2) F: 4.54, 4.83, 3.10, 2.87, 0.85, 1.51, 0.24, 0.41. The qualitative parallel between values for RuDP carboxylase and CO(2) fixation suggests that one major control point in fixation is the step catalyzed by RuDP carboxylase.     

Dynamics of energy charge and adenine nucleotides during uncoupling of catabolism and anabolism in Penicillium ochrochloron

Filamentous fungi are able to spill energy when exposed to energy excess by uncoupling catabolism from anabolism, e.g. via overflow metabolism. In current study we tested the hypothesis that overflow metabolism is regulated via the energetic status of the hyphae (i.e. energy charge, ATP concentration). This hypothesis was studied in Penicillium ochrochloron during the steady state of glucose- or ammonium-limited chemostat cultures as well as during three transient states ((i) glucose pulse to a glucose-limited chemostat, (ii) shift from glucose-limited to ammonium-limited conditions in a chemostat, and (iii) ammonium exhaustion in batch culture). Organic acids were excreted under all conditions, even during exponential growth in batch culture as well as under glucose-limited conditions in a chemostat. Partial uncoupling of catabolism and anabolism via overflow metabolism was thus constitutively present. Under all tested conditions, overflow metabolism was independent of the energy charge or the ATP concentration of the hyphae. There was a reciprocal correlation between glucose uptake rate and intracellular adenine nucleotide content. During all transients states a rapid decrease in energy charge and the concentrations of nucleotides was observed shortly after a change in glycolytic flux (“ATP paradoxon”). A possible connection between the change in adenine nucleotide concentrations and the purine salvage pathway is discussed.