Intracellular accumulation of AMP as a cause for the decline in rate of ethanol production by Saccharomyces cerevisiae during batch fermentation

A general hypothesis is presented for the decline in the rate of ethanol production (per unit of cell protein) during batch fermentation. Inhibition of ethanol production is proposed to result from the intracellular accumulation of AMP during the transition from growth to the stationary phase. AMP acts as a competitive inhibitor of hexokinase with respect to ATP. When assayed in vitro in the presence of ATP and AMP concentrations equivalent to those within cells at different stages of fermentation, hexokinase activity declined in parallel with the in vivo decline in the rate of ethanol production. The coupling of glycolytic flux and fermentation to cell growth via degradation products of RNA may be of evolutionary advantage for Saccharomyces cerevisiae. Such a coupling would reduce the exposure of nongrowing cells to potentially harmful concentrations of waste products from metabolism and would conserve nutrients for future growth under more favorable conditions.     

Intracellular accumulation of AMP as a cause for the decline in rate of ethanol production by Saccharomyces cerevisiae during batch fermentation.

A general hypothesis is presented for the decline in the rate of ethanol production (per unit of cell protein) during batch fermentation. Inhibition of ethanol production is proposed to result from the intracellular accumulation of AMP during the transition from growth to the stationary phase. AMP acts as a competitive inhibitor of hexokinase with respect to ATP. When assayed in vitro in the presence of ATP and AMP concentrations equivalent to those within cells at different stages of fermentation, hexokinase activity declined in parallel with the in vivo decline in the rate of ethanol production. The coupling of glycolytic flux and fermentation to cell growth via degradation products of RNA may be of evolutionary advantage for Saccharomyces cerevisiae. Such a coupling would reduce the exposure of nongrowing cells to potentially harmful concentrations of waste products from metabolism and would conserve nutrients for future growth under more favorable conditions.     

Further studies on the properties of the rabbit reticulocyte adenosine 3',5'-cyclic monophosphate-dependent protein kinase I

The properties of cyclic AMP-dependent protein kinase I isolated from rabbit reticulocytes were further investigated. The enzyme catalyzes the phosphorylation of histone in the presence of ATP and Mg²⁺ and this reaction is stimulated by cyclic AMP. The pH optimum of the reaction was between 8.5 and 9.0, when assayed in the presence of cyclic AMP. No distinct pH optimum was observed in the absence of the cyclic nucleotide. The K_m values for ATP appeared to be very similar whether it was determined in the presence (K_m = 1.7 × 10⁻⁴m) or absence (K_m = 2.5 × 10⁻⁴m) of cyclic AMP. The rate of heat inactivation of the catalytic activity and the cyclic AMP binding activity of kinase I were found to be dependent on the presence of Mg²⁺, ATP, and/or cyclic AMP. In the presence of cyclic AMP, the rate of inactivation of the catalytic activity of kinase I at 53 ° was accelerated. On the other hand, the cyclic AMP binding activity appeared to be protected from heat inactivation by the cyclic nucleotide. When both ATP and Mg²⁺ were present in the heating mixture, no loss of catalytic and binding activities of kinase I were observed even up to 8 min of heating at 53 °. The cyclic AMP binding activity of kinase I was almost completely inhibited by mercuric acetate at a concentration of 1 mm, while the loss in catalytic activity was only 50%. These results substantiate our previous observation that kinase I contains two nonidentical subunits, a catalytic subunit and a cyclic AMP binding subunit.     

Oscillatory starch degradation and fermentation in the green alga Chlamydomonas reinhardii

After transition to anaerobiosis an oscillatory fermentation of plastidic starch is found in the green alga Chlamydomonas reinhardii. Oscillations are investigated by analysis of fluctuations in starch, fermentation products and metabolites. Period length, amplitudes and phase relationships are determined by a biometrical processing of the biochemical signals, which allows statistical treatment even under limitation of data points. The mean period length is 59 min and oscillations are detectable within 3 h after the onset of anaerobiosis. Metabolized starch carbon is found by 90% in the accumulating fermentation products as formate, acetate and ethanol. From the intermediates only pyruvate increases during fermentative oscillations. A phase shift of 180° between ATP and both, AMP and ADP, indicates activity of adenylate kinase and values for energy charge fluctuates between 0.70 and 0.89. Phase relations of glucose-6-P to both AMP and pyruvate are 190° and 56°, respectively, whereas that of pyruvate to ATP is 134°. Glucose-6-P concentration does not follow the rate of starch degradation. Phase relations of fermentation products to pyruvate indicates enzyme activities of formate pathway. High period length as observed during algal fermentation is thought to be generated by a complex rate control in a different compartmentalized glycolytic pathway.     

The HPr(Ser) Kinase of Streptococcus salivarius: Purification, Properties, and Cloning of the hprK Gene

In gram-positive bacteria, HPr, a protein of the phosphoenolpyruvate:sugar phosphotransferase system, is phosphorylated on a serine residue at position 46 by an ATP-dependent protein kinase. The HPr(Ser) kinase of Streptococcus salivarius ATCC 25975 was purified, and the encoding gene (hprK) was cloned by using a nucleotide probe designed from the N-terminal amino acid sequence. The predicted amino acid sequence of the S. salivarius enzyme showed 45% identity with the Bacillus subtilis enzyme, the conserved residues being located mainly in the C-terminal half of the protein. The predicted hprK gene product has a molecular mass of 34,440 Da and a pI of 5.6. These values agree well with those found experimentally by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, molecular sieve chromatography in the presence of guanidine hydrochloride, and chromatofocusing using the purified protein. The native protein migrates on a Superdex 200 HR column as a 330,000-Da protein, suggesting that the HPr(Ser) kinase is a decamer. The enzyme requires Mg²⁺ for activity and functions optimally at pH 7.5. Unlike the enzyme from other gram-positive bacteria, the HPr(Ser) kinase from S. salivarius is not stimulated by FDP or other glycolytic intermediates. The enzyme is inhibited by inorganic phosphate, and its K_ms for HPr and ATP are 31 μM and 1 mM, respectively.     

Ethanol production during batch fermentation with Saccharomyces cerevisiae: changes in glycolytic enzymes and internal pH

During batch fermentation, the rate of ethanol production per milligram of cell protein is maximal for a brief period early in this process and declines progressively as ethanol accumulates in the surrounding broth. Our studies demonstrate that the removal of this accumulated ethanol does not immediately restore fermentative activity, and they provide evidence that the decline in metabolic rate is due to physiological changes (including possible ethanol damage) rather than to the presence of ethanol. Several potential causes for the decline in fermentative activity have been investigated. Viability remained at or above 90%, internal pH remained near neutrality, and the specific activities of the glycolytic and alcohologenic enzymes (measured in vitro) remained high throughout batch fermentation. None of these factors appears to be causally related to the fall in fermentative activity during batch fermentation.     

Ethanol production during batch fermentation with Saccharomyces cerevisiae: changes in glycolytic enzymes and internal pH.

During batch fermentation, the rate of ethanol production per milligram of cell protein is maximal for a brief period early in this process and declines progressively as ethanol accumulates in the surrounding broth. Our studies demonstrate that the removal of this accumulated ethanol does not immediately restore fermentative activity, and they provide evidence that the decline in metabolic rate is due to physiological changes (including possible ethanol damage) rather than to the presence of ethanol. Several potential causes for the decline in fermentative activity have been investigated. Viability remained at or above 90%, internal pH remained near neutrality, and the specific activities of the glycolytic and alcohologenic enzymes (measured in vitro) remained high throughout batch fermentation. None of these factors appears to be causally related to the fall in fermentative activity during batch fermentation.     

Glycolytic Flux Is Adjusted to Nitrogenase Activity in Nodules of Detopped and Argon-Treated Alfalfa Plants

To investigate the short-term (30–240 min) interactions among nitrogenase activity, NH₄⁺ assimilation, and plant glycolysis, we measured the concentrations of selected C and N metabolites in alfalfa (Medicago sativa L.) root nodules after detopping and during continuous exposure of the nodulated roots to Ar:O₂ (80:20, v/v). Both treatments caused an increase in the ratios of glucose-6-phosphate to fructose-1,6-bisphosphate, fructose-6-phosphate to fructose-1,6-bisphosphate, phosphoenolpyruvate (PEP) to pyruvate, and PEP to malate. This suggested that glycolytic flux was inhibited at the steps catalyzed by phosphofructokinase, pyruvate kinase, and PEP carboxylase. In the Ar:O₂-treated plants the apparent inhibition of glycolytic flux was reversible, whereas in the detopped plants it was not. In both groups of plants the apparent inhibition of glycolytic flux was delayed relative to the decline in nitrogenase activity. The decline in nitrogenase activity was followed by a dramatic increase in the nodular glutamate to glutamine ratio. In the detopped plants this was coincident with the apparent inhibition of glycolytic flux, whereas in the Ar:O₂-treated plants it preceded the apparent inhibition of glycolytic flux. We propose that the increase in the nodular glutamate to glutamine ratio, which occurs as a result of the decline in nitrogenase activity, may act as a signal to decrease plant glycolytic flux in legume root nodules.     

Ethanol Production by Thermophilic Bacteria: Relationship Between Fermentation Product Yields of and Catabolic Enzyme Activities in Clostridium thermocellum and Thermoanaerobium brockii

Significant quantitative differences in end-product yields by two strains of Clostridium thermocellum and one strain of Thermoanaerobium brockii were observed during cellobiose fermentation. Most notably, the ethanol/H₂ and lactate/acetate ratios were drastically higher for T. brockii as compared with C. thermocellum strains LQRI and AS39. Exogenous H₂ addition (0.4 to 1.0 atm) during culture growth increased the ethanol/acetate ratio of both T. brockii and AS39 but had no effect on LQRI. All strains had an operative Embden-Meyerhof glycolytic pathway and displayed catabolic activities of fructose-1,6-diphosphate–activated lactate dehydrogenase, coenzyme A acetylating pyruvate and acetaldehyde dehydrogenase, hydrogenase, ethanol dehydrogenase, and acetate kinase. Enzyme kinetic properties (apparent K_m, V_max, and Q₁₀ values) and the specificity of electron donors/acceptors for different oxidoreductases involved in pyruvate conversion to fermentation products were compared in the three strains. Both species contained ferredoxin-linked pyruvate dehydrogenase and pyridine nucleotide oxidoreductases. Ferredoxin-nicotinamide adenine dinucleotide (NAD) reductase activity was significantly higher in T. brockii than in AS39 and was not detectable in LQRI. H₂ production and hydrogenase activity were inversely related to ferredoxin-NAD reductase activity in the three strains. Ferredoxin-NAD phosphate reductase activity was present in cell extracts of both species. Alcohol dehydrogenase activity in C. thermocellum was NAD dependent, unidirectional, and inhibited by low concentrations of NAD and ethanol. Ethanol dehydrogenase activity of T. brockii was both NAD and NADP linked, reversible, and not inhibited by low levels of reaction products. The high lactate yield of T. brockii correlated with increased fructose-1,6-diphosphate. The relation of catabolic enzyme activity and quantitative differences in intracellular electron flow and fermentation product yields of these thermophilic bacteria is discussed.     

Adenine nucleotide levels in Rhodospirillum rubrum during switch-off of whole-cell nitrogenase activity.

Adenine nucleotide pools were measured in Rhodospirillum rubrum cultures that contained nitrogenase. The average energy charge [([ATP] + 1/2[ADP])/([ATP] + [ADP] + [AMP])] was found to be 0.66 and 0.62 in glutamate-grown and N-limited cultures respectively. Treatment of glutamate-grown cells with darkness, ammonia, glutamine, carbonyl cyanide m-chlorophenylhydrazone, or phenazine methosulphate resulted in perturbations in the adenine nucleotide pools, and led to loss of whole-cell nitrogenase activity and modification in vivo of the Fe protein. Treatment of N-limited cells resulted in similar changes in adenine nucleotide pools but not enzyme modification. No correlations were found between changes in adenine nucleotide pools or ratios of these pools and switch-off of nitrogenase activity by Fe protein modification in vivo. Phenazine methosulphate inhibited whole-cell activity at low concentrations. The effect on nitrogenase activity was apparently independent of Fe protein modification.     

Effects of Salinity, Diurnal Cycle and Age on Nucleotide Pools of Bean Leaves

In the present study, we investigated the relationship betweensalt stress and nucleotide levels in the shoot of Phaseolusvulgaris L. cv. Stringless Green Pod to determine if reducedgrowth was correlated with reduced nucleotide levels. Overallfresh weight of 25-d-old plants after having been on full salttreatment for 7 d was 33% lower compared with untreated plants.Shoot fresh weight decreased by 40% compared with 22% for theroots thus increasing the root to shoot ratio from 0·7to 0·9. We examined young and juvenile leaves as wellas mature leaves in order to compare growing tissue to fullyenlarged tissue. To ascertain whether the effects of salt stresson nucleotide pools were more severe during the day than atnight, we studied the combined effects of diurnal cycle andsalt stress on these nucleotide pools. Salt treatment selectivelyaffected certain nucleotide pools with the adenine nucleotides(AdN) being the most affected. We found large diurnal fluctuationsof AdN pools in all leaves. During the day, AMP and ADP increasedwhile ATP decreased. The sum, ATP + ADP, tended to remain constantand in mature leaves total AdN increased with AMP, an indicationof net synthesis. At night, ATP increased in all leaves. However,salt stress prevented this night-time increase in mature leaveswhile enhancing it in juvenile and young leaves. In the daytime,salt stress caused a nearly 2-fold increase in AMP of youngleaves and a large increase in the adenylate kinase mass actionratio (K). At night, the excess AMP disappeared with no changein total AdN. It is clear from these results that salt stressdid not reduce shoot growth by depleting ATP in growing leaves.It did, however, reduce the ATP level of mature leaves and perhapstheir ability to supply essential metabolites for growing regions. Key words: Phaseolus, nucleotides, salt stress, salinity, growth     

Metabolic and physiological studies of Corynebacterium glutamicum mutants

The physiology and central carbon metabolism of Corynebacterium glutamicum was investigated through the study of specific disruption mutants. Mutants deficient in phosphoenolpyruvate carboxylase (PPC) and/or pyruvate kinase (PK) activity were constructed by disrupting the corresponding gene(s) via transconjugation. Standard batch fermentations were carried out with these mutants and results were evaluated in the context of intracellular flux analysis. The following were determined. (a) There is a significant reduction in the glycolytic pathway flux in the pyruvate kinase deficient mutants during growth on glucose, also evidenced by secretion of dihydroxyacetone and glyceraldehyde. The resulting metabolic overflow is accommodated by the pentose phosphate pathway (PPP) acting as mechanism for dissimilating, in the form of CO(2), large amounts of accumulated intermediates. (b) The high activity through the PPP causes an overproduction of reducing power in the form of NADPH. The overproduction of biosynthetic reducing power, as well as the shortage of NADPH produced via the tricarboxylic acid cycle (as evidenced by a reduced citrate synthase flux), are compensated by an increased activity of the transhydrogenase (THD) enzyme catalyzing the reaction NADPH + NAD(+)<-->NADP(+) + NADH. The presence of active THD was also confirmed directly by enzymatic assays. (c) Specific glucose uptake rates declined during the course of fermentation and this decline was more pronounced in the case of a double mutant strain deficient in both PPC and PK. Specific ATP consumption rates similarly declined during the course of the batch. However, they were approximately the same for all strains, indicating that energetic requirements for biosynthesis and maintenance are independent of the specific genetic background of a strain. The above results underline the importance of intracellular flux analysis, not only for producing a static set of intracellular flux estimates, but also for uncovering changes occurring in the course of a batch fermentation or as result of specific genetic modifications. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:864-879, 1997.     

Glycosis in the eggs of the echiuroid, Urechis unicinctus and the oyster, Crassostrea gigas. Rate-limiting steps and activation at fertilization.

The content of glycolytic intermediates and of adenine nucleotides was measured in eggs of the echiuroid, Urechis unicinctus and the oyster, Crassostrea gigas, before and after fertilization. On the whole, the profile of the change in each glycolytic intermediate in Urechis eggs upon fertilization was found to be essentially similar to that in oyster eggs. Calculation of the mass action ratio for each glycolytic step from the amounts of glycolytic intermediates determined suggests that there are at least three limiting enzymes in the glycolysis system in unfertilized and fertilized eggs of each species examined. Phosphorylase (EC 2.4.1.1), phosphofructokinase (EC 2.7.1.11), and pyruvate kinase (EC 2.7.1.40) may be rate-limiting enzymes for the glycolysis system in Urechis eggs as well as in oyster eggs. These enzymes are thought to be activated upon fertilization, though even the reactions of the enzymes in fertilized eggs do not reach a state of equilibrium. In eggs of Urechis and oyster, phosphorylase is the first enzyme to be activated following fertilization. In Urechis eggs, pyruvate kinase is activated after the instant increase in the phosphorylase activity upon fertilization, followed by phosphofructokinase activation. In oyster eggs, however, pyruvate kinase and phosphofructokinase seem to be stimulated simultaneously, subsequent to phosphorylase activation upon fertilization. The mechanism controlling phosphorylase and pyruvate kinase activity is unknown, but the phosphofructokinase activity in both species may be regulated by the intracellular concentration of adenine nucleotides, since the enzyme activity is enhanced along with a decline in the phosphate potential in the eggs of both Urechis and of oyster.     

Pyruvate Kinase of Streptococcus lactis

The kinetic properties of pyruvate kinase (ATP:pyruvate-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis have been investigated. Positive homotropic kinetics were observed with phosphoenolpyruvate and adenosine 5′-diphosphate, resulting in a sigmoid relationship between reaction velocity and substrate concentrations. This relationship was abolished with an excess of the heterotropic effector fructose-1,6-diphosphate, giving a typical Michaelis-Menten relationship. Increasing the concentration of fructose-1,6-diphosphate increased the apparent V_max values and decreased the K_m values for both substrates. Catalysis by pyruvate kinase proceeded optimally at pH 6.9 to 7.5 and was markedly inhibited by inorganic phosphate and sulfate ions. Under certain conditions adenosine 5′-triphosphate also caused inhibition. The K_m values for phosphoenolpyruvate and adenosine 5′-diphosphate in the presence of 2 mM fructose-1,6-diphosphate were 0.17 mM and 1 mM, respectively. The concentration of fructose-1,6-diphosphate giving one-half maximal velocity with 2 mM phosphoenolpyruvate and 5 mM adenosine 5′-diphosphate was 0.07 mM. The intracellular concentrations of these metabolites (0.8 mM phosphoenolpyruvate, 2.4 mM adenosine 5′-diphosphate, and 18 mM fructose-1,6-diphosphate) suggest that the pyruvate kinase in S. lactis approaches maximal activity in exponentially growing cells. The role of pyruvate kinase in the regulation of the glycolytic pathway in lactic streptococci is discussed.     

Transient changes in cyclic AMP and in the enzymic activity of protein kinase and phosphorylase during the cardiac cycle in the canine myocardium and the effect of propranolol

Summary Oscillation of cyclic AMP and in the activity ratio of cyclic AMP-dependent protein kinase and of glycogen phosphorylase with the cardiac cycle were demonstrated in the canine heart in situ. For tissue sampling an ECG (R-wave)-triggered, automatically working push-freeze-drill apparatus was developed which allows intraventricular cryobiopsies from the left ventricular muscle of anaesthetized open-chest dogs. The nucleotide cyclic AMP oscillated with the cardiac cycle during normal working condition, the higher cyclic AMP level occuring during systole. Cyclic GMP was assayed to be without oscillatory changes during the contraction-relaxation cycle. The rise in the activity ratio of protein kinase was found to coincide with the maximum in the level of cyclic AMP. Propranolol pretreatment prevents the transient in the level of the nucleotide as well as in the activity ratio of the kinase indicating i) a causal relationship between these changes and ii) a neurohumoral, beat-to-beat regulation by catecholamines released from the sympathetic nerve endings within the heart. Contrary the activity ratio of phosphorylase retains its transient changes during the cardiac cycle in the presence of propranolol, indicating a Ca-mediated activation of phosphorlase kinase during the contraction process.     

Alterations of the glycolytic pathway and adenine nucleotide state in livers of clofibrate treated rats.

The influence of clofibrate on the glycolytic pathway in liver was studied. The changes in the activity of glucokinase and hexokinase were not significant. A reduction of phosphofructokinase (p less than 0.05) and pyruvate kinase activity was found (p less than 0.0005) during clofibrate feeding. An in vitro inhibition of these enzymes could not be demonstrated by clofibrate up to a concentration of 2.5 mM. Crossover plots of glycolytic intermediates indicate that the reduced pyruvate kinase activity may influence the glycolytic pathway in vivo. Clofibrate feeding induces a lower ATP:ADP ratio, a lower adenylate energy charge and elevates AMP levels in rat liver. This may possibly stimulate the hepatic glycogenolysis and the glucose utilisation by this organ.     

Differential Effects of Two Protein Kinase C Inhibitors, Calphostin C and Gö6976, on Pineal Cyclic Nucleotide Accumulation

Abstract: In rat pinealocytes, protein kinase C (PKC) is involved in the α₁-adrenergic-mediated potentiation of β-adrenergic-stimulated cyclic nucleotide responses; however, the specific PKC isozyme(s) involved in the potentiation mechanism remain unknown. In the present study, we compared the effects of two PKC inhibitors, calphostin C, a specific inhibitor of PKC, and Gö6976, a selective inhibitor of PKCα and PKCβ₁, on the adrenergic-stimulated cyclic nucleotide accumulation in rat pinealocytes. Surprisingly, Gö6976 was found to have an enhancing effect on basal cyclic GMP and isoproterenol-stimulated cyclic AMP and cyclic GMP accumulation, an effect not shared by calphostin C. Gö6976 also increased the norepinephrine- and ionomycin-induced potentiation of isoproterenol-stimulated cyclic AMP and cyclic GMP accumulation, whereas the effect of calphostin C was inhibitory. The enhancing effect of Gö6976 was abolished in the presence of isobutylmethylxanthine or zaprinast, but not rolipram, suggesting that this effect of Gö6976 may be mediated through type V or the retinal type of phosphodiesterase. Based on these observations, we propose that some of the PKC isozyme(s) inhibited by calphostin C are involved in the potentiation of β-adrenergic-stimulated cyclic nucleotide responses and that they act by enhancing synthesis. However, PKC isozymes inhibited by Gö6976 appear to be basally active and tonically inhibit cyclic nucleotide accumulation through their stimulatory action on phosphodiesterase.     

Potentiation of Agonist-Stimulated Cyclic AMP Accumulation by Tyrosine Kinase Inhibitors in Rat Pinealocytes

Abstract: To study cross-talk mechanisms in rat pinealocytes, the role of tyrosine kinase or kinases in the regulation of adrenergic-stimulated cyclic AMP production was investigated. Both norepinephrine- and isoproterenol-stimulated cyclic AMP accumulation were increased by two distinct tyrosine kinase inhibitors, genistein or erbstatin, in a concentration-dependent manner. A similar increase was observed with two other inhibitors, tyrphostin B44 and herbimycin. In contrast, daidzein, an inactive analogue of genistein, was ineffective; whereas vanadate, a phosphotyrosine phosphatase inhibitor, reduced the adrenergic-stimulated cyclic AMP accumulation. The tyrosine kinase inhibitors were effective in potentiating the cholera toxin-or forskolin-stimulated cyclic AMP accumulation, indicating that their sites of action are at the postreceptor level. Neither an activator nor inhibitors of protein kinase C influenced the potentiation of the cyclic AMP responses by genistein, suggesting that the potentiation effect by tyrosine kinase inhibitors does not involve the phospholipase C/protein kinase C pathway. However, when the phosphodiesterase was inhibited by isobutylmethylxanthine, genistein failed to potentiate and vanadate did not inhibit the adrenergic-stimulated cyclic AMP accumulation, indicating that the phosphodiesterase is a probable site of action for these inhibitors. These results suggest that cyclic AMP metabolism in the pinealocytes is tonically inhibited by tyrosine kinase acting on the cyclic AMP phosphodiesterase.     

Protein Kinase A Regulates Growth, Sporulation, and Pigment Formation in Aspergillus fumigatus

Aspergillus fumigatus is an opportunistic human pathogenic fungus causing severe infections in immunocompromised patients. Cyclic AMP (cAMP) signal transduction plays an important role in virulence. A central component of this signaling cascade is protein kinase A (PKA), which regulates cellular processes by phosphorylation of specific target proteins. Here we describe the generation and analysis of A. fumigatus mutants expressing the gene encoding the catalytic subunit of PKA, pkaC1, under control of an inducible promoter. Strains overexpressing pkaC1 showed high PKA activity, reduced growth, sporulation deficiency, and formation of a dark pigment in the mycelium. These data indicate that cAMP-PKA signaling is involved in the regulation of important processes, such as growth, asexual reproduction, and biosynthesis of secondary metabolites. Furthermore, elevated PKA activity led to increased expression of the pksP gene. The polyketide synthase PksP is an essential enzyme for production of dihydroxynaphthalene-melanin in A. fumigatus and contributes to virulence. Our results suggest that increased pksP expression is responsible for pigment formation in the mycelium. Comparative proteome analysis of the pkaC1-overexpressing strain and the wild-type strain led to the identification of proteins regulated by the cAMP-PKA signal transduction pathway. We showed that elevated PKA activity resulted in activation of stress-associated proteins and of enzymes involved in protein biosynthesis and glucose catabolism. In contrast, proteins which were involved in nucleotide and amino acid biosynthesis were downregulated, as were enzymes involved in catabolism of carbon sources other than glucose.     

Purine uptake and release in rat C6 glioma cells: nucleoside transport and purine metabolism under ATP-depleting conditions

Adenosine, through activation of membrane-bound receptors, has been reported to have neuroprotective properties during strokes or seizures. The role of astrocytes in regulating brain interstitial adenosine levels has not been clearly defined. We have determined the nucleoside transporters present in rat C6 glioma cells. RT-PCR analysis, (3)H-nucleoside uptake experiments, and [(3)H]nitrobenzylthioinosine ([(3)H]NBMPR) binding assays indicated that the primary functional nucleoside transporter in C6 cells was rENT2, an equilibrative nucleoside transporter (ENT) that is relatively insensitive to inhibition by NBMPR. [(3)H]Formycin B, a poorly metabolized nucleoside analogue, was used to investigate nucleoside release processes, and rENT2 transporters mediated [(3)H]formycin B release from these cells. Adenosine release was investigated by first loading cells with [(3)H]adenine to label adenine nucleotide pools. Tritium release was initiated by inhibiting glycolytic and oxidative ATP generation and thus depleting ATP levels. Our results indicate that during ATP-depleting conditions, AMP catabolism progressed via the reactions AMP --> IMP --> inosine --> hypoxanthine, which accounted for >90% of the evoked tritium release. It was surprising that adenosine was not released during ATP-depleting conditions unless AMP deaminase and adenosine deaminase were inhibited. Inosine release was enhanced by inhibition of purine nucleoside phosphorylase; ENT2 transporters mediated the release of adenosine or inosine. However, inhibition of AMP deaminase/adenosine deaminase or purine nucleoside phosphorylase during ATP depletion produced release of adenosine or inosine, respectively, via the rENT2 transporter. This indicates that C6 glioma cells possess primarily rENT2 nucleoside transporters that function in adenosine uptake but that intracellular metabolism prevents the release of adenosine from these cells even during ATP-depleting conditions.