Phosphodiesterase activity and cyclic AMP content during early germination ofMucor rouxii spores

Incubation ofMucor rouxii spores in complex medium under aerobic conditions resulted in a very rapid transient increase in the level of soluble, low-K_m cAMP phosphodiesterase. Maximum activity was reached after 2–4 min. Simultaneously, the cAMP content increased quickly, reproducing the profile exhibited by phosphodiesterase. Activation of the enzymein vitro by a cAMP-dependent phosphorylation process showed that its stimulation was minimal at those times when its activity was highest. The correlation between cellular cAMP content, phosphodiesterase activity, and sensitivity of the enzyme to activation by phosphorylation suggests that thein vivo regulation of phosphodiesterase activity by cAMP-dependent phosphorylation may be the mechanism for shutoff of the cAMP signal elicited by glucose.     

Genetic and biochemical evidence that trehalase is a substrate of cAMP-dependent protein kinase in yeast

In Saccharomyces cerevisiae, trehalase activity in crude extracts obtained from wild type cells was activated about 3-fold by preincubation with cAMP and ATP. The inactive trehalase fractionated by DEAE-Sephacel chromatography was activated by the addition of the cAMP-dependent protein kinase fraction from wild type cells in the presence of cAMP and ATP. Using the crude extract obtained from bcy1 mutant cells which were deficient in the regulatory subunit of cAMP-dependent protein kinase, the stimulation of trehalase activity was observed in the absence of cAMP. The cAMP-dependent protein kinase of CYR3 mutant cells which had a high Ka value for cAMP in the phosphorylation reaction required a high cAMP concentration for activation of trehalase. Increased activation of partially purified inactive trehalase (Mr = 320,000) was observed to correlate with increased phosphorylation of a protein (Mr = 80,000) identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The assay results using various mutants altered in cAMP metabolism indicated that the activation and phosphorylation of inactive trehalase fractions depended on the cAMP concentration accumulated in mutant cells. Inactivation and dephosphorylation of active trehalase fractions were observed by treatment with alkaline phosphatase or crude cell extracts. The results indicated that the conversion of inactive form of trehalase to the active form is regulated by cAMP through cAMP-dependent protein kinase.     

Effect of growth conditions on catabolite repression and cyclic AMP synthesis in Escherichia coli 3000A1

Catabolite repression of beta-galactosidase synthesis in E. coli 3000A1 (adenine-) was studied under a variety of growth conditions. The differential rate of induced beta-galactosidase synthesis was maximal at the growth rate of 0.75 division per h, irrespective of whether growth conditions were aerobic or anaerobic. The addition of cyclic AMP (cAMP) to the medium partly restored the repressed synthesis of beta-galactosidase under some growth conditions, but showed little or no effect on the enzyme synthesis under other conditions. Although growth rate and profile of beta-galactosidase synthesis in glucose-grown cells were similar to those in arabinose-grown cells, the acceleration of beta-galactosidase synthesis upon the addition of cAMP was found only in glucose-grown cells. The cells aerobically grown in the presence of glycerol, xylose, or arabinose showed a high synthetic rate of cAMP and were insensitive to exogenously supplied cAMP as regards beta-galactosidase synthesis. Although the cells grown with glucose showed similar rates of cAMP synthesis under aerobic and anaerobic conditions, the differential rate of beta-galactosidase synthesis was much higher in the anaerobic state than in the aerobic state. These findings support the idea that catabolite repression found in the strain is caused through two mechanisms, i.e., cAMP-mediated and cAMP-independent ones.     

Activation of neutral trehalase by fermentable sugars and cAMP in the fission yeast Schizosaccharomyces pombe

Abstract Derepressed cells of Schizosaccharomyces pombe 972 h⁻ suspended in the presence of glucose or other fermentable sugars displayed a transient activation of trehalase which was not blocked by cycloheximide. Repressed cells were unable to show glucose-induced trehalase stimulation. Nitrogen sources, protonophores or uncouplers failed to produce direct trehalase activation but increased the activity of the enzyme in the presence of glucose. Exogenous cAMP induced a rapid and pronounced stimulation of trehalase in both repressed and derepressed cells suggesting that the response to glucose includes activation of adenylate cyclase as part of a cAMP signalling pathway that increases the catalytic activity of trehalase by enzyme modification.     

Relationship between the production of spirosomes and anaerobic glycolysis activity in Escherichia coli B

The effects of culture conditions (aerobic or anaerobic) and glucose in the medium on the production of spirosomes in Escherichia coli B were studied by SDS-PAGE and electron microscopy. The Mr of the spirosome of E. coli B was estimated to be 97,000. Electron microscopy revealed that the amount of spirosomes derived from anaerobic cultures was about eightfold larger than that from aerobic cultures. In SDS-PAGE, the bands of spirosome protein derived from anaerobic cultures were more intense than those derived from aerobic cultures, either in peptone water or in Davis-Mingioli's minimal medium. With increased glucose concentration under aerobic conditions, the intensity of the band of spirosome protein was similar to that observed under anaerobic conditions in basal media. These results suggest that spirosome production by E. coli B is related to its anaerobic glycolysis activity.     

Trehalose mobilization during early germination ofPilobolus longipes sporangiospores

Pilobolus longipes spores were activated by either exogenous glucose or 6-deoxyglucose. Trehalose content of glucose-activated spores increased and the substrate for trehalose synthesis was exogenous glucose. Addition of 6-deoxyglucose resulted in mobilization of trehalose, with about 20% of the reserve being consumed in the first hour. Little or no change in trehalase activity occurred during spore activation. Most of the trehalase activity associated with spores could be removed by washing with phosphate buffer. This extracellular enzyme was relatively stable, had a pH optimum of 5.6 and a K_m of about 0.5 mM and was estimated to be 66,000 in molecular weight. The specific activity of the crude enzyme extracts fromP. longipes was not influenced by cAMP, but, under the same conditions, the regulatory trehalase fromSaccharomyces cerevisiae became activated. These experiments indicate that trehalase activity in germinatingP. longipes spores may not be regulated by cAMP-dependent phosphorylation. Instead, the results suggest that trehalose is mobilized by a decompartmentation process.     

pH, pCO2 and NADP changes during aerobic and anaerobic incubation of the brain cortex of young and adult rats

Wistar rats of both sexes, aged 5 and 90-110 days, bred in the author's department, were used for experiments in which pH, pCO2 and NADP changes induced by incubating brain cortex homogenate (30 min at 37 degrees C) under aerobic and anaerobic conditions were studied. The medium was S?rensen's buffer solution, with succinate as substrate. NADP was assayed by determining glucose-6-phosphate dehydrogenase activity, which is NADP-dependent. The pH and pCO2 were determined with an ABL-2 radiometer (Denmark). The NADP extinction curves showed that the adult rat cortex, with or without substrate, always contained significantly less NADP under anaerobic conditions (the amount of its reduced form increased). In 5-day-old rats, the course of changes in the extinction curves was completely analogous to that for adult rats if the tissue was incubated without substrate; in the presence of substrate, the amount of the oxidized form, i.e. of NADP, decreased and there was no difference between the course of extinction changes under anaerobic and aerobic conditions. Similarly, the pH fell significantly less in 5-day-old rats during anaerobic incubation than in adult rats and was practically no different from the pH changes found during aerobic incubation. In addition, we found that the increase in pCO2 during anaerobic incubation of the brain cortex of 5-day-old rats was very small (also compared with aerobic conditions) whereas the increase in pCO2 under aerobic and anaerobic conditions in adult rats was the same.(ABSTRACT TRUNCATED AT 250 WORDS)     

Control of biodegradative threonine dehydratase inducibility by cyclic AMP in energy-restricted Escherichia coli.

To explain the requirement for anaerobic conditions in the induction of biodegradative L-threonine dehydratase in Escherichia coli, Crookes strain, measurements of cyclic AMP (cAMP) were made during aerobic and anaerobic growth and upon an aerobic-to-anaerobic transition. Internal cAMP levels were similar (5 to 10 muM) throughout exponential growth, whether aerobic or anaerobic, but only during anaerobiosis was threonine dehydratase synthesized. When an exponentially growing aerobic culture was made anaerobic, a sharp increase in internal cAMP was noted, reaching 300 muM within 10 min and declining thereafter to normal anaerobic levels. Threonine dehydratase synthesis was detected immediately after the attainment of peak cAMP levels and continued for several generations. A similar pattern but with less accumulation of cAMP and less threonine dehydratase production was also noted upon treatment of an aerobically growing culture with KCN. Pyruvate addition at the time of anaerobic shock severely affected both cAMP accumulation and threonine dehydratase synthesis; however, externally added cAMP could partially counter the pyruvate effect on enzyme synthesis. The conclusion was reached that conditions which resulted in a temporary energy deficit brought about the major accumulation of cAMP, and this elevated level served as a signal for initiation of threonine dehydratase synthesis to supply energy by the nonoxidative degradation of threonine.     

Nitrogen-source-induced activation of neutral trehalase in Schizosaccharomyces pombe and Pachysolen tannophilus: role of cAMP as second messenger

Abstract Resting cells of the fission yeast Schizosaccharomyces pombe , suspended in buffer with glucose, responded to the addition of asparagine by increasing trehalase activity. This response was preceded by a peak in cAMP concentration. The addition of the nitrogen source to resting cells, devoid of the catalytic subunit of cAMP-dependent protein kinase, produced the transient increase in cAMP but did not promote any change in trehalase activity. In the budding yeast Pachysolen iannophilus , the activation of trehalase by nitrogen source was also accompanied by a sharp peak in cAMP. These results suggest that in the two yeasts cAMP acts as a second messenger in the transduction of the nitrogen-source-induced signal causing the activation of trehalase.     

Trehalase activation in yeasts is mediated by an internal acidification

It has been reported that the addition of glucose, uncouplers and nystatin to yeast cells grown in a sugarfree medium causes trehalase activation; it has been postulated that this activation might be mediated by the depolarization of the plasma membrane. In this article the values of membrane potential and pH gradient across the plasma membrane of Saccharomyces cerevisiae have been determined under the same conditions as those in which trehalase is activated. Membrane potential was evaluated from the distribution of triphenylmethylphosphonium, the pH gradient from the distribution of benzoic acid across the plasma membrane. When the effect of several agents on the two components of the electrochemical proton gradient across the plasma membrane of ethanol-grown yeast cells were studied, under trehalase activation conditions, the following observations were made. (a) The addition of glucose activated trehalase and caused internal acidification of the cells, but had practically no effect on the membrane potential. (b) The addition of 200 mM KCl depolarized the cell membrane but did not affect the internal pH, nor trehalase activity. (c) Although carbonyl cyanide m-chlorophenylhydrazone depolarized the cells at external pH 6.0 and 7.0, it only activated trehalase at an external pH 6.0, leading to the acidification of the internal medium at this pH. (d) Nystatin caused an increase in the triphenylmethylphosphonium accumulation at external pH 6.0 and 7.0, but only activated trehalase at external pH 6.0, causing acidification of the cell interior at this pH. (e) Activation of trehalase was also observed when the internal acidification was caused by addition of a weak acid such as acetate. It is concluded that trehalase activation is mediated by an intracellular acidification and is independent of the membrane potential.     

Fermenting Escherichia coli Is Able to Grow in Media of High Osmolarity, But Is Sensitive to the Presence of Sodium Ion

Escherichia coli is able to grow at increased NaCl concentrations that provides an increase in medium osmolarity and cellular Na⁺ content. The addition of 0.5 M NaCl to the growth medium led to a substantial decrease in growth rate during anaerobic fermentation on glucose at pH of 7.3 or 9.0. This inhibitory effect of 0.5 M NaCl was at least threefold stronger than that seen under aerobic conditions, and stronger than equivalent concentrations of sucrose, KCl, or potassium glutamate under anaerobic conditions. Further, proline was found to stimulate the growth rate at high NaCl concentration under anaerobic and to a lesser extent, under aerobic conditions. Wild-type cells and mutants having a functional NhaA or ChaA alone grown under anaerobic conditions at pH 9.0 and subsequently loaded with Na⁺ were shown to extrude Na⁺ at a rate that were lower than the extrusion rate reported for appropriate aerobically grown bacteria (Sakuma et al. [1998] Biochim Biophys Acta 1363:231–237). The growth rate and Na⁺ extrusion activity of a mutant having a functional NhaA were similar to that of the wild type and higher than that of a mutant with an active ChaA. A mutant defective for both NhaA and ChaA was unable to grow under anaerobic conditions at pH 9.0 in the presence of 0.15 M Na⁺. It is suggested that the observed strong inhibition in the growth of E. coli during fermentation under anaerobic conditions in the presence of increased NaCl concentration could be due to a decrease in Na⁺ extrusion activity. Received: 18 September 1998 / Accepted: 2 April 1999     

Biochemical characterization of neutral trehalase activity in Saccharomyces boulardii

Lyophilized cells of the non-pathogenic yeast Saccharomyces boulardii are used in many countries for the treatment of several types of diarrhoea and other gastrointestinal diseases. Although the cells must be viable, their mechanism of action is unknown. The disaccharide trehalose is a protectant against several forms of environmental stress in yeast and is involved in maintaining cell viability. There is no information on the enzymes involved in degradation of trehalose in S. boulardii. The aim of the present study was to characterize trehalase activity in this yeast. Cells of S. boulardii grown in glucose exhibited neutral trehalase activity only in the exponential phase. Acidic trehalase was not detected in glucose medium. Cells grown in trehalose exhibited acid and neutral trehalase activities at all growth stages, particularly in the exponential phase. The optimum pH and temperature values for neutral trehalase activity were determined as 6.5 and 30 °C respectively, the half-life being approximately 3 min at 45 °C. The relative molecular mass of neutral trehalase is 80 kDa and the K _m 6.4 mM (±0.6). Neutral trehalase activity at pH 6.5 was weakly inhibited by 5 mM EDTA and strongly inhibited by ATP, as well as the divalent ions Cu⁺⁺, Fe⁺⁺ and Zn⁺⁺. Enzyme activity was stimulated by Mg⁺⁺ and Ca⁺⁺ only in the absence of cAMP. The presence of cAMP with no ion additions increased activity by 40%. This revised version was published online in July 2006 with corrections to the Cover Date.     

Regulation of Nitrate Assimilation and Nitrate Respiration in Aerobacter aerogenes

The influence of growth conditions on assimilatory and respiratory nitrate reduction in Aerobacter aerogenes was studied. The level of nitrate reductase activity in cells, growing in minimal medium with nitrate as the sole nitrogen source, was much lower under aerobic than anaerobic conditions. Further, the enzyme of the aerobic cultures was very sensitive to sonic disintegration, as distinct from the enzyme of anaerobic cultures. When a culture of A. aerogenes was shifted from anaerobic growth in minimal medium with nitrate and NH(4) (+) to aerobiosis in the same medium, but without NH(4) (+), the production of nitrite stopped instantaneously and the total activity of nitrate reductase decreased sharply. Moreover, there was a lag in growth of about 3 hr after such a shift. After resumption of growth, the total enzymatic activity increased again slowly and simultaneously became gradually sensitive to sonic disintegration. These findings show that oxygen inactivates the anaerobic nitrate reductase and represses its further formation; only after a de novo synthesis of nitrate reductase with an assimilatory function will growth be resumed. The enzyme in aerobic cultures was not significantly inactivated by air, only by pure oxygen. The formation of the assimilatory enzyme complex was repressed, however, by NH(4) (+), under both aerobic and anaerobic conditions. The results indicate that the formation of the assimilatory enzyme complex and that of the respiratory enzyme complex are regulated differently. We suggest that both complexes have a different composition, but that the nitrate reductase in both cases is the same protein.     

Effect of the activity of primary proton pumps on the growth of Escherichia coli in the presence of acetate

Escherichia coli batch cultures were grown under aerobic and anaerobic conditions on glucose with the substrate addition at pH 7.0. The cultures accumulated acetate in the medium at concentrations sufficient to inhibit the growth. This inhibitory effect of acetate was mediated apparently via its action on the intracellular pH. The inhibition of E. coli growth by acetate increased when the redox proton pump was switched off in the course of transition from aerobiosis to anaerobiosis and when the regulation of K+ fluxes was disordered in the presence of valinomycin. H+-ATPase was not essentially involved in maintaining the high rate of E. coli growth in the presence of acetate under aerobic conditions. If the activity of H+-ATPase was inhibited under anaerobic conditions at pH 7.0, the growth ceased after the dissipation of ionic gradients on the membrane. When CCCP was added under aerobic conditions, the growth did not stop at once if the medium had a pH of 7.6, but ceased immediately at pHout 7.0 in the glucose-salt medium.     

Protein tyrosine phosphorylation during prolonged in vitro incubation of ejaculated bovine spermatozoa is regulated by the oxidative state of the medium

Protein tyrosine phosphorylation plays a regulatory role in a multitude of physiological processes in sperm. Changes in protein tyrosine phosphorylation, viability, and motility were studied as a function of extended incubation of bovine sperm in vitro at ambient temperature (18-20 degrees C). Fresh ejaculates were incubated after dilution for 8 days. On Days 0, 2, 5, and 8, an aliquot of sperm was incubated with or without theophylline at 37 degrees C for 30 min prior to assessing sperm viability, motility, and tyrosine phosphorylation of soluble and whole-cell proteins. There was a time-dependent decline in sperm motility, which was to some extent reversed by incubation with theophylline. The sum of the phosphotyrosine signal from two soluble proteins (M(r) 67 000 and 36 000) declined with incubation time in both theophylline-treated and untreated sperm. There were major differences in the pattern of tyrosine phosphorylation during incubation between ejaculates from different bulls. Tyrosine phosphorylation of a number of proteins from whole-cell extracts increased in a time-dependent manner during in vitro incubation and was unaffected by the presence of theophylline in the medium. The oxygenation state of the incubation medium had profound effects on sperm motility, viability, and tyrosine phosphorylation of proteins from whole-cell extracts. Sperm motility and viability declined more rapidly under aerobic compared with anaerobic conditions. Tyrosine phosphorylation of proteins from whole-cell extracts increased considerably during anaerobic incubation, while there was no significant change during aerobic incubation. This increase in phosphorylation due to anaerobic incubation was reversed when sperm were transferred from an anaerobic to an aerobic environment, indicating that the oxygenation state of the medium regulates both protein tyrosine kinases and phosphatases. In addition, sperm incubated under aerobic conditions for 5 days retained the ability to phosphorylate proteins when transferred to an anaerobic environment. The increase in protein tyrosine phosphorylation during in vitro incubation took place in a medium that did not contain capacitating substances such as heparin, sodium bicarbonate, or BSA. It transpired over a time scale of days and was not augmented by an increase in intracellular cAMP concentration through phosphodiesterase inhibition. Protein tyrosine phosphorylation during extended in vitro incubation at ambient temperature was significantly inhibited by the presence of oxygen in the medium.     

Induction of citrate lyase in Enterobacter cloacae grown under aerated conditions and its effect on citrate metabolism.

Growth of Enterobacter cloacae on K+ citrate under aerated conditions (no detectable oxygen tension in the medium even though it was aerated) was slower (mean generation time, 130 min) than under aerobic conditions (mean generation time, 72 min), but with a faster utilization of citrate, resulting in a molar growth yield of 10.6 g (dry weight) of cells per mol of citrate utilized versus 40 g (dry weight) of cells per mol of citrate utilized for aerobic growth. The rapid utilization of citrate under aerated conditions was apparently due to the induction of citrate lyase and was supported by the finding that cells excreted acetate and a small amount of oxalacetate under aerated conditions, but not under aerobic conditions when the cells were devoid of citrate lyase activity. The activity of oxalacetate decarboxylase in aerated cells was slightly lower than in aerobic cells, indicating that little of the oxalacetate produced by the citrate lyase was metabolized by the decarboxylase. Oxalacetate was probably metabolized by malate dehydrogenase, previously shown to be present in anaerobic and aerobic cells. Thus, about 70% of the citrate was cleaved by the citrate lyase, resulting in little or no production of energy for growth. The remaining citrate was metabolized via the citric acid cycle under aerated conditions, since the cells contained alpha-ketoglutarate dehydrogenase at the same level as in aerobically grown cells. The presence of the other enzymes of the cycle was shown in earlier studies.     

Physiological response to anaerobicity of glycerol-3-phosphate dehydrogenase mutants of Saccharomyces cerevisiae.

Mutants of Saccharomyces cerevisiae, in which one or both of the genes encoding the two isoforms of NAD-dependent glycerol-3-phosphate dehydrogenase had been deleted, were studied in aerobic batch cultures and in aerobic-anaerobic step change experiments. The respirofermentative growth rates under aerobic conditions with semisynthetic medium (20 g of glucose per liter) of two single mutants, gpd1 delta and gpd2 delta, and the parental strain (mu = 0.5 h-1) were almost identical, whereas the growth rate of a double mutant, gpd1 delta gpd2 delta, was approximately half that of the parental strain. Upon a step change from aerobic to anaerobic conditions in the exponential growth phase, the specific carbon dioxide evolution rates (CER) of the wild-type strain and the gpd1 delta strain were almost unchanged. The gpd2 delta mutant showed an immediate, large (> 50%) decrease in CER upon a change to anaerobic conditions. However, after about 45 min the CER increased again, although not to the same level as under aerobic conditions. The gpd1 delta gpd2 delta mutant showed a drastic fermentation rate decrease upon a transition to anaerobic conditions. However, the CER values increased to and even exceeded the aerobic levels after the addition of acetoin. High-pressure liquid chromatographic analyses demonstrated that the added acetoin served as an acceptor of reducing equivalents by being reduced to butanediol. The results clearly show the necessity of glycerol formation as a redox sink for S. cerevisiae under anaerobic conditions.     

In vitro assembly of cytochrome oxidase from separately accumulated subunits: a reconsideration

Trehalase activity in a yeast protoplast lysate increased 40-times upon preincubation with cAMP and ATP. The activity present without the preincubation could all be sedimentated at 8000 × g, for 10 min confirming the previously reported localization of the active trehalase (Ta) in the vacuoles. Virtually all the trehalase activity newly formed upon the preincubation, however, was found in the soluble fraction, indicating that a trehelase-zymogen (Tz) is located in the cytosol. This raises the possibility that a cAMP-dependent phosphorylation not only transforms Tz to Ta but also initiates the transfer of trehalase from the cytosol into the vacuoles.