H+ and K+ fluxes and attendant processes inCandida utilis induced by inhibitory ethanol concentration

In oxygen-aerated non-growing cells a subinhibitory ethanol concentration (1 g/L) causes an H⁺/K⁺ exchange. An inhibitory ethanol concentration (30 g/L) slows down acidification but has no effect on its extent. The transmembrane ΔpH depends directly on the ethanol level in the medium and is not lowered at high ethanol concentrations. Changes in membrane potential induced by ethanol are also concentration dependent. The high ethanol level does not increase the passive permeability of cell membrane to H⁺.     

Comparative study of plant alcohol dehydrogenases

Alcohol dehydrogenase was isolated both from monocotyledons and dicotyledons, some of them with proteins (bean, pea), others with lipids (rape, sunflower) and still others with sugars (rice) as reserve substances. Molecular weights of the isolated dehydrogenases ranged from 53 000 to 80 000. Plant alcohol dehydrogenases (ADH) catalyze the oxidation of ethanol as well as the reduction of acetaldehyde. pH optimum for the oxidation is in the alkaline region, for the reduction it is near neutrality. The Michaelis constants for ethanol oxidation are, with the exception of rice, higher than those for reduction of acetaldehyde. The specificity of plant ADH toward alcohols is relatively broad and only quantitatively different in the individual plants. Inhibitors of the ADH’s studied are oximes, amides and intermediates of sugar metabolism, such as malate, acetate or succinate. The degree of inhibition brought about by the inhibitors studied differs from plant to plant but the inhibition type is the same.     

初始pH值对产氢产乙酸/耗氢产乙酸两段耦合工艺定向生产乙酸的影响

以葡萄糖为底物,以经加热预处理并活化过的厌氧污泥为种泥,研究了初始pH值对产氢产乙酸/耗氢产乙酸两段耦合工艺厌氧发酵定向生产乙酸的影响。实验考察了7个初始pH值(5、6、7、8、9、10、11)条件下的底物降解、产物产生和发酵过程pH值的变化。结果表明:产氢产乙酸段初始pH值的变化不仅影响本阶段产酸,而且影响耗氢产乙酸段产酸。初始pH=5时主要进行乙醇型发酵;pH=6和7时主要进行丁酸型发酵;pH=8时混合酸型发酵类型逐渐占优势,pH=8~11时均以乙酸为主要产物,耦合系统生产乙酸最优初始pH值为10。在初始pH=8~11范围内,产氢产乙酸段初期的乙醇浓度一般较高,但到后期因乙醇被微生物进一步代谢转化成乙酸而使其含量下降。     

The apparent oxidation of NADH by whole cells of the methylotrophic bacterium Methylophilus methylotrophus

Previous reports that whole cells of Methylophilus methylotrophus oxidase exogenous NADH have been investigated. Essentially identical rates of oxygen consumption were observed following the addition of methanol or NADH to whole cells. Both activities were inhibited by EDTA and hydroxylamine, but not by HQNO, and exhibited similar pH optima. Analyses of the reaction stoichiometry with NADH as substrate showed that the expected amount of oxygen was consumed, but also revealed acidification (instead of alkalinisation) and no oxidation of NADH. Further studies showed that commerical NADH is contaminated with ethanol which is oxidised to acetic acid by the low specificity methanol oxidase system present in this organism. The oxidation of exogenous NADH by whole cells of M. methylotrophus reported previously is therefore spurious.Abbreviations EDTA = ethylenediaminetetracetate - HQNO = 2–n-heptyl-4-hydroxy-quinoline-N-oxide - DEAE = diethylaminoethyl     

The mechanism of intracellular acidification induced by glucose in Saccharomyces cerevisiae

Addition of glucose or fructose to cells of Saccharomyces cerevisiae adapted to grow in the absence of glucose induced an acidification of the intracellular medium. This acidification appeared to be due to the phosphorylation of the sugar since: (i) glucose analogues which are not efficiently phosphorylated did not induce internal acidification; (ii) glucose addition did not cause internal acidification in a mutant deficient in all the three sugar-phosphorylating enzymes; (iii) fructose did not affect the intracellular pH in a double mutant having only glucokinase activity; (iv) glucose was as effective as fructose in inducing the internal pH drop in a mutant deficient in phosphoglucose isomerase activity; and (v) in strains deficient in two of the three sugar-phosphorylating activities, there was a good correlation between the specific glucose- or fructose-phosphorylating activity of cell extracts and the sugar-induced internal acidification. In addition, in whole cells any of the three yeast sugar kinases were capable of mediating the internal acidification described. Glucose-induced internal acidification was observed even when yeast cells were suspended in growth medium and in cells suspended in buffer containing K+, which supports the possible signalling function of the glucose-induced internal acidification. Evaluation of internal pH by following fluorescence changes of fluorescein-loaded cells indicated that the change in intracellular pH occurred immediately after addition of sugar. The apparent Km for glucose in this process was 2 mM. Changes in both the internal and external pH were determined and it was found that the internal acidification induced by glucose was followed by a partial alkalinization coincident with the initiation of H+ efflux. This reversal of acidification could be due to the activity of the H+-ATPase, since it was inhibited by diethylstilboestrol. Coincidence between internal alkalinization and the H+ efflux was also observed after addition of ethanol.     

Effects of ethanol on Saccharomyces cerevisiae as monitored by in vivo 31P and 13C nuclear magnetic resonance

Cell suspensions of a respiratory deficient mutant of Saccharomyces cerevisiae were monitored by in vivo ³¹P and ¹³C Nuclear Magnetic Resonance in order to evaluate the effect of ethanol in intracellular pH and metabolism. In the absence of an added energy source, ethanol caused acidification of the cytoplasm, as indicated by the shift to higher field of the resonance assigned to the cytoplasmic orthophosphate. Under the experimental conditions used this acidification was not a consequence of an increase in the passive influx of H⁺. With cells energized with glucose, a lower value for the cytoplasmic pH was also observed, when ethanol was added. Furthermore, lower levels of phosphomonoesters were detected in the presence of ethanol, indicating that an early event in glycolysis is an important target of the ethanol action. Acetic acid was identified as responsible for the acidification of the cytoplasm, in experiments where [¹³C]ethanol was added and formation of labeled acetic acid was detected. The intracellular and the extracellular concentrations of acetic acid were respectively, 30 mM and 2 mM when 0.5% (120 mM) [¹³C]ethanol was added.Abbreviations P_i inorganic phosphate - P_ic inorganic phosphate in the cytoplasm - P_iv inorganic phosphate in the vacuole - tP terminal phosphate in polyphosphate     

Rapid and direct effects of pH on connexins revealed by the connexin46 hemichannel preparation

pH is a potent modulator of gap junction (GJ) mediated cell-cell communication. Mechanisms proposed for closure of GJ channels by acidification include direct actions of H+ on GJ proteins and indirect actions mediated by soluble intermediates. Here we report on the effects of acidification on connexin (Cx)46 cell-cell channels expressed in Neuro-2a cells and Cx46 hemichannels expressed in Xenopus oocytes. Effects of acidification on hemichannels were examined macroscopically and in excised patches that permitted rapid (<1 ms) and uniform pH changes at the exposed hemichannel face. Both types of Cx46 channel were found to be sensitive to cytoplasmic pH, and two effects were evident. A rapid and reversible closure was reproducibly elicited with short exposures to low pH, and a poorly reversible or irreversible loss occurred with longer exposures. We attribute the former to pH gating and the latter to pH inactivation. Half-maximal reduction of open probability for pH gating in hemichannels occurs at pH 6.4. Hemichannels remained sensitive to cytoplasmic pH when excised and when cytoplasmic [Ca2+] was maintained near resting ( approximately 10(-7) M) levels. Thus, Cx46 hemichannel pH gating does not depend on cytoplasmic intermediates or a rise in [Ca2+]. Rapid application of low pH to the cytoplasmic face of open hemichannels resulted in a minimum latency to closure near zero, indicating that Cx46 hemichannels directly sense pH. Application to closed hemichannels extended their closed time, suggesting that the pH sensor is accessible from the cytoplasmic side of a closed hemichannel. Rapid closure with significantly reduced sensitivity was observed with low pH application to the extracellular face, but could be explained by H+ permeation through the pore to reach an internal site. Closure by pH is voltage dependent and has the same polarity with low pH applied to either side. These data suggest that the pH sensor is located directly on Cx46 near the pore entrance on the cytoplasmic side.     

Transfer and reoxidation of reducing equivalents as the rate-limiting steps in the oxidation of ethanol by liver cells isolated from fed and fasted rats

A study was made of factors regulating the oxidation of ethanol in liver cells isolated from fed and fasted rats. The rate of ethanol oxidation was greater in liver cells from fed rats than from fasted rats. Inhibitors of the malate-aspartate shuttle decreased the rate of ethanol oxidation, suggesting that this shuttle contributes to the reoxidation of cytosolic NADH produced during the oxidation of ethanol. The greater inhibition of ethanol oxidation by antimycin than by rotenone suggests that the α-glycerophosphate shuttle also plays an important role in transporting reducing equivalents. The components of the malate-aspartate and α-glycerophosphate shuttles stimulated ethanol oxidation to a greater extent in liver cells from fasted rats than those from fed rats, suggesting that in the fasted state, ethanol oxidation is regulated by the intracellular concentrations of substrate shuttle components which transfer reducing equivalents into the mitochondria. Therefore, uncoupling agents, which stimulate oxygen consumption, do not stimulate ethanol oxidation, and concentrations of antimycin which depress oxygen uptake are much less effective in decreasing ethanol oxidation. By contrast, in liver cells from fed rats, the rate of ethanol oxidation was increased by uncoupling agents. Such stimulation was not observed when cells were prepared in the absence of albumin, probably due to leakage of shuttle substrates which leads to abnormally low intracellular levels. Indeed, when the shuttle substrates were added back to these preparations, uncouplers were effective in stimulating the rate of ethanol oxidation beyond the stimulation produced by the shuttle substrates alone. Thus, under conditions of sufficient intracellular levels of the intermediates of the substrate shuttles, ethanol oxidation is regulated by the capacity of the mitochondrial respiratory chain to reoxidize reducing equivalents generated by the alcohol dehydrogenase reaction.     

Comparison of CSTR and UASB reactor configuration for the treatment of sulfate rich wastewaters under acidifying conditions

The effects of lowering the operational pH from 6 to 5 on mesophilic (30 °C) sulfate reduction during the acidification of sucrose at an organic loading rate of 5 gCOD (l_reactor d)⁻¹ and at a COD/SO₄²⁻ ratio of 4 were evaluated in a CSTR and in a UASB reactor. The HRT was 24 h and 10 h, respectively. Acidification was complete in both reactors at pH 6 and the lowering of the operational pH to 5 did not affect the acidification efficiency in the CSTR but decreased the acidification efficiency of the UASB to 72%. The decrease to pH 5 caused an increase in the effluent butyrate and ethanol concentrations in both reactors. Lowering the pH from 6 to 5 caused a decrease in sulfate reduction efficiencies in both reactors, from 43% to 25% in the CSTR and from 95% to 34% in the UASB reactor. The acidification and sulfate reduction efficiencies at pH 5 could be increased to 94% and 67%, respectively, by increasing the HRT of the UASB reactor to 24 h.     

Combined effect of acetic acid, pH and ethanol on intracellular pH of fermenting yeast

Summary The internal pH of Saccharomyces cerevisiae IGC 3507 III (a respiratory-deficient mutant) was measured by the distribution of [¹⁴C]propionic acid, when the yeast was fermenting glucose at pH 3.5, 4.5 and 5.5 in the presence of several concentrations of acetic acid and ethanol. Good correlation was obtained between fermentation rates and internal pH. For all external pH values tested, the internal pH was 7.0–7.2 in the absence of inhibitors. The addition of acetic acid and/or ethanol resulted in a decrease of fermentation rate together with a drop in internal pH. Internal pH did not depend on the concentration of total external acetic acid but only on the concentration of the undissociated form of the acid. Ethanol potentiated the effect of acetic acid both with respect to inhibition of fermentation and internal acidification.     

Electrochemical study of proton translocation function of hydrogenase 3

The oxidation of formate associated with fast acidification of medium by whole Escherichia coli cells lacking both hya and hyb hydrogenases was studied. The extent of acidification was dependent on the amount of formate added. An average H+/formate ratio of 1.3 was obtained. The proton release was inhibited by carbonyl cyanide m-chlorophenylhydrazone. Inverted vesicles of E. coli were found to translocate protons upon oxidation of formate at pH 6.5. The extent of alkalization was also dependent on the amount of formate added. The maximum H+/formate ratio for this reaction was close to 0.6. Formate oxidation by inverted vesicles from E. coli (delta hya delta hyb) was sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone. It was supposed that the hydrogenase 3 (hyc) component of E. coli formate hydrogen lyase is responsible for the translocation of protons at low pH.     

Oxygen radical generation by microsomes: role of iron and implications for alcohol metabolism and toxicity

Experiments were carried out to evaluate whether the molecular mechanism for ethanol oxidation by microsomes, a minor pathway of alcohol metabolism, involved generation of hydroxyl radical (.OH). Microsomes oxidized chemical .OH scavengers (KMB, DMSO, t-butyl alcohol, benzoate) by a reaction sensitive to catalase, but not SOD. Iron was required for microsomal .OH generation in view of the potent inhibition by desferrioxamine; however, the chelated form of iron was important. Microsomal .OH production was effectively stimulated by ferric EDTA or ferric DTPA, but poorly increased with ferric ATP, ferric citrate, or ferric ammonium sulfate. By contrast, the latter ferric complexes effectively increased microsomal chemiluminescence and lipid peroxidation, whereas ferric EDTA and ferric DTPA were inhibitory. Under conditions that minimize .OH production (absence of EDTA, iron) ethanol was oxidized by a cytochrome P-450-dependent process independent of reactive oxygen intermediates. Under conditions that promote microsomal .OH production, the oxidation of ethanol by .OH becomes more significant in contributing to the overall oxidation of ethanol by microsomes. Experiments with inhibitors and reconstituted systems containing P-450 and NADPH-P-450 reductase indicated that the reductase is the critical enzyme locus for interacting with iron and catalyzing production of reactive oxygen species. Microsomes isolated from rats chronically fed ethanol catalyzed oxidation of .OH scavengers, light emission, and inactivation of added metabolic enzymes at elevated rates, and displayed an increase in ethanol oxidation by a .OH-dependent and a P-450-dependent pathway. It is possible that enhanced generation of reactive oxygen intermediates by microsomes may contribute to the hepatotoxic effects of ethanol.     

Guanine versus deoxyribose damage in DNA oxidation mediated by vanadium(IV) and vanadium(V) complexes

Vanadyl sulfate reacts with the peroxy acid oxidant KHSO5 to produce guanine-selective oxidation of a 167-bp restriction fragment of DNA. The oxidized lesions result in strand scission after hot piperidine treatment. Although several reactive intermediates are possible, quenching studies with ethanol and tert-butyl alcohol suggest that a monoperoxysulfate radical or a caged sulfate radical are the likely species responsible for oxidation of guanine. Several oxidants and various vanadium complexes (including insulin mimetic compounds) were studied with DNA for comparison. None of the other vanadium complexes showed modification of the double-stranded 167-bp fragment of DNA in the presence of KHSO5. The reactivity of VOSO4 may be due to its irreversible oxidation potential of 0.77 V (vs. Ag+/AgCl, pH 7.0, 10 mM phosphate), making it an appropriate catalyst for decomposition of monoperoxysulfate.     

Vacuolar acidification in Saccharomyces cerevisiae induced by elevated hydrostatic pressure is transient and is mediated by vacuolar H+-ATPase

We analyzed the vacuolar acidification in response to elevated hydrostatic pressure in Saccharomyces cerevisiae. The vacuolar pH, defined using 6-carboxyfluorescein, was directly measured in a hyperbaric chamber with a transparent window under high hydrostatic pressure. The vacuole of strain X2180 became acidified at the onset of pressurization to an extent dependent on the magnitude of pressure applied. A pressure of 40–60 MPa transiently reduced the vacuolar pH by about 0.33 within 4 min. The transient acidification was observed in the presence of D-glucose, D-fructose, or D-mannose as a carbon source, but not 3-o-methyl-D-glucose, ethanol, or glycerol, suggesting that the generation of CO₂ was involved in the process. A vma3 mutant defective in vacuolar acidification showed no reduction of vacuolar pH when hydrostatic pressure was applied. This result indicates that the transient vacuolar acidification induced by elevated hydrostatic pressure is mediated through the function of the vacuolar H+-ATPase. Received: August 21, 1996 / Accepted: November 11, 1996     

Comparison of CSTR and UASB reactor configuration for the treatment of sulfate rich wastewaters under acidifying conditions

The effects of lowering the operational pH from 6 to 5 on mesophilic (30 °C) sulfate reduction during the acidification of sucrose at an organic loading rate of 5 gCOD (l_reactor d)⁻¹ and at a COD/SO₄²⁻ ratio of 4 were evaluated in a CSTR and in a UASB reactor. The HRT was 24 h and 10 h, respectively. Acidification was complete in both reactors at pH 6 and the lowering of the operational pH to 5 did not affect the acidification efficiency in the CSTR but decreased the acidification efficiency of the UASB to 72%. The decrease to pH 5 caused an increase in the effluent butyrate and ethanol concentrations in both reactors. Lowering the pH from 6 to 5 caused a decrease in sulfate reduction efficiencies in both reactors, from 43% to 25% in the CSTR and from 95% to 34% in the UASB reactor. The acidification and sulfate reduction efficiencies at pH 5 could be increased to 94% and 67%, respectively, by increasing the HRT of the UASB reactor to 24 h.     

Mechanism of oxidation of inorganic sulfur compounds by thiosulfate-grown Thiobacillus thiooxidans

Thiobacillus thiooxidans was grown at pH 5 on thiosulfate as an energy source, and the mechanism of oxidation of inorganic sulfur compounds was studied by the effect of inhibitors, stoichiometries of oxygen consumption and sulfur, sulfite, or tetrathionate accumulation, and cytochrome reduction by substrates. Both intact cells and cell-free extracts were used in the study. The results are consistent with the pathway with sulfur and sulfite as the key intermediates. Thiosulfate was oxidized after cleavage to sulfur and sulfite as intermediates at pH 5, the optimal growth pH on thiosulfate, but after initial condensation to tetrathionate at pH 2.3 where the organism failed to grow. N-Ethylmaleimide (NEM) inhibited sulfur oxidation directly and the oxidation of thiosulfate or tetrathionate indirectly. It did not inhibit the sulfite oxidation by cells, but inhibited any reduction of cell cytochromes by sulfur, thiosulfate, tetrathionate, and sulfite. NEM probably binds sulfhydryl groups, which are possibly essential in supplying electrons to initiate sulfur oxidation. 2-Heptyl-4-hydroxy-quinoline N-oxide (HQNO) inhibited the oxidation of sulfite directly and that of sulfur, thiosulfate, and tetrathionate indirectly. Uncouplers, carbonyl cyanide-m-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol (DNP), inhibited sulfite oxidation by cells, but not the oxidation by extracts, while HQNO inhibited both. It is proposed that HQNO inhibits the oxidation of sulfite at the cytochrome b site both in cells and extracts, but uncouplers inhibit the oxidation in cells only by collapsing the energized state of cells, delta muH+, required either for electron transfer from cytochrome c to b or for sulfite binding.     

Effect of intersubunit interaction in horse liver alcohol dehydrogenase on the kinetics of ethanol oxidation]

The kinetics of enzymatic oxidation of ethanol in the presence of alcohol dehydrogenase within a wide range of ethanol and NAD concentrations (pH 6.0--11.5) were studied. It was shown that high concentrations of ethanol (greater than 0.7--5 mM, depending on pH) and NAD (greater than 0.4--0.8 mM) activate alcohol dehydrogenase from horse liver within the pH range of 6.0--7.9. A mechanism of activation based on negative cooperativity of ADH subunits for binding of ethanol and NAD was proposed. The catalytic and Michaelis constants for alcohol dehydrogenase were calculated from ethanol and NAD at all pH values studied. The changes resulting from the subunit cooperativity were revealed. The nature of ionogenic groups of alcohol dehydrogenase, which affect the formation of complexes between the enzyme and NAD and ethanol, and the rate constants for catalytic oxidation of ethanol was assumed. The biological significance of the enzyme capacity for activation by high concentrations of ethanol within the physiological range of pH in the blood under excessive use of alcohol is discussed.     

Intracellular acidification does not account for inhibition of Saccharomyces cerevisiae growth in the presence of ethanol

Abstract Intracellular acidification has been considered one of a number of mechanisms underlying the inhibition of growth and fermentation by ethanol in yeast. However, most of the studies on the effect of ethanol on yeast intracellular pH (pH_i) were carried out by using unadapted cells to which ethanol was added. In this paper we show that the pH_i of exponential cells of Saccharomyces cerevisiae IGC 3507 III grown in a medium with glucose and inhibitory concentrations of ethanol only decreased to values below those in unstressed cells (6.9) for concentrations equal to or above 7% (v/v). Only at these supracritical levels (7–10% (v/v)) was pH homeostasis in ethanol-adapted yeast affected. This is consistent with the significant increase of plasma membrane permeability and decrease of plasma membrane H⁺-ATPase in comparison with the corresponding values in unstressed cells. These deleterious effects were only observed with those high concentrations of toxin. These results indicate that intracellular acidification does not account for inhibition of yeast growth in the presence of ethanol. In fact, growth was inhibited by ethanol concentrations (3–6% (v/v)) that did not lead to the decrease of pH_i. Furthermore, even for supracritical concentrations, close to the maximal that allowed growth (10% (v/v)), the dedrease of pH_i was not important reaching, at the most, values of 6.5–6.6.     

Kinetics of the reaction catalysed by rape alcohol dehydrogenase

The kinetics of the enzyme reaction of ethanol oxidation and acetaldehyde reduction catalysed by alcohol dehydrogenase (ADH) (EC 1.1.1.1) isolated from germinating rape seeds obeys the bi-bi ordered mechanism of Theorell and Chance. The enzyme reaction depends on the pH and temperature. The K_m values for the basic substrates have the lowest values around the pH optimum of the reaction. The enzyme is most stable at pH 6.5–7. The K_m values for ethanol and NAD increase with increasing temperature. The maximum rate of the ethanol oxidation satisfies the Arrhenius equation. The activation energy for the given temperature range is 40.11 kJ/mol. The rape ADH is denatured by heating above 60° but the enzyme-NAD complex is thermally more stable than the enzyme alone.