The Action of Nitrophenols on Respiration and on Glucose Assimilation in Yeast

The consumption of 2·25 mg. glucose by a resting suspensionof baker's yeast in the presence of finely graded concentrationsof 3:5 -dinitro-o-cresol was followed at pH 2·4 and 5·0by measuring oxygen uptake and carbon-dioxide output; at pH8·5 oxygen uptake alone was measured. At each pH certainconcentrations of dinitrocresol stimulated respiration and increasedthe ratio of glucose oxidized to glucose assimilated; at higherconcentrations, assimilation was reduced to zero, but respirationbecame inhibited and the whole of the glucose was then metabolizedby aerobic fermentation. Dinitrocresol is believed to be actingin these experiments as an uncoupling agent, lowering the netrate of formation of energy-rich phosphate and so allowing respirationto proceed faster while assimilation, an endergonic process,is inhibited. The maximum respiratory stimulation obtainable at pH 8·5,325 per cent, of the control, is greater than can be obtainedat pH 2·6 or 5·0, which may be due to the slowerrespiration rate of controls at pH 8's. The slow endogenousrespiration can also be stimulated more than the exogenous.With o- and p-nitrophenols similar relations are obtained, butphenol does not stimulate respiration, although it inhibitsassimilation in lower concentrations than respiration. Undernitrogen, a stimulation of fermentation can be obtained withdinitrocresol at pH 5·0 but not at 2·6. At bothpH levels, assimilation is more easily suppressed than fermentationrate.     

Glucose Respiration in Zygorhyncus moelleri; the Entry of Glucose into the Cells

When glucose was added to carbohydrate-starved cells of Zygorhyncus,moelleri the rate of oxygen uptake did not immediately riseto a constant value, but there was a lag period of 2 or 3 hoursbefore it reached its maximum level. The length of this lagperiod increased from a few minutes for short periods of starvationto 2–3 hours after 12 hours in a carbohydrate-free medium.Factors believed to affect cellular permeability (a cationicdetergent, adjustments of the pH, and of the potassium/calciumratio) reduced the length of the lag period by not more than40 per cent. of the original value without affecting the finalrate of oxygen uptake. Investigation of the entry of glucoseinto the cells showed that the rate of oxygen uptake was notlimited by the concentration of intra-cellular glucose for morethat about 11 per cent. of the lag period in starved cells.The reasons for this difference in the percentage of the lagperiod apparently due to a permeability barrier are tentativelydiscussed in connexion with the route by which glucose entersthe cells.     

The Effect of Cyanide on the Respiration and the Oxidative Assimilation of Glucose by Chlorella vulgaris

It is shown that low concentrations of cyanide stimulate theendogenous respiration of Chlorella vulgaris. When glucose isadded the respiration rate is much increased but is now reversiblyinhibited by cyanide. Some 30–60 per cent. of the totalrespiration remains uninhibited. One-eighth to one-ninth ofthe glucose added is completely oxidized. Most of the remainderis assimilated to di- or polysaccharide. Low concentrationsof cyanide which inhibit the rate of glucose oxidation alsoinhibit the assimilation of glucose. Two possible interpretationsof this fact are discussed. It is suggested that the assimilationof glucose is coupled with the oxidation of glucose by a cyanide-sensitiverespiratory system. The mathematical consequences of this theoryare considered and shown to agree with the experimental results.The effect of cyanide on the respiratory quotient is also discussed.     

Brachiopod Metabolism and Enzymes

Brachiopods consume oxygen at relatively low rates, and sometimesconsume none at all for hours. Specimens of Terebratulina septentrionalissurvived total anoxia for 3.5 days at 3°C. Isolated tissuesconverted ¹⁴Cµglucose into eight carboxylic acids at anaverage rate of 1.5 x 10^–10 mole/SOL;hr per g tissue.Carbon from labelled glucose flowed steadily into citric acidand into an unknown acid for 2 hours under both aerobic andanaerobic conditions. In the first hour, more label was foundin malic acid after aerobic incubation, and more label in succinicacid after anaerobic incubation, while the fraction in lacticacid was the same. Terebratulina carried on a mixed fermentationboth in the presence and in the absence of oxygen. The inarticulate Glottidia pyramidata has a succinate dehydrogenasewith kinetic properties favoring succinate oxidation, whilethe Terebratulina enzyme is more likely to operate in the reversedirection. Lactate metabolism is relatively unimportant in bothspecies. Information on nitrogen-compound metabolism is limited to theinarticulate Lingula reevii, which is ammonotelic. Arginaseand urease activities exceeded those of bivalve mollusks, whileaspartate and alanine aminotransferase rates were both muchlower. Some unique features of DNA, RNA and hemerythrin fromLingula have been discovered in the last few years.     

Links between Glucose Uptake and Metabolism in Nitella translucens

The uptake of ¹⁴C-glucose into cells of Nitella translucenshas been investigated under experimental conditions previouslyused in studies of the ionic relations of these cells. Glucoseentry was considerably stimulated by light, and under aerobicconditions the fluxes remained constant for many hours. Theinflux of glucose was inhibited by over 80 per cent at low temperature(4° C) and by over 90 per cent by the uncoupler carbonylcycanide-m-chlorophenylhydrazone. 2-Deoxy-D-glucose was a non-competitiveinhibitor of glucose uptake both in light and darkness. Cyclicphotophosphorylation promoted the influx (with decreasing efficiency)for several hours. It is suggested that an ATP-dependent transportprocess controls glucose entry to the cells, and that passivediffusion is of little significance.     

Changes in cytochrome contents and respiratory activity of Bacillus cereus Strain T during germination, vegetative growth and sporulation

Vegetative cells of Bacillus cereus Strain T contain cytochromeb-562, a minor b-type component, in addition to known components,cytochrome a+a₃, cytochrome b-557 and cytochrome c-551. Also,the spores contain low but definite amounts of cytochromes b-562and c-551, which were oxidized when the spores were shaken withair. Contents of cytochromes a, b and c per cell and per cellnitrogen, and the activity of glucose oxidation increased duringspore germination and elongation. During the stage precedingfirst cell division, cytochrome contents per cell increasedin parallel with the increase of cell nitrogen, while the activityof glucose oxidation decreased. During early exponential growth,the content of cytochrome b per cell nitrogen and respiratoryactivity with glucose again increased. When cells entered thesporulation stage, characterized by structural changes insidethe cells, the activity of glucose oxidation began to decrease,while that of acetate or succinate oxidation started to increase.During the sporulation process, the contents of the three cytochromecomponents continued to increase and reached the highest levelin cells containing completed spores, but the activity of respirationwith endogenous or added substrates was negligible in thesecells. (Received November 10, 1975; )     

Studies on the Preparation of a Respiratory Cell-Free Extract of the Alga Chlorella

Various techniques used for the preparation of extracts wereinvestigated, with a view to obtaining a respiratory preparationof Chlorella. Of the methods investigated, drying the cells at 18–20°in vacuo over P₂O₅ for 20–22 hr. was the only successfulone. Suggestions are made to account for the failure of cell-disintegrationtechniques. The rates of oxidation of organic acids relative to glucosewere such that the tricarboxylic-acid cycle may be presumedto occur, but the dependence of rates of oxidation on pH indicatedthat drying did not eliminate permeability barriers entirely.Simultaneous addition of malate or citrate with pyruvate didnot have a catalytic effect on the rate of oxidation. The resultsindicated, however, that the oxidation pathways of these substanceshad common steps. Malonate inhibition of succinic acid oxidationcould not be demonstrated. Experiments with glucose oxidation showed that the drying processhad affected the permeability of the cells rather than theirability to synthesize polysaccharide.     

Effects of glucose and other modifiers of insulin release on the oxidative metabolism of amino acids in micro-dissected pancreatic islets

The oxidation of alanine, arginine, leucine, glucose, and pyruvate was studied in microdissected pancreatic islets of obese-hyperglycaemic mice. The following main observations were made. The oxidation of glucose was enhanced severalfold when its concentration was raised from 3 to 20mm. At the latter concentration the rate was about 65mmol/h per kg dry wt. The oxidation of 17mm-pyruvate amounted to 20mmol/h per kg dry wt. indicating a significant entry of this compound into the beta-cells. Leucine oxidation was little affected by concentration changes above 5mm, the rate at 20mm corresponding to about 25% of that obtained with 20mm-glucose. In the absence of glucose, the oxidation of alanine or arginine was barely significant. Glucose stimulated the oxidation of alanine but depressed that of leucine. These effects of glucose were blocked by mannoheptulose or iodoacetamide but were not influenced by adrenaline, diazoxide, dibutyryl 3':5'-cyclic AMP, or glibenclamide. The rate of alanine oxidation was doubled in the presence of 17mm-pyruvate but was unaffected by citrate or succinate. Succinate depressed the oxidation of leucine. Neither alanine nor leucine significantly affected the oxidation of glucose. It is suggested that the effects of glucose on the oxidation of alanine and leucine were mediated by metabolism of the sugar, and that amino acids do not act as insulin secretagogues by serving as fuels for the beta-cells. The results are consistent with the existence of mechanisms auxiliary to glucose metabolism for control of insulin release.     

The Heterotrophic Nutrition of Chlorella vulgaris (Brannon No. 1 Strain): With two Figures In the Text

A range of sugars, sugar alcohols, sugar phosphates, organicacids, and monohydric alcohols have been tested as carbon sourcesfor growth and as respiratory substrates using Chlorella vulgaris,Brannon I, grown in darkness. Much higher rates of growth and respiration were obtained withd-glucose than with any other substance tested. Ethanol (at0·005 M.) sustained both growth and respiration at c.50 per cent, of the level with glucose (0·028 M. or higher).Evidence was obtained that the organism can become ‘adapted’to utilize d-galactose and sucrose as effective carbon sources.Sustained growth was not obtained with any of the other substancestested. The glucose monophosphates, methanol and certain organic acids(oxalacetate, -ketoglutarate, cis-aconitate, and pyruvate) clearlystimulated oxygen uptake but to a less extent than ethanol.The other substances tested were either inhibitory to respirationor inactive or of very low activity as substrates. The growth in darkness and in liquid culture of Chlorella whensupplied with d-glucose was insensitive to pH over the range4·5 to 7·0 and was markedly enhanced by a highlevel of aeration. Gains in cellular dry weight ranging from45 to 90 per cent, of the weight of d-glucose disappearing fromthe culture medium were recorded in growth experiments; measurementsof CO₂ evolution in the Warburg indicated retention of up totwo-thirds of the glucose-C in cell material.     

The Disappearance of Intermediates involved in Glucose Oxidation during the Starvation of the Fungus Zygorhyncus moelleri

The respiration of carbohydrate-starved cells of Zygorhyncusmoelleri showed a lag period of 2 to 3 hours when glucose wasadded before the rate of oxygen consumption became constant.Analysis of the rates of oxygen uptake from the time of additionof the sugar until they became constant showed that the lagperiod could not be ascribed entirely to a low concentrationof free glucose in the cells in the period immediately followingthe addition of glucose to the medium. The analysis was, however,consistent with the supposition that the synthesis of an intermediarymetabolite on the glucose oxidation pathway was necessary beforethe oxidation could proceed with a maximum speed. The lengthof the lag period could be reduced by adding extracts of cellstogether with glucose; extracts of unstarved cells were moreeffective than those of starved cells in shortening the lagperiod. Various known substances were also effective in thisrespect, acetate and ethanol being the most active. These resultsare discussed together with earlier work on the lag period.     

Studies in the Physiology of Lichens: With one Figure in the Text: Absorption and Utilization of Some Simple Organic Nitrogen Compounds by Peltigera polydactyla

Peltigera discs can accumulate relatively very large amountsof asparagine from solution by a process which involves theentry and accumulation of asparagine in the tissues as the completemolecule. Utilization of absorbed asparagine occurs at a relativelyslow rate and involves the preliminary deamidation of it toammonia. Asparagine absorption is completely inhibited by sodiumfluoride, and partially inhibited by glucose. Absorption isaccompanied by a release of ammonia in the medium and also byincreases in respiration rates and a change in R.Q. None ofthe absorbed asparagine can be washed out of the tissues. Discsalso show strong powers of absorption from solutions of glutamine,and glutamic and aspartic acids. There is more organic thaninorganic nitrogen in solution in the liquids available to thelichen in its habitat. The amounts of glucose and of phosphate which Peltigera andsome other lichens can accumulate from solution are also verylarge considering the slow growth-rates of these plants. Itis suggested that the possession of strong powers of absorptionof substances from solution may be a general characteristicof lichens and plants of similar habitats: it may representan adaptation to existence in barren conditions where the concentrationof nutrients in solution is very low. Utilization of absorbednutrients at a slow rate may be an adaptation to the need tosurvive long periods of starvation. The ecological implicationsof these suggestions are also discussed.     

Sugar Absorption and Surface Carbohydrase Activity of Peltigera polydactyla (Neck.) Hoffm.

A method suitable for the study of the physiology of absorptionof substances from solution and of respiration of Peltigerapolydactyla was elaborated making use of discs of tissue whichcould be floated on solutions. The rate of absorption of hexosesugars by the discs was sensitive to temperature, the presenceof inhibitors, such as sodium fluoride and silver nitrate, butrelatively insensitive to hydrogen ion concentration. Glucosewas absorbed preferentially to fructose from equimolar solutions.Absorption of sugar from sucrose solutions involved inversionby an enzyme system held on the cell surface. This system wasrelatively unaffected by exposure to moderate temperature, tosodium fluoride or silver nitrate but sensitive to hydrogenion concentration. Sucrose did not seem to be absorbed as suchand the glucose was absorbed preferentially from the productsof inversion. All parts of the thallus were equally effectivein inversion but absorption was mainly into the algal layer.The entry into the tissues was associated with a respiratoryrise.     

Succinoxidase and Cytochrome Oxidase in Mitochondria from the Spadix of Arum

1. Mitochondria prepared from young Arum spadix have enoughcytochrome c oxidase to account for the rate at which succinateis oxidized, but succinoxidase activity increases markedly asthe plants mature so that in old material cytochrome oxidaseactivity is only 10 per cent, of succinoxidase. 2. Disintegration of the mitochondria by vibration with ballotini,treatment with digitonin or incubation in the warm reveals anintra-mitochondrial cytochrome c oxidase probably active enoughto account for the fastest rates of succinate oxidation. 3. Succinic dehydrogenase activity is demonstrated and experimentswith p-chloromercuribenzoate indicate that it plays a part inthe oxidation of succinate. 4. Cyanide completely inhibits both external and internal cytochromeoxidase but even at the earliest stages it only reduces succinoxidaseby about 50 per cent. Antimycin A also inhibits succinoxidaseby about 50 per cent.     

Studies on the metabolism of a sulfur-oxidizing bacterium IV.1 Growth and oxidation of sulfur compounds in Thiobacillus thiooxidans

By immersing a few small cellophane bags containing BaCO³ powderin STARKEY's medium, the duration of lag phase in the growthof Thiobacillus thiooxidans is minimized and the yield of cellsis increased ten times that of the previous method. The activitiesof oxidation for sulfur and sulfite change with growth. Sulfiteis oxidized at a comparable rate to that of sulfur oxidationat pH values between 6.0 and 6.5. In the presence of cysteineor glutathione, thiosulfate can be oxidized at a pH above 5.0.At pH values below 4.5, apparent oxidation of thiosulfate andtetrathionate to sulfate is observed. This result is accountedfor by the facts that thiosulfate is decomposed to sulfur andsulfite under the acidic condition at pH values below 4.5, andthat tetrathionate is reduced to thiosulfate enzymatically.In the oxidation of tetrathionate, oxygen uptake begins aftera lag phase, the duration of which depends on the concentrationsof cells and of tetrathionate. Cysteine is oxidized to cystine.The oxidation is strongly inhibited by metal-chelating agents.The cysteine oxidizing activity is, however, quite stable andis not lost by treating cells with organic solvents, sonic oscillation,by heating or lyophilization. ¹III=References (11). ²Partly supported by a grant from the Ministry of Education.     

Wheat Viability at High Moisture Content under Hermetic and Aerobic Storage Conditions

Wheat seeds (Triticum durum) were stored under both hermeticand aerobic conditions at 25 °C with moisture contents from15 to 33 per cent. Under hermetic storage, seeds lost viabilitymore rapidly the higher the moisture content, whereas in aerobicstorage, seed longevity was enhanced as the moisture contentwas increased from 24 to 31 per cent, and over this range ofmoisture content the seeds survived longer under aerobic thanhermetic storage. On the contrary, an apparent reversal of thistrend occurred when moisture content was increased above 31per cent. The possibility that the changes in longevity occurring at highermoisture contents might be due to the activation of seed metabolismwas supported by the enhanced incorporation of [³H]leucine intoTCA insoluble material (indicating increased protein synthesis)and the reduced leakage of glucose (indicating increased membranerestitution) when seeds were allowed to achieve higher moisturecontents during the prehydration period. The highest level ofseed activation was found in seeds preconditioned to about 31per cent moisture content. Moreover, these seeds, when subsequentlystored under aerobic conditions, maintained a higher rate ofprotein synthesis and lower membrane permeability during thestorage period than seeds at lower water contents. It is suggested that seeds stored at a sufficient hydrationlevel in the presence of oxygen can sustain an effective metabolismduring extended storage, thus permitting the repair of cellulardamage. However, it might be possible that at about 33 per centmoisture content seed could suffer from an excessive advancementof metabolism. Triticum durum, seed storage, effects of high moisture content and oxygen     

Production of carbonyl reductase by Geotrichum candidum in a laboratory scale bioreactor

Fungal fermentation is very complex in nature due to its nonlinear relationship with the time, especially in batch culture. Growth and production of carbonyl reductase by Geotrichum candidum NCIM 980 have been studied in a laboratory scale stirred tank bioreactor at different pH (uncontrolled and controlled), agitation, aeration and dissolved oxygen concentration. The yield of the process has been calculated in terms of glucose consumed. Initial studies showed that fermenter grown cells have more than 15 times higher activity than that of the shake flask grown cells. The medium pH was found to have unspecific but significant influence on the enzyme productivity. However, at controlled pH 5.5 the specific enzyme activity was highest (306U/mg). Higher agitation had detrimental effect on the cell mass production. Dissolved oxygen concentration was maintained by automatic control of the agitation speed at an aeration rate of 0.6 volume per volume per minute (vvm). Optimization of glucose concentration yielded 21g/l cell mass with and 9.77x10(3)U carbonyl reductase activity/g glucose. Adaptation of different strategies for glucose feeding in the fermenter broth was helpful in increasing the process yield. Feeding of glucose at a continuous rate after 3h of cultivation yielded 0.97g cell mass/g glucose corresponding to 29.1g/l cell mass. Volumetric oxygen transfer coefficient (K(L)a) increased with the increasing of agitation rate.     

Effect of pH and Monensin on Glucose Transport by Fibrobacter succinogenes, a Cellulolytic Ruminal Bacterium

Fibrobacter succinogenes S85, a cellulolytic ruminal bacterium, required sodium for growth and glucose uptake. Cells which were deenergized with iodoacetate (500 μM) could not take up [¹⁴C]glucose. However, deenergized cells which were treated with valinomycin, loaded with potassium, and diluted into sodium or sodium plus potassium to create an artificial electrical gradient (ΔΨ) plus a chemical gradient of sodium (ΔpNa) or ΔpNa alone transported glucose at a rapid rate. Cells which were loaded with potassium plus sodium and diluted into sodium (ΔΨ with sodium, but no ΔpNa) also took up glucose at a rapid rate. Potassium-loaded cells that were diluted into buffers which did not contain sodium (ΔΨ without sodium) could not take up glucose. An artificial ZΔpH which was created by acetate diffusion could not drive glucose transport even if sodium was present. The maximum rate and affinity of glucose transport (pH 6.7) were 62.5 nmol/mg of protein per min and 0.51 mM, respectively. S85 was unable to grow at a pH of less than 5.5, and there was little glucose transport at this pH. When the extracellular pH was decreased, the glucose carrier was inhibited, intracellular pH declined, the cells were no longer able to metabolize glucose, and ΔΨ declined. Monensin (1 μM) or lasalocid (5 μM) decreased intracellular ATP and dissipated both the ΔΨ and ΔpNa. Since there was no driving force for transport, glucose transport was inhibited. These results indicated that F. succinogenes used a pH-sensitive sodium symport mechanism to take up glucose and that either a ΔΨ or a ΔpNa was required for glucose transport.     

Ribonuclease and Other Factors Involved in the Respiratory Senescence of Maize Scutellum

The scutella from seedlings of Zea mays L. germinated at 28–30°C increase in respiration rate to the third day, followed bya decline which is quite noticeable by the fifth day. A searchhas been made for factors responsible for the respiratory decline.The electronmicroscope shows the five-day mitochondria to benormal in appearance. Very active preparations are obtainedby isolating the mitochondria at pH 7.6 with inclusions of bovineserum albumin and ethylenediamine-tetraacetic acid. A solubleribonuclease (RNase A) which increases rapidly with age impairsboth oxidation and phosphorylation. The largely particle-boundribonuclease (RNase B) is not inhibitory. Plant ribonucleaseis resistant to the proteolysis occurring during senescence.It is suggested that the soluble ribonuclease contributes tothe respiratory decline, but that other factors may also beinvolved.     

The metabolic properties of spermatozoa from the epididymis of the tammar wallaby,Macropus eugenii

The utilization of various substrates by sperm from the cauda epididymidis of the tammar was examined because the major naturally occurring sugar in the semen of this species is N-acetyl-D-glucosamine (NAG) and not fructose, as in eutherian mammals. The sperm displayed a high level of endogenous respiration that supported motility for relatively prolonged periods of time in vitro. They also metabolised exogenous ¹⁴C-labelled glucose, NAG, sucrose, and acetate through glycolytic and/or oxidative processes to produce lactate and ¹⁴CO₂ at varying rates. The rate of uptake of NAG by tammar sperm was about four times greater than that of other substrates. Glucose and/or NAG stimulated the rate of oxygen consumption by about 20%, but acetate stimulated oxygen consumption by more than 40%. The most striking findings were that NAG almost completely inhibited the oxidation of glucose and sucrose by the sperm and depressed the uptake of glucose, 3-O-methylglucose, and sucrose. Acetate oxidation also was inhibited by NAG, but only by about 50%. Tammar sperm generated substantial amounts of free glucose during incubation with NAG, but this and the inhibitory effects of NAG on glucose oxidation were not mimicked by rat sperm. It is proposed that tammar sperm fail to oxidise glucose in the presence of NAG because of the rapid cellular uptake of NAG relative to glucose. Also, the intracellular glucose and acetate liberated from NAG would compete with exogenous glucose for processing in the Embden-Meyerhof and tricarboxylic acid (TCA) cycle pathways. It is also suggested that tammar sperm oxidise sucrose after extracellular hydrolysis into its glucose and fructose components. The biological implications of these metabolic and transport properties of tammar sperm have as yet to be determined. Mol. Reprod. Dev. 49:92–99, 1998. © 1998 Wiley-Liss, Inc.     

The effect of digitoxose on insulin release, glucose oxidation and oxygen consumption by islets of lean and genetically obese diabetic (ob/ob) mice

Digitoxose specifically and competitively inhibited glucose stimulated insulin release from islets of both lean and obese mice without affecting either the rate of glucose oxidation or the rate of glucose stimulated oxygen consumption. Obese mouse islets were marginally more resistant to the inhibitory effect of digitoxose than lean mouse islets. Digitoxose provides a means for dissociating glucose stimulated insulin release by isolated islets from their metabolism of glucose confirming that glucose metabolism per se is not a necessary prerequisite for the initiation of insulin release but is required to fuel the insulin secretory process.