Iodoacetate inhibits the biosynthesis of alanine in glial cells and its utilization in photoreceptors of the honeybee drone (Apis mellifera) retina

The aim of this work was to study the effects of iodoacetate on the metabolism of the honeybee drone retina. In the superfused retina, iodoacetate only at high concentration (3 mmol·1^-1) causes a 77% decrease in the O₂ consumption induced by a flash of light. Chromatographic analysis showed that 3 mmol·1^-1 iodoacetate strongly inhibited glycolysis in the retinal glial cells and consequently suppressed the biosynthesis of alanine, which is the fuel transferred from the glia to the photoreceptors. However, the synthesis of ¹⁴C-alanine from [1-¹⁴C]-pyruvate was not affected by iodoacetate. It was therefore surprising to find that superfusion of the retina with 10 mmol·1^-1 pyruvate had no protective effect on the decrease in O₂ consumption, and that the ¹⁴CO₂ production from [1-¹⁴C]-pyruvate was inhibited 60% by iodoacetate. Also, no protection from the effect of iodoacetate was obtained by adding 10 and 20 mmol·1^-1 alanine in the superfusate, even though the transport of ¹⁴C-alanine in the photoreceptor cells was not significantly affected by 3 mmol·1^-1 iodoacetate. However, exposure to iodoacetate strongly inhibited the production of ¹⁴C-glutamate from ¹⁴C-alanine. In contrast, the transformation of ¹⁴C-proline to ¹⁴C-glutamate was not affected by iodoacetate. Indeed, in the presence of iodoacetate, photostimulation caused a decrease in the total concentration of proline and glutamate. It appears therefore that 3 mmol·1^-1 iodoacetate inhibits not only glycolysis and, consecutively, the formation of alanine, but also its use in the photoreceptors. Possibly a large intracellular store of proline, whose mitochondrial use was not affected, contributed in slowing down the inhibition of O₂-consumption by iodoacetate.Abbreviations DNP dinitrophenol - HPLC high pressure liquid chromatography - IAA iodoacetate - QO ₂ change in oxygen consumption - QO ₂ oxygen consumption - PO ₂ partial pressure of O₂     

Penicillin recovery from aqueous solutions by continuous foam flotation

Summary Penicillin G recovery is investigated in a continuous flotation column in the presence of different collectors which form a complex with penicillin. The performance of the penicillin recovery was investigated as a function of the mole ratio () of collector-to-penicillin and the aliphatic chain length of the collector. At =1 and low penicillin concentrations (e.g., 20 mg·1^-1), high foam liquid concentrations (680 mg·l^-1), low residue concentrations (12 mg·l^-1) and high penicillin separation (56) can be attained. At =4 the separation increases to 150, and 95% of the penicillin can be recovered.Symbols C_p penicillin concentration in feed (mg·l^-1) - C_R penicillin concentration in outlet liquid (mg·l^-1) - C_S penicillin concentration in foam liquid (mg·l^-1) - C_S/C_P penicillin enrichment (-) - C_S/C_R penicillin separation (-) - % Pen in S penicillin yield in foam liquid (%) - VV}_S foam liquid volume flow (ml·min^-1) - VV}_P feed (ml·min^-1) - VV_{N 2} nitrogen flow rate (ml·s^-1) - temperature     

Biochemical documentation of allelic variation of the I-E antigens of the d,k, p,r, and u haplotypes

The extent of allelic variation of the E and E polypeptide chains of the I-E antigens from the H-2> ^d ,H-2 ^k , H-2 ^p , H-2 ^r , and H-2 ^u haplotypes is described. E and E chains were individually labeled with arginine or lysine and compared by tryptic peptide analysis. The results indicate minimum variability among the E polypeptides encoded by the d, k, p, and r haplotypes. However, the E ^u chain differed significantly from the other allelic E gene products. On the other hand, the E alleles demonstrated substantial variability with the E ^d being notably less similar to the other alleles than they are to each other. These findings are consistent with a number of observations regarding the serology and functions of the I-E antigens.Abbreviations MHC major histocompatibility complex - NMS normal mouse serum - NP-40 Nonidet P-40 - NTS 0.25% NP-40, 10 mM Tris-Cl, 0.15 M NaCl (pH 7.4) - SDS sodium dodecylsulfate - SDS-PAGE polyacrylamide gel electrophoresis in the presence of SDS     

Is there a chloride ATPase in the gills of the shore crab Carcinus maenas?

Summary In gills of the shore crab Carcinus maenas an ATPase activity was found which was stimulated by bicarbonate and inhibited by low concentration of oligomycin and thiocyanate. This ATPase was activated by small hydrated alkali cations, i.e., activation was absent in the presence of Li⁺, small in the presence of Na⁺, and highest in the presence of K⁺ (K _m=4 mM). Inhibitor studies using ouabain, NEM, and vanadate suggest that this ATPase is different from (Na⁺+K⁺)-ATPase, the H⁺-ATPase of organelles, or an E ₁ E ₂-type ATPase represented by the H⁺/K⁺-ATPase in gastric mucosa. Results obtained by differential and density gradient centrifugation indicate that this ATPase is located in crab gill mitochondria, a location ruling out its direct participation in transepithelial ion transport. Since the ATPase lacked specific Cl^--activation it is not considered to be a Cl^- pump but a mitochondrial F ₁ F ₀-ATPase. Specific activities of mitochondrial ATPase and (Na⁺+K⁺)-ATPase were of comparable magnitude. Both ATPases were greatly increased in gills of crabs acclimated to brackish water (salinity 10) compared to crabs maintained in sea water (30). These results imply that low salinity-induced modifications in branchial tissues include mechanisms for active ion uptake as well as the elements for provision of cellular energy.Abbreviations ATPase adenosine triphosphatase - HEPES N-(2-hydroxyethyl)-1-piperazine-N(2-ethanesulfonic acid) - LDH lactate dehydrogenase - NADH reduced nicotinamide adenine dinucleotide - NEM Niethylmaleimide - PEP phosphoenolpyruvate - PK pyruvate kinase - TRIS TRIS (hydroxymethyl)aminomethane - S salinity     

Effect of divalent cations on the short-term NH 4 + inhibition of nitrogen fixation in Azotobacter chroococcum

Incubation of Azotobacter chroococcum in the presence of micromolar concentrations of MnCl₂, but not MgCl₂, prevented nitrogenase activity from NH ₄ ⁺ inhibition. Mg(II), at a 100-fold concentration with respect to Mn(II), counteracted the protective effect of Mn(II) on nitrogenase activity. When Mn(II) was added to cells that had been given NH₄Cl, stopping of NH ₄ ⁺ uptake and recovery of nitrogenase activity took place, and a raise of NH ₄ ⁺ concentration in medium developed. Furthermore, incubation of A. chroococcum cells with 20 M Mn(II) under air, but not under an argon: oxygen (79%:21%) gas mixture, resulted in NH ₄ ⁺ excretion to the external medium. The Mn(II)-mediated uncoupling of nitrogen fixation from ammonium assimilation leads us to conclude that Mn(II) may act as a physiological inhibitor of glutamine synthetase.Abbreviations Hepes N-2-Hydroxyethylpiperazine-N-ethanesulfonic acid - Mops 3-(N-Morpholino)propanesulfonic acid     

Kinetics of inhibition in propionic acid fermentation

The inhibitory effect of propionic acid P and biomass concentration X is studied in batch and continuous fermentations with cell recycle.In batch fermentations, the specific growth rate decreases and cancels out at a critical propionic acid concentration Pc ₁; the formerly decreasing specific production rate becomes constant after Pc ₁ and cancels out when a second critical propionic acid concentration Pc ₂ is reached.In continuous fermentation with cell recycle, a similar inhibition is observed with biomass. The specific rates decrease and become constant at a critical biomass concentration Xc. They cancel out at different high biomass concentrations.In both cases, the specific production rate can be related to the specific growth rate by the Luedeking and Piret expression: =+, [1], where the constants and are determined by the fermentation parameters.List of Symbols t h time - X kg/m³ biomass concentration - P kg/m³ propionic acid concentration - A kg/m³ acetic acid concentration - S kg/m³ lactose concentration - dX/dt kg/(m³h) instantaneous rate of cell growth - dP/dt kg/(m³h) instantaneous rate of propionic acid production - h^–1 specific growth rate - h^–1 specific propionic acid production rate - D h^–1 dilution rate     

PO2 and pH changes in the retina of the crab Ocypode ryderi: evidence for aerobic glycolysis

Summary The isolated retina of the terrestrial crab Ocypode ryderi exhibits a pronounced lactate production in spite of being supplied with sufficient O₂ (140 torr). To determine whether this lactate production is caused by hypoxic areas in the tissue or represents aerobic glycolysis, oxygen partial pressure and pH measurements with two-channel glass microelectrodes and additional biochemical analyses were carried out on this organ. Distinct profiles were obtained for O₂ partial pressure and pH inside the tissue. At a depth of 200 m different O₂ partial pressure levels could be observed depending on the O₂ partial pressure in the medium (85 torr at 280 torr and 36 torr at 130 torr, respectively). The extracellular pH displays a similar pattern; it reaches a stable value of 7.15 at 100 m inside the tissue. Lowering bath O₂ partial pressure from 280 torr to about 15 torr (hypoxia) induces a decrease of the O₂ partial pressure in the tissue with different time-courses for different tissue depths. However, hypoxia did not change the extracellular pH. Addition of antimycin A (100 mol · 1^-1) to the medium abolishes the O₂ partial pressure gradient and the delayed recovery of the tissue O₂ partial pressure after hypoxia. These results and the biochemical data suggest that in the crab retina a high glycolytic activity occurs simultaneously with oxydative carbohydrate degradation (aerobic glycolysis).Abbreviations AEC Atkinson energy charge - DC bioelectric potential - dw dry weight - HEPES N-[2-Hydroxyethyl]piperazine-N-[2-ethanesulphonic acid] - PCO₂ carbon dioxide partial pressure - PO₂ oxygen partial pressure - P _tO₂ oxygen partial pressure inside the tissue - P _mO₂ oxygen partial pressure in the medium - pH_t pH inside the tissue - pH_m pH in the superfusion medium     

Leaf and canopy responses to elevated CO2 in a pine forest under free-air CO2 enrichment

Physiological responses to elevated CO₂ at the leaf and canopy-level were studied in an intact pine (Pinus taeda) forest ecosystem exposed to elevated CO₂ using a free-air CO₂ enrichment (FACE) technique. Normalized canopy water-use of trees exposed to elevated CO₂ over an 8-day exposure period was similar to that of trees exposed to current ambient CO₂ under sunny conditions. During a portion of the exposure period when sky conditions were cloudy, CO₂-exposed trees showed minor (7%) but significant reductions in relative sap flux density compared to trees under ambient CO₂ conditions. Short-term (minutes) direct stomatal responses to elevated CO₂ were also relatively weak (5% reduction in stomatal aperture in response to high CO₂ concentrations). We observed no evidence of adjustment in stomatal conductance in foliage grown under elevated CO₂ for nearly 80 days compared to foliage grown under current ambient CO₂, so intrinsic leaf water-use efficiency at elevated CO₂ was enhanced primarily by direct responses of photosynthesis to CO₂. We did not detect statistical differences in parameters from photosynthetic responses to intercellular CO₂ (A _net-C _i curves) for Pinus taeda foliage grown under elevated CO₂ (550 mol mol^–1) for 50–80 days compared to those for foliage grown under current ambient CO₂ from similar-sized reference trees nearby. In both cases, leaf net photosynthetic rate at 550 mol mol^–1 CO₂ was enhanced by approximately 65% compared to the rate at ambient CO₂ (350 mol mol^–1). A similar level of enhancement under elevated CO₂ was observed for daily photosynthesis under field conditions on a sunny day. While enhancement of photosynthesis by elevated CO₂ during the study period appears to be primarily attributable to direct photosynthetic responses to CO₂ in the pine forest, longer-term CO₂ responses and feedbacks remain to be evaluated.     

Cutaneous ion exchange,and renal and extrarenal partitioning of acid and ammonia excretion in the larval tiger salamander,Ambystoma tigrinum,following ingestion of ammonium salts

Summary The skin/gills and the kidneys of aquatic amphibians are potential sites of acid-base regulation. The roles of these organs in acid-base balance were examined in larval Ambystoma tigrinum following gastric infusion of ammonium salts. A single dose of 1.75 mEq NH₄Cl·100 g^-1 produced a mixed acidosis by 1 h after gavage. By 8 h after ingestion, pH and HCO ₃ ^– had increased and PCO₂ had decreased as the animals recovered. A prolonged acidosis was developed in a second group by gavage of an initial dose (1.5 mEq·100 g^-1), followed by periodic maintenance doses (0.25 mEq·100 g^-1) to prolong the disturbance for 8 h. The magnitude of the acidosis during this period was similar to that seen at 1 h after ingestion in the time-course study. A third group of larvae were given NaCl as a control for salt loading, which induced a small but significant respiratory acidosis. Unidirectional fluxes of Na⁺ and Cl^- were examined during these serial ingestions. Salt loading inhibited the influx of the ingested ion. Na⁺ influx increased during the NH₄Cl-induced acidosis. A fourth group of larvae were used to partition acid and ammonia excretion between the skin and the kidneys. These animals were given (NH₄)₂SO₄ to allow re-examination of Cl^- flux rates under non-Cl^--loaded conditions. The ensuing acidosis had a reduced respiratory component and, therefore, pH did not decrease as much. Cl^- influx rates did decrease significantly under these conditions. In both control and acidotic conditions, the majority of the acid efflux was as ammonia and the skin was the primary site of acid excretion. However, both the skin and the kidneys increased total acid excretion, although the efflux across the skin showed a much greater increase. This suggests a primary role for the skin in acid-base regulation in aquatic amphibians.Abbreviations GFR glomerular filtration rate - PO₂ partial pressure of oxygen - PCO₂ partial pressure of carbon dioxide - SITS 4-acetamido-4-isothiocynanatostilbene-2,2-disulfonic acid - TA titratible acidity Present address: Department of Organismal Biology and Anatomy, University of Chicago, 1025 E. 57th St., Chicago, IL 60637, USA     

Different ammonium-ion uptake,metabolism and detoxification efficiencies in two Lemnaceae

¹⁵N-Nuclear magnetic resonance spectroscopy was used to follow nitrogen assimilation and amino-acid production in Wolffia arrhiza (L.) Hork. ex. Wimmer, clone Golan exposed to 4.0 mM ¹⁵NH₄Cl solutions for 24 h. The main ¹⁵N-labelled metabolites were asparagine and glutamine, as well as substantial amounts of unreacted, intracellular NH ₄ ⁺ . These results were compared with those of a previous study on Lemna gibba L. clone Hurfeish (Monselise et al., 1987, New Phytol. 10, 341–345) with regard to NH ₄ ⁺ uptake, assimilation and detoxification efficiencies. Both species, grown under continuous white light, were capable of preferential uptake of NH ₄ ⁺ in the presence of nitrate. Relative growth rates indicate that both species tolerate increased levels of NH ₄ ⁺ , up to 10^–2 mol · 1^–1, with L. gibba showing a slightly greater tolerance. No ¹⁵N-labelled free NH ₄ ⁺ was detectable in L. gibba, while in W. arrhiza excess NH ₄ ⁺ was found within the cells. This fact indicates that L. gibba is more efficient in detoxification than W. arrhiza, presumably because of inability of W. arrhiza to regenerate the NH ₄ ⁺ traps, glutamate and aspartate, rapidly enough. This is also evident from the observation that addition of -ketoglutarate to the medium caused nearly complete assimilation of intracellular NH ₄ ⁺ in W. arrhiza. In both plants, addition of -ketoglutarate increased both NH ₄ ⁺ uptake and assimilation. Addition of l-methionine dl-sulfoximine, an inhibitor of glutamine synthetase inhibited NH ₄ ⁺ assimilation, while addition of azaserine, an inhibitor of glutamate synthase, resulted in ¹⁵N incorporation into the glutamine-amide position only. These results are consistent with the glutamine synthetase-glutamate synthase pathway being the major route of NH ₄ ⁺ assimilation in the two plants under the conditions used.Abbreviations AZA azaserine (O-diazoacetyl-l-serine) - GOGAT glutamine oxoglutarate amine transferase=]glutamate synthase E.C. 1.4.7. and E.C. 1.4.1.13. - GS glutamine synthetase E.C. 6.3.1.2. - -KG -ketoglutarate=2-oxoglutarate - MSO l-methionine dl-sulphoximine - NMR nuclear magnetic resonance - RGR relative growth rate This article is dedicated to Professor Bernhard Schrader on the occasion of his 60th birthdayWe wish to thank Professor Robert Glaser for helpful discussions, and Mrs. Aliza Levkoviz and Mr. Gideon Raziel for skillful assistance.     

Effects of dinitrophenol and oligomycin on the coupling between anaerobic metabolism and anaerobic sodium transport by the isolated turtle bladder

Dinitrophenol (1 x 10^-5 M) has been found to inhibit anaerobic sodium transport by the isolated urinary bladder of the fresh water turtle. Concurrently, anaerobic glycolysis was stimulated markedly. However, tissue ATP levels diminished only modestly, remaining at approximately 75% of values observed under anaerobic conditions without DNP. The utilization of glucose (from endogenous glycogen) corresponded closely to that predicted from the molar quantities of lactate formed. Thus the glycolytic pathway was completed in the presence of DNP and if ATP were synthesized normally during glycolysis, synthesis should have been increased. On the other hand, the decrease in Na transport should have decreased ATP utilization. Oligomycin did not block sodium transport either aerobically or anaerobically, but ATP concentrations did decrease. When anaerobic glycolysis was blocked by iodoacetate, pyruvate did not sustain sodium transport thus suggesting that no electron acceptors were available in the system. Two explanations are entertained for the anaerobic effect of DNP: (a) Stimulation by DNP of plasma membrane as well as mitochondrial ATPase activity; (b) inhibition of a high energy intermediate derived from glycolytic ATP or from glycolysis per se. The arguments relevant to each possibility are presented in the text. Although definitive resolution is not possible, we believe that the data favor the hypothesis that there was a high energy intermediate in the anaerobic system and that this intermediate, rather than ATP, served as the immediate source of energy for the sodium pump.     

Seasonal variation in the microbial contamination of game carcasses in an Austrian hunting area

In 2003, 50 game carcasses (ungulates) originating from one Austrian hunting ground were subject to visual examination for (fecal) contamination of the body cavities and microbiological testing of the body cavities in order to assess variations in microbial surface contamination in the season June–August compared to October–December. No carcass tested positive for the bacterial pathogens Salmonella or Listeria. Bacterial surface counts in October–December (median values: total aerobic count: 4.12 log₁₀ colony-forming-units (cfu)/cm²; Enterobacteriaceae: 2.48 log₁₀ cfu/cm²) were significantly lower than those in June–August (median values: total aerobic count: 5.65 log₁₀ cfu/cm²; Enterobacteriaceae: 3.45 log₁₀ cfu/cm²). The cooling regime (0.4 °C, 62% relative humidity) allowed no microbial growth for 96 h but was associated with weight loss of the carcasses. All carcasses had undergone a precooling phase of 8–12 h, with temperatures of 17.8±1.2 °C in the season June–August and 9.8±1.2 °C in October–December. This temperature difference was identified as the most probable effector for the observed seasonal variation. The results demonstrate the need for a continuous cool chain after evisceration of game carcasses.     

Liver metabolism in cold hypoxia: a comparison of energy metabolism and glycolysis in cold-sensitive and cold-resistant mammals

The effects of cold hypoxia were examined during a time-course at 2 °C on levels of glycolytic metabolites: glycogen, glucose, glucose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, phosphoenolpyruvate, pyruvate, lactate and energetics (ATP, ADP, AMP) of livers from rats and columbian ground squirrels. Responses of adenylate pools reflected the energy imbalance created during cold hypoxia in both rat and ground squirrel liver within minutes of organ isolation. In rat, ATP levels and energy charge values for freshly isolated livers were 2.54 mol·g^-1 and 0.70, respectively. Within 5 min of cold hypoxia, ATP levels had dropped well below control values and by 8 h storage, ATP, AMP, and energy charge values were 0.21 mol·g^-1, 2.01 mol·g^-1, and 0.17, respectively. In columbian ground squirrels the patterns of rapid ATP depletion and AMP accumulation were similar to those found in rat. In rat liver, enzymatic regulatory control of glycolysis appeared to be extremely sensitive to the decline in cellular energy levels. After 8 h cold hypoxia levels of fructose-6-phosphate decreased and fructose-1,6-bisphosphate increased, thus reflecting an activation of glycolysis at the regulatory step catalysed by phospho-fructokinase fructose-1,6-bisphosphatase. Despite an initial increase in flux through glycolysis over the first 2 min (lactate levels increased 3.7 mol·g^-1), further flux through the pathway was not permitted even though glycolysis was activated at the phosphofructokinase/fructose-1,6-bisphosphatase locus at 8 h, since supplies of phosphorylated substrate glucose-1-phosphate or glucose-6-phosphate remained low throughout the duration of the 24-h period. Conversely, livers of Columbian ground squirrels exhibited no activation or inactivation of two key glycolytic regulatory loci, phosphofructokinase/fructose-1,6-bisphosphatase and pyruvate kinase/phosphoenolpyruvate carboxykinase and pyruvate carboxylase. Although previous studies have shown similar allosteric sensitivities to adenylates to rat liver phospho-fructokinase, there was no evidence of an activation of the pathway as a result of decreasing high energy adenylate, ATP or increasing AMP levels. The lack of any apparent regulatory control of glycosis during cold hypoxia may be related to hibernator-specific metabolic adaptations that are key to the survival of hypothermia during natural bouts of hibernation.Abbreviations DHAP dihydroxyacetonephosphate - EC energy charge - F1,6P₂ fructose-1,6-bisphosphate - F2,6P₂ fructose-2,6-bisphosphate - F6P fructose-6-phosphate - FBP fructose-1,6-bisphosphatase - G1P glucose-1-phosphate - G6P glucose-6-phosphate - GAP glyceraldehyde-3-phosphate - GAPDH glyceraldehyde-3-phosphate dehydrogenase - L/R lactobionate/raffinose-based solution - MR metabolic rate - PDH pyruvate dehydrogenase - PEP phosphoenolpyruvate - PEPCK & PC phosphoenolpyruvate carboxykinase and pyruvate carboxylase - PFK phosphofructokinase; PK, pyruvate kinase - Q ₁₀ the effect of a 10 °C drop in temperature on reaction rates (generally, Q ₁₀=2–3) - TA total adenylates - UW solution University of Wisconsin solution (L/R-based)     

Blood-gas,acid-base and catecholamine responses to lactic acid infusion in larval and adult Ambystoma tigrinum

Larval and adult Ambystoma tigrinum were subjected to acidosis by infusing lactic acid (2 M·g^-1) into the peritoneal cavity. Blood was sampled at intervals to establish the time-course of the ensuing acidosis. Both larvae and adults became significantly acidotic after 1 h. The larval acidosis was more pronounced (-4 pH units versus-2 pH units) than adults due to greater extracellular buffering capacity (higher [HCO₃ ^-]) in adults. Both forms recovered in about 8 h. Larvae showed a typical increase in circulating norepinephrine during the initial stages of the acidosis; adults did not, having significantly lower norepinephrine titer than larvae during the acidosis. Both larvae and adults showed transient increases in PO₂ during the acidosis. The ₁ and ₂ antagonists, timolol and butoxamine respectively, (0.2 g·g^-1) were administered to separate groups of larvae. Butoxamine (₂) delayed the recovery from the acidosis by prolonging the increase in arterial PCO₂ and reversing the recovery of [HCO₃ ^-]. Timolol (₁) did not delay recovery. We conclude that ₂ receptors are involved in the catecholamine responses to acidosis in larvae. Catecholamines appear not to play the same role in adult acid-base disturbances as they seem to in larvae.Abbreviations RBC red blood cell     

Degradation of diphenylether by Pseudomonas cepacia Et4: enzymatic release of phenol from 2,3-dihydroxydiphenylether

2,3-Dihydroxybiphenyl dioxygenase from Pseudomonas cepacia Et 4 was found to catalyze the ring fission of 2,3-dihydroxydiphenylether in the course of diphenylether degradation. The enzyme was purified and characterized. It had a molecular mass of 240 kDa and is dissociated by SDS into eight subunits of equal mass (31 kDa). The purified enzyme was found to be most active with 2,3-dihydroxybiphenyl as substrate and showed moderate activity with 2,3-dihydroxydiphenylether, catechol and some 3-substituted catechols. The K _m-value of 1 M for 2,3-dihydroxydiphenylether indicated a high affinity of the enzyme towards this substrate. The cleavage of 2,3-dihydroxydiphenylether by 2,3-dihydroxybiphenyl dioxygenase lead to the formation of phenol and 2-pyrone-6-carboxylate as products of ring fission and ether cleavage without participation of free intermediates. Isotope labeling experiments carried out with ¹⁸O₂ and H₂ ¹⁸O indicated the incorporation of ¹⁸O from the atmosphere into the carboxyl residue as well as into the carbonyl oxygen of the lactone moiety of 2-pyrone-6-carboxylate. Based on these experimental findings the reaction mechanism for the formation of phenol and 2-pyrone-6-carboxylate is proposed in accordance with the mechanism suggested by Kersten et al. (1982).Non-standard abbreviations DPE diphenylether - 2,3-dihydroxy-DPE 2,3-dihydroxydiphenylether - PCA 2-pyrone-6-carboxylic acid - 2,3-dihydroxy-BP dioxygenase 2,3-dihydroxybiphenyl dioxygenase - GC gas chromatography     

The effect of hypoxia on carbohydrate metabolism in flounder (Platichthys flesus L.)—II. High energy phosphate compounds and the role of glycolytic and gluconeogenetic enzymes

• 1.1. ATP, ADP, AMP, energy charge potential and total adenylates in heart, kidney and muscle are relatively unaffected by environmental hypoxia. In the liver, hypoxia causes a 90% drop in ATP, a rise in ADP and AMP, and a drop in energy charge potential and total adenylates. In the muscle tissue ATP concentration is stabilized by a large creatine phosphate pool.
• 2.2. Hexokinase activity in the heart is 20 times higher than in the swimming muscle, and thus the heart has a high potential for utilizing exogenous glucose as an anaerobic substrate.
• 3.3. The role of creatine phosphate in regulating muscle glycolysis is discussed on background of the strong inhibition of muscle phosphofructokinase by physiological concentrations of creatine phosphate.
• 4.4. Flounder heart has a dominating M-type lactate dehydrogenase which is identical to the muscle enzyme by electrophoretic and kinetic criteria. This improves the anaerobic capabilities of the flounder heart compared to other fish hearts.
• 5.5. Both liver and kidney have high activities of the gluconeogenetic enzymes glucose-6-phosphatase, fructose-1,6-diphosphatase, and phosphoenolpyruvate carboxykinase and both are capable of synthesizing glucose from [¹⁴C]lactate. Because of more favorable energy conditions in the kidney this organ may substitute the liver as a gluconeogenetic organ during hypoxia.

     

The menaquinol oxidase of Bacillus subtilis W23

The quinol oxidase appears to be mainly responsible for the oxidation of bacterial MKH₂ in Bacillus subtilis W23 growing with either glucose or succinate. The activity of the enzyme was maximum with dimethylnaphthoquinol, a water-soluble analogue of the bacterial menaquinol. Menadiol or duroquinol were less actively respired, and naphthoquinol was not oxidized at all. After fourtyfold purification the isolated enzyme contained 5.3 mol cytochrome aa ₃ per gram of protein and negligible amounts of cytochrome b and c. The turnover number based on cytochrome aa ₃ was about 10³ electrons · s^-1 at pH 7 and 37°C. The preparation consisted mainly of a M _r 57000 and a M _r 36000 polypeptide. The N-terminal amino acid sequence of the latter polypeptide differed from that predicted by the qoxA gene of B. subtilis strain 168 (Santana et al. 1992), in that asp-14 predicted by qoxA was missing in the M _r 36000 polypeptide.Abbreviations DMN 2,3-dimethyl-1,4-naphthoquinone - DMNH₂ 2,3-dimethyl-1,4-naphthoquinol - Duroquinol 2,3,5,6-tetramethyl-1,4-benzoquinol - MK menaquinone - MKH₂ menaquinol - NBH₂ 2,3-dimethoxy-5-methyl-6-(n-nonyl)-1,4-benzoquinol - TMPD N,N,N, N,-tetramethyl-1,4-phenylenediamine     

Genetic manipulation of 6-phosphofructokinase in potato tubers

The aim of this work was to discover whether genetic manipulation of 6-phosphofructokinase [EC 2.7.1.11; PFK(ATP)] influenced the rate of respiration of tuber tissue of Solanum tuberosum L. Transgenic plants were produced that contained the coding sequence of the Escherichia coli pfkA gene linked to a patatin promoter. Expression of this chimaeric gene in tubers resulted in a 14to 21-fold increase in the maximum catalytic activity of PFK(ATP) without affecting the activities of the other glycolytic enzymes. Tubers, and aged disks of tuber tissue, from transformed plants showed no more than a 30% fall in the content of hexose 6-monophosphates; the other intermediates of glycolysis increased threeto eightfold. Fructose-2,6-bisphosphate was barely detectable in aged disks of transformed tubers. The relative rates of ¹⁴CO₂ production from [1-¹⁴C]-and [6-¹⁴C]-glucose supplied to disks of transformed and control tubers were similar. Oxygen uptake and CO₂ production by aged disks of transformed tubers did not differ significantly from those from control tubers. The same was true of CO₂ production, in air, and in nitrogen, for tuber tissue. It is concluded that PFK(ATP) does not dominate the control of respiration in potato tubers.Abbreviations Fru2,6bisP fructose-2,6-bisphosphate - FW freshweight - GUS -glucuronidase - PFK(ATP) 6-phosphofructokinase - PFK(PPi) pyrophosphate: fructose-6-phosphate 1-phosphotransferase     

Acid-base and electrolyte regulation,and haemolymph gas transport in crayfish,Astacus astacus,exposed to soft,acid water with and without aluminium

Summary Intermoult crayfish (Astacus astacus) were exposed to acid (pH 4), soft water ([Ca⁺⁺]=100 mol·l^–1) in the absence and presence of aluminium (25 mol·l^–1) for variable time periods (up to 21 days) in order to assess the consequences for acid-base and electrolyte balance and haemolymph gas transport. Haemolymph osmolality and concentration of major ions decreased drastically and to a similar extent in acid and acid-aluminium water. Muscle tissue ion concentrations were, however, regulated at an almost constant level. A severe metabolic acidosis was gradually developed, attaining a haemolymph metabolic acid load of 6–7 mequiv·l^–1 after 12–21 days. The acidosis was partially compensated by ventilatory means, with the postbranchial haemolymph PCO₂ decreasing earlier in acidaluminium-exposed than in acid-exposed specimens. Hyperventilation seemed to be a direct acid-base regulatory response, since the rise in postbranchial PCO₂ had only minimal influence on haemolymph O₂ transport. The Bohr effect of Astacus astacus haemocyanin was low (log P₅₀/GdpH=-0.24), and the mean P₅₀ only increased from 15 to 19 mmHg after 21 days of acid exposure. The decrease in O₂ affinity with decreasing pH was accompanied by a decrease in the cooperativity of O₂ binding. The haemolymph haemocyanin concentration was not affected by acid and acid-aluminium exposure, but decreased after 21 days due to starvation. Muscle tissue aluminium concentrations were unaffected, whereas gill tissue concentrations increased in acid-aluminium exposed crayfish, most likely due to accumulation of aluminium on the gill surface. Mortality was low, and an internal hypoxia and lactacidosis was not developed in either of the experimental groups. This suggests that the gas transfer qualities of the chitincovered gills of crayfish are much less sensitive to acid and acid-aluminium stress than the gills of teleost fish.Abbreviations Hc haemocyanin - SO₂ saturation of Hc with O₂ - P ₅₀ oxygen tension of haemolymph at 50% SO₂ - n ₅₀ Hills coelficient around 50% SO₂     

Graded intracellular acidosis produces extensive and reversible reductions in the effective free energy change of ATP hydrolysis in a molluscan muscle

Phosphorus nuclear magnetic resonance spectroscopy was used to evaluate the impact of experimental reductions of intracellular pH on in vitro preparations of the radula protractor muscle of the marine gastropod, Busycon canaliculatum. The intracellular pH of radula refractor muscle bundles superfused with buffered artificial sea water (pH=7.8) was 7.29. It was possible to clamp muscle intracellular pH at various acidotic states by changing the superfusate to 5, 10, and 15 mmol·l^-1 5,5-dimethyl-oxazolidine-2,4-dione in buffered artifical sea water (pH=6.5). Consistent and temporally stable reductions of intracellular pH were achieved (intracellular pH=6.98, 6.79, and 6.62, respectively). During the acidotic transitions, arginine phosphate concentrations decreased and inorganic phosphate concentrations increased in a reciprocal manner and remained essentially constant after the intracellular pH stabilized. The extent of changes in arginine phosphate and inorganic phosphate was directly proportional to the magnitude of the imposed acidosis. Total adenosine triphosphate concentrations remained unchanged in all treatments. However, the magnesium adenosine triphosphate to total adenosine triphosphate ratio declined in direct relation to the extent of the acidosis. Intracellular free Mg²⁺ fell incrementally with reduced intracellular pH. All of the above effects were rapidly reversed when the 5,5-dimethyl-oxazolidine-2,4-dione was washed out by changing the superfusate to buffered artificial sea water (pH=7.8). Mg-adenosine diphosphate concentrations were calculated in all treatments using equilibrium constants for the arginine kinase reaction corrected for pH and intracellular free [Mg²⁺]. The metabolite, intracellular pH, and [Mg²⁺] data were used to estimate the effective free energy of hydrolysis of adenosine triphosphate (dG/d_ATP) under most experimental conditions. Experimental acidosis resulted in dramatic reductions in dG/d_ATP which were fully reversible upon wash-out of 5,5-dimethyl-dioxazolidine-2,4-dione and recovery to normal intracellular pH conditions. Acidosis resulted in net hydrolysis of arginine phosphate, likely via a complex mechanism involving enhancement of rate of adenosine triphosphate hydrolysis and/or inhibition of adenosine triphosphate synthesis.Abbreviations ABRM anterior byssus retractor muscle - ADP, ATP adenosine di- and triphosphate - AP arginine phosphate - BASW buffered artificial sea-water - CP creatine phosphate - DMO 5, 5-dimethyl-oxazolidine-2, 4-dione - G _o ^obs standard free energy change of ATP hydrolysis - dG/d_ATP effective free energy change of ATP hydrolysis - HEPES 4-(2-hydroxyethyl)-piperazine-1-ethanesulphonic acid - K _obs equilibrium constant under specified conditions - MES 2-[N-morpholino] ethanesulphonic acid - [Mg²⁺]_i intracellular free magnesium concentration - NMR nuclear magnetic resonance - pH _i intracellular pH·P _i inorganic phosphate - PIPES piperazine-N,N-bis-2-ethane sulphonic acid - RPM radula protractor muscle - SR sarcoplasmic reticulum