Uptake and turnover of acetate in hypersaline environments

Abstract: Acetate uptake and turnover rates were determined for the heterotrophic community in hypersaline environments (saltern crystallizer ponds, the Dead Sea) dominated by halpphilic Archaea. Acetate was formed from glycerol, which is potentially the major available carbon source for natural communities of halophilic Archaea. Values of [ K _t+ S _n] (the sum of the substrate affinity and the substrate concentration present in situ) for acetate measured in saltern crystallizer ponds were around 4.5–11.5 μM, while in the Dead Sea during a Dunaliella bloom values up to 12.8 μM were found. Maximal theoretical rates ( V _max) of acetate uptake in saltern crystallizer ponds were 12–56 nmol l⁻¹ h⁻¹, with estimated turnover times for acetate ( T _t) between 127–730 h at 35°C. V _max values measured in the Dead Sea were between 0.8 and 12.8 nmol l⁻¹ h⁻¹, with turnover times in the range of 320–2190 h. V _max values for acetate were much lower than those for glycerol. Comparisons with pure cultures of halophilic Archaea grown under different conditions showed that the natural communities were not adapted for preferential use of acetate. Both in natural brines and in pure cultures of halophilic Archaea, acetate incorporation rates rapidly decreased above the optimum pH value, probably since acetate enters the cell only in its unionized form. The low affinity for acetate, together with low potential utilization rates result in the long acetate turnover times, which explains the accumulation of acetate observed when low concentrations of glycerol are supplied as a nutrient to natural communities of halophilic Archaea.     

The role of glycerol in the nutrition of halophilic archaeal communities: a study of respiratory electron transport

Abstract Respiratory electron transport activity in the Dead Sea and saltern crystallizer ponds, hypersaline environments inhabited by dense communities of halophilic archaea and unicellular green algae of the genus Dunaliella , was assayed by measuring reduction of 2-( p -iodophenyl)-3( p -nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan. Typical rates obtained were in the order of 5.5–17.7 nmol INT reduced h ⁻¹ per 10⁶ cells at 35 ° C. In Dead Sea water samples, respiratory activity was stimulated more than two-fold by addition of glycerol, but not by any of the other carbon compounds tested, including sugars, organic acids, and amino acids, or by addition of inorganic nutrients. Stimulation by glycerol had a half-saturation constant of 0.75 μM. A similar respiratory activity was also found when Dead Sea water samples were diluted with distilled water and incubated in the light. As Dunaliella cells did not reduce INT, it is suggested that photosynthetically produced glycerol leaking from the algae is the preferred carbon and energy source for the development of halophilic archaea in hypersaline environments. In samples from saltern crystallizer pond stimulation of INT reduction by glycerol was much less pronounced, probably because the community was less severely carbon-limited.     

Activity of methanotrophic bacteria in Green Bay sediments

Abstract Sediment pore water samples obtained from a 19 m station in Green Bay in Lake Michigan were examined for levels of ambient dissolved methane and copper, and for the potential for in situ methane oxidation by methanotrophs found within surface sediments. The in situ methane concentration in the upper oxic sediment layer ranged from 20–150 μmol · 1⁻¹ at this station. The activity of methanotrophs and the kinetics of methane oxidation in these sediments were demonstrated by the uptake of radiolabeled methane. K_s values varied between 4.1–9.6 nmol · cm³ of sediment slurry. High V_max values (12.7–35.2 nmol · cm⁻³ · h⁻¹) suggest a large population of methanotrophs in the sediments. An average methane flux to the oxic sediments of 0.24 mol · m⁻² · year⁻¹ was calculated from the pore water methane gradients. Pore water concentrations of copper in the upper sediment layer ranged from 10–120 nmol · 1⁻¹. Based upon the copper concentration, other measured parameters, and equilibrium conditions defined by WATEQF4, an estimate for dissolved free Cu²⁺ concentration of 5–38 nmol · 1⁻¹ pore water was obtained. Several factors control the rate of methane oxidation, including oxygen, methane, and the bioavailability of free Cu²⁺.     

Gas exchange in intact isolated chloroplasts from Chlamydomonas reinhardtii during starch degradation in the dark

During starch degradation in intact isolated chloroplasts from Chlamydomonas reinhardtii gas exchange was studied with a mass spectrometer. Oxygen uptake by intact chloroplasts in the dark never exceeded 1.5% of the starch degradation rate [maximum 15 nmol O₂ (mg Chl)⁻¹ h⁻¹ consumed. 1 000 nmol glucose (mg Chl)⁻¹h⁻¹ degraded]. Evolution of CO₂ under aerobic conditions [9.8–28 nmol (mg Chl)⁻¹ h⁻¹] was stimulated by addition of 0.1–0.5 m M oxaloacetate [393–425 nmol CO₂ (mg Chl)⁻¹ h⁻¹]. Pyridoxal phosphate (5 m M ) inhibited starch degradation by more than 80%, but had no effect on O₂ uptake. Starch degradation rates and CO₂ evolution did not differ under acrobic and anaerobic conditions. Increasing P_i in the reaction medium from 0.5 m M to 5.0 m M stimulated starch degradation by 230 and 260% under aerobic and anaerobic conditions, respectively. A rapid autooxidation of reduced ferredoxin was observed in a reconstituted system consisting of purified Chlamydomonas ferredoxin, purified Chlamydomonas NADP-ferredoxin oxidoreductase (EC 1.6.7.1) and NADPH. Addition of isolated thylakoids from C. reinhardtii did not affect the rate of O₂ uptake. Our results clearly indicate the absence of any oxygen requirement during starch degradation in isolated chloroplasts.     

Evidence for cytochrome P-450 and P-450-mediated benzo(a) pyrene hydroxylation in the white rot fungus Phanerochaete chrysosporium

Abstract The presence of cytochrome P-450 and P-450-mediated benzo(a)pyrene hydroxylase activity in both microsomal and soluble fractions of the white rot fungus Phanerochaete chrysosporium was shown. The reduced carbon monoxide difference spectrum showed maxima at 448–450 and 452–454 nm for microsomal and cytosolic fractions, respectively. Both P-450 fractions produced a Type I substrate binding spectrum on addition of benzo(a)pyrene. Activity for benzo(a)pyrene hydroxylation was NADPH-dependent and inhibited by carbon monoxide. K _m values for activity showed a difference between the cellular fractions with a K _m of 89 μM for microsomal P-450 and 400 μM for cytosolic P-450. The V _max values observed were 0.83 nmol min⁻ (nmol microsomal P-450) ⁻¹ and 0.4 nmol min⁻¹ (nmol cytosolic P-450)⁻¹. The results indicate that P-450-mediated benzo(a)pyrene hydroxylase activity could play a role in xenobiotic transformation by this fungus beside the known ligninolytic exocellular enzymes.     

Characterization of ATPase activities in rice root plasmalemma vesicles isolated by two-phase partitioning

Plasma membrane vesicles were isolated from the roots of 7-day-old rice plants ( Oryza sativa L. cv. Bahía) by utilizing an aqueous polymer two-phase system with 6.2%:6.2% (w/w) Dextran T500 and polyethylene glycol 3350 (PEG) at pH 7.6. Plasmalemma vesicles of high purity were obtained as indicated by the vanadate-sensitive K⁺, Mg²⁺-ATPase activity that was 18 times higher in the upper (PEG-rich) phase than in the lower (Dextran-rich) phase and by specific staining with sodium silicotungstate. Two peaks of ATPase activity were found. One showed a pH optimum at 6.0 in the presence of 150 m M KCl and 3 m M ATP with apparent K_m (ATP) and V_max of 0.75 m M and 79 μmol (mg protein)⁻¹ h⁻¹, respectively. With 50 m M KCl and 7 m M ATP a pH optimum of 6.5, an apparent K_m (ATP) of 6.3 m M and V_max of 159 μmol (mg protein)⁻¹ h⁻¹ were determined. Both activities were specific for ATP, unspecific for monovalent cations, sensitive to sodium vanadate and Ca²⁺ but insensitive to azide and nitrate.     

Effects of copper on active and passive Rb+ influx in roots of winter wheat

The effects of copper (CuCl₂) on active and passive Rb⁺(⁸⁶Rb⁺) influx in roots of winter wheat grown in water culture for 1 week were studied. External copper concentrations in the range of 10–500 μ M in the uptake nutrient solution reduced active Rb⁺ influx by 20–70%, while passive influx was unaffected (ca 10% of the Rb⁺ influx in the Cu-free solution). At external Rb⁺ concentrations of up to 1 m M , Cu exposure (50 μ M decreased V_max to less than half and increased K_m to twice the value of the control. Short Cu exposure reduced the K⁺ concentration in roots of low K⁺ status. Pretreatment for 5 min in 50 μ M CuCl₂ prior to uptake experiments reduced Rb⁺ influx by 26%. After 60 min pretreatment with Cu, the corresponding reduction was 63%. Cu in the cultivation solution impeded growth, especially of the roots. The Cu concentration in the roots increased linearly with external Cu concentration (0–100 μ M ) while Cu concentration in the shoots was relatively unchanged. The K⁺ concentration in both roots and shoots decreased significantly with increased Cu in the cultivation solutions. Possible effects of Cu on membranes and ion transport mechanisms are discussed.     

Characteristics of ammonium absorption by excised root and leaf tissues of maize

Absorption of ammonium from solutions of ammonium chloride by maize ( Zea mays L. cv. GS-2) tissue was studied. In contrast to an initial rapid phase of absorption in root tissue, a one hour lag period was recorded in leaf tissue. The maximum rate of uptake was observed at 5–10 m M NH₄Cl in both tissues. Roots had a K_m value of 1.0 m M and V_max of 24.3 μmol ammonium (g fresh weight)⁻¹ h⁻¹, whereas the leaf tissue had a higher K_m (4.1 m M ) and a lower V_max (8.7 μmol). There was a concentration dependent increase in ethanol soluble and insoluble fractions of organic nitrogen during ammonium supply. The optimum pH for ammonium absorption for both tissues was 7.4. The optimal concentration of CaCl₂ for ammonium absorption was 5 m M whereas that of KCl was only 1 m M . In both tissues, the absorption was inhibited substantially by DCMU, DNP, cycloheximide, lincomycin, sodium tungstate, sodium arsenate and to some extent also by the anions nitrate and sulfate. It is suggested that a carrier is involved in an active uptake of ammonium in the leaf tissues.     

Production of d-lactate, acetate, and pyruvate from glycerol in communities of halophilic archaea in the Dead Sea and in saltern crystallizer ponds

Abstract When glycerol is added to cultures of halophilic archaea, especially representatives of the genera Haloferax and Haloarcula , massive amounts of acids are formed. HPLC and enzymatic analyses of supernatants of Haloferax cultures grown in the presence of glycerol showed that all produced d -lactate and acetate. Cultures of two Haloarcula species tested produced pyruvate and acetate from glycerol. In all cases only a small fraction of the added glycerol was converted to organic acids. Both lactate, pyruvate, and acetate can be used as substrates for the growth of many halophilic archaea, including those that produce them, and acid production is possibly an overflow phenomenon, due to the limited capacity of the enzymatic systems responsible for their dissimilation. To test whether lactate is formed also by natural communities of halophilic archaea at low glycerol concentrations such as may be encountered in situ, we incubated samples from the Dead Sea and from the saltern crystallizer ponds at Eilat with 1.5–3 μM [U-¹⁴C]glycerol. After depletion of the glycerol, around 10% of the label was found in lactate and acetate in both brine samples. In addition, pyruvate was formed in Dead Sea water. Upon further incubation of the Dead Sea samples after depletion of the glycerol, pyruvate disappeared rapidly, while acetate and lactate concentrations decreased only very slowly. In saltern brines the lactate formed was degraded after depletion of the glycerol, but the concentration of labelled acetate decreased only very slowly.     

Uptake kinetics of iron-phytosiderophores in two maize genotypes differing in iron efficiency

Iron inefficiency in the maize ( Zea mays L.) mutant ysl is caused by a defect in the uptake system for Fe-phytosiderophores. To characterize this defect further, the uptake kinetics of Fe-phytosiderophores in ysl was compared to the Fe-efficient maize cultivar Alice. Short-term uptake of ⁵⁹Fe-labeled Fe-deoxymugineic acid (Fe-DMA) was measured over a concentration range of 0.03 to 300 μM. Iron uptake in Fe-deficient plants followed Michaelis-Menten kinetics up to about 30 μM and was linear at higher concentrations, indicating two kinetically distinct components in the uptake of Fe-phytosiderophores. The saturable component had similar K_m (∼ 10 μM) in both genotypes. In contrast. V_max was 5.5 μmol Fe-DMA g⁻¹ dry weight [30 min]⁻¹ in Alice, but only 0.6 μmol Fe-DMA g⁻¹ dry weight [30 min]⁻¹ in _ysl. Uptake experiments with double-labeled ⁵⁹Fe-[¹⁴C]DMA suggest that in both cultivars Fe-DMA was taken up by the roots as the intact chelate. The results indicate the existence of a high-affinity and a low-affinity uptake system mediating Fe-phytosiderophore transport across the root plasma membrane in maize. Apparently, the mutation responsible for Fe inefficiency in ysl affected high-affected uptake and led to a decrease in activity and/or number of Fe-phytosiderophore transporters.     

Nickel and rubidium uptake by whole oat plants in solution culture

Nickel and rubidium uptake by oat plants ( Avena sativa L. cv. Victory) were examined in relation to solution temperature, solution concentrations, metabolic inhibitors, anaerobic root conditions, transpiration and time. Over a 4-h period, uptake rates for both Ni²⁺ and Rb⁺ remained constant at 23°C. Decreasing temperatures to 2°C, 20 μ M concentrations of 2,4-dinitrophenol (DNP), or anaerobic root conditions decreased Ni²⁺ and Rb⁺ uptake rates by 97 to 86% in whole plants. Treatment of excised roots with 20 μ M DNP decreased Ni²⁺ uptake by 93%. Nickel and Rb⁺ uptake rates measured as a function of the external solution concentration followed a typical parabolic curve. K_m (0.012 m M ) and V_max [2.72 μmol (g dry weight)^-1 h^-1] values for Ni²⁺ were nearly 7 times lower than those for Rb⁺ [0.09 m M and 19.2 μmol (g dry weight)^-1 h^-1]. In all experiments, Ni²⁺ and Rb⁺ showed qualitatively similar uptake patterns, but Rb⁺ uptake was quantitatively more sensitive than Ni²⁺ to experimental manipulations.     

γ-Glutamyl-transpeptidase in tobacc suspension cultures: Catalytic properties and subcellular localization

γ-Glutamyl-transpeptidase activity (EC 2.3.2.2) was found in ammonium sulfate precipitates of extracts from cultured cells of Nicotiana tabacum L. var. Samsun. Specific activity up to 3.2 nmol (mg protein)⁻¹ min⁻¹ was achieved, using the artificial substrate γ-glutamyl- p -nitroanilide (K_m 0.6 m M ) instead of glutathione. Optimal enzyme activity was obtained at pH 8.0–8.5 and 45°C. The enzyme reaction was inhibited competitively by γ-glutamyl analogs (6-diazo-5-oxo-L-norleucine: K_i 0.76 μ M ; L-azaserine: K_i 0.23 m M ) or the inorganic ion m -periodate (K_i 0.43 m M ). Cell fractionation and in vivo experiments revealed that 77% of the γ-glutamyl-transpeptidase activity is localized in the soluble cytoplasmic fraction, while 20–23% of the enzyme is found on the outer surface of the plasmalemma.     

Extracellular exo-polygalacturonase secreted from carrot cell cultures. Its purification and involvement in pectic polymer degradation

Exo-polygalacturonase (exo-PGase, EC 3.2.1.67) activity has been detected in a culture filtrate of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The extracellular exo-PGase was purified to electrophoretic homogeneity using DEAE-Sephadex A-50 ion-exchange chromatography, Sephadex G-150 gel filtration, and preparative polyacrylamide gel electrophoresis (PAGE). The molecular mass of the purified enzyme was calculated to be 48 kDa from Sephadex G-200 gel filtration, and 50 kDa from sodium dodecyl sulfate (SDS)-PAGE after treatment with SDS and 2-mercaptoethanol. The isoelectric point was at pH 6.2. The K_m and V_max values for polygalacturonate (degree of polymerization: 52) were 14.4 μ M and 25.6 μmol (mg protein)⁻¹ h⁻¹, respectively. The optimal activity in McIlvaine's buffer occurred at pH 4.6. The enzyme activity was inhibited by Ba²⁺, Cu²⁺, Mn²⁺ and Hg²⁺. The enzyme was involved in ca 15% hydrolysis of the acidic polymer purified from carrot pectic polysaccharides, and connected with the release of galacturonic acid. Even after an exhaustive reaction the enzyme had, however, little or no effect on cell walls from carrot cell cultures.     

A limnological investigation of the tarn Syslaktjønn, W Norway

An investigation covering hydrography, chemistry, vascular and cryptogamic plants, nitrogen fixation, phytoplankton biomass and production, and zooplankton was carried out from April to November 1976 in tarn in W Norway, The volume of the tarn was 18000 m³ and the turnover time about 30 d. Temperature ranged between 3.6 and 23.4°C and pH between 4.8 and 5.5. Nuphar luteum and Carex rostrata were the two dominating vasculars-with biomasses of 117 and 97 g m⁻², respectively The biomass of the bryophytes ( Sphagnum spp.) was about 510 g m⁻² and the production of the order 0.2–2.1 μg (mg d.w.)⁻¹h⁻¹. Nitrogen fixation in association with Sphagnum spp. was estimated at 25 g yr⁻¹ for the whole tarn. Phytoplankton was dominated by diatoms, green algae and chrysophyceans. The chlorophyll a content ranged from 2 to 20 mg m⁻³ and the carbon assimilation rates from 0.03 to 20 mg C m⁻³h⁻¹ at 0–4 m depths. Production in the period was of the magnitude 22 g C m⁻². The copepod Eudiaptomus gracilis was the most common netzooplankter. Large numbers of rotationrians were found during summer.     

Cadmium accumulation in the chloroplast of Euglena gracilis

Intracellular distribution of Cd, cysteine, glutathione, and Cd-induced thiol peptides in Euglena gracilis cultured under photoheterotrophic conditions was studied. After 3 days of culture with 0.2 m M CdCl₂, 62% of the Cd accumulated by cells was equally distributed between the cytosolic and chloroplastic fractions. However, after 8 days, metal content increased in the crude chloroplastic fraction to 40% of total and decreased to 19% in the cytosol; in Percoll-purified chloroplasts the estimated content of Cd raised to 62%. Accumulation of Cd in chloroplasts could be mediated by a transporter of free Cd²⁺, since uptake of added CdCl₂ in isolated chloroplasts exhibited a hyperbolic type of kinetics with a K_m of 57 µ M and V_max of 3.7 nmol (mg protein)⁻¹ min⁻¹. The contents of cysteine and glutathione markedly increased in both chloroplasts (7–19 times) and cytosol (4–9 times) by exposure to Cd²⁺, although they were always higher in the cytosol. Thiol-containing peptides induced by Cd were mainly located in the cytosol after 3 days, and in the chloroplasts after 8 days of culture. The data suggested that Cd was compartmentalized into chloroplasts in a process that may involve the transport of free Cd and the participation of thiol-peptides.     

Attraction of zebrafish, Brachydanio rerio, to alanine and its suppression by copper

Preference responses of zebrafish to 10⁻³, 10⁻⁴ and 10⁻⁵M alanine (Ala) were concentration- dependent. Behavioural responses to copper (Cu) and Cu + Ala mixtures were also assessed. Zebrafish avoided 100 and 10 μg Cu l⁻¹, but not 1 μg l⁻¹. Mixtures of 10⁻³ m Ala+ 100 μg Cu l⁻¹ and 10 ⁴ M Ala + 10 μg Cu 1⁻¹ were avoided as intensely as was Cu alone. Responses to 10⁻³ M Ala + 10 or 1 μg Cu l⁻¹ and 10 ⁴ M Ala +1 μg Cu l⁻¹ did not differ statistically from controls (no detectable preference or avoidance). These results demonstrate, firstly, that a concentration of a pollutant avoided by itself (10 μg Cu l⁻¹) may not be avoided when encountered with an attractant chemical stimulus (Ala) and may suppress the preference for an attractant stimulus, and secondly, that a concentration of a pollutant not avoided by itself and not considered deleterious (1 μg Cu l⁻¹) suppresses attraction to Ala (an important constituent of prey odours for many fishes).     

Field estimates of oxygen consumption for the brine shrimp Parartemia zietziana Sayce (Crustacea: Anostraca) in two salt lakes in Victoria, Australia

SUMMARY. Oxygen consumption of P. zietziana was measured monthly in two saline (>60‰ salinity) lakes from November 1973 to November 1975 with short (<2 h) in situ incubations in BOD bottles. Tests in which oxygen decline was monitored continuously showed that there was no handling effect and respiratory rate was constant down to 1.8–1.9 mg O₂ 1⁻¹, about 40% of the usual initial concentration. Incubations over 24 h demonstrated no diurnal fluctuations in oxygen consumption. Multiple regression analysis indicated that 90% of the variance in respiratory rate ( R in mg O₂x10⁻⁴h⁻¹ individual⁻¹) was accounted for by changes in salinity (3%; S in ‰), temperature (7%; T in °C) and dry weight (8%; W in mg × 10⁻³): log R =−1.123+0.0025+0.021 T+ 0.756 log W. From this equation and data on population density, population respiration was calculated: 91864.5 mg O₂ m⁻² year⁻¹ in Pink Lake and 12367.5 mg O₂ m⁻²year⁻¹ in Lake Cundare.     

Subject index volume 144

Abstract β-xylosidase (EC 3.2.1.37) has been purified from Aspergillus nidulans mycelium grown on oat-spelt xylan as sole carbon source. Its pH optimum for activity was found to be 5.0 and the optimum temperature was 50 °C. Its molecular mass was estimated by gel filtration to be 180000. Using p-nitrophenyl-β-d-xylopyranoside as substrate, the K _m and V _max values have been found to be 1.1 mM and 25.6 μmol min⁻¹(mg protein)⁻¹, respectively. Enzyme activity was inhibited by Hg²⁺, Ag²⁺, and Cu²⁺ at a concentration of 1 × 10⁻³ M. The synthesis of β-xylosidase in A. nidulans is strongly induced by arabinose and xylose and is subject to carbon catabolite repression mediated by the cre A gene product.     

High-Affinity Uptake of (RS)-Nipecotic Acid in Astrocytes Cultured from Mouse Brain. Comparison with GABA Transport

Abstract: (RS)-Nipecotic acid is taken up into cultured astrocytes by a saturable high-affinity transport system with a K_m, of 28.8 ± 2.8 μM and a V_max of 0.294 ± 0.022 nmol × min⁻¹× [mg cell protein]⁻¹. The uptake which represents a net inward transport was sodium-dependent, requiring translocation of one sodium ion for each molecule of nipecotic acid taken up. The most potent inhibitors of GABA uptake into astrocytes (GABA, (R)-nipecotic acid, (3RS,4SR)-4-hydroxynipecotic acid, and guvacine) were shown to be potent inhibitors of nipecotic acid uptake (IC₅₀) 20, 25, 25, and 50 μm respectively), GABA being a competitive inhibitor. (S)-2,4-Diaminobutyric acid was a more efficient inhibitor than β-alanine of glial uptake of (RS)-nipecotic acid. It is concluded that astroglial uptake of (RS)-nipecotic acid and GABA is mediated by the same transport system.     

Characterization of ammonium and methylamine transport systems in phototrophic sulfur bacteria

Abstract Transport of ammonium and methylamine into the cells of green sulfur bacterium Chlorobium limicola and purple sulfur bacterium Thiocapsa roseopersicina is carried out by a common transport system. This system has (for C. limicola and T. roseopersicina , respectively) pH optimum 7.0 and 7.5; V _max 0.6 and 4.2 nmol min⁻¹ (mg protein)⁻¹; K_m 5.9 × 10⁻⁵ M and 1.3 × 10⁻⁵ M, and is capable of forming 120- and 600-fold methylamine gradients. The methylamine transport can be energized by the artificially imposed transmembrane K⁺ diffusive potential and is inhibited by tetraphenylphosphonium or valinomycin and K⁺. The data presented indicate that methylamine transport in both studied species is exclusively driven by the membrane potential gradient (ΔΨ).