Accumulation of technetium by cyanobacteria

Accumulation of pertechnetate ions (⁹⁹TcO₄ ^–) by the cyanobacterial speciesSynechocystis PCC 6803,Synechococcus PCC 6301,Plectonema boryanum,Anabaena variabilis and a redOscillatoria sp. consisted solely of a single rapid energy-independent phase (biosorption); no energy-dependent uptake was detected. Biosorption of TcO₄ ^– was concentration-dependent and could be described by a Freundlich adsorption isotherm for each cyanobacterial species examined. Decreasing pH increased the accumulation of TcO₄ ^– by all the species as did an increase in external NaCl concentration. Accumulation of TcO₄ ^– was also increased inA. variabilis, P. boryanum and the redOscillatoria by an increased external osmotic potential. Concentrations of cations affected TcO₄ ^– accumulation; K⁺ increased accumulation in all the species, Mg²⁺, Ca²⁺, Sr²⁺ and Cs⁺ increased accumulation inSynechococcus PCC 6301 and Ca²⁺ increased accumulation by the redOscillatoria. Some anions decreased TcO₄ ^– accumulation; CO₃ ^2– inA. variabilis and the redOscillatoria, SO₄ ^2– inSynechocystis PCC 6803, and HCO₃ ^– inP. boryanum. The majority of TcO₄ ^– accumulated by all the cyanobacteria was easily desorbed, with no difference in the amounts desorbed between desorption agents of different pH or cation concentration.(*author for correspondence)     

Water treatment of land-based fish farm effluents by outdoor culture of marine diatoms

The feasibility of using fish farm effluents was evaluated as a source of inorganic nutrients for mass production of marine diatoms. Batch cultures were conducted from May to July 1995 in 16-L outdoor rectangular tanks, homogenized by gentle aeration (0.2 L _air L^–1 h^–1). The effluents from the two fish farms studied were both characterized by high concentrations of inorganic materials (NH₄-N, PO₄-;P, Si(OH)₄-Si) and were shown to support production of marine diatoms. Moreover, periodic measurements of inorganic matter levels in the cultures showed that clearance was efficacious (90% in 3–5 days). Water purification efficiency and culture productivity were further increased through appropriate nutrient balancing. When effluents were limited in silicate, addition of Na₂SiO₃ induced a significant increase in both diatom biomass and nutrient removal efficiency. In this case, up to 720 000 cell mL^–1 were produced dominated bySkeletonema costatum. By contrast, in effluents loaded with silicate, adjustment of the N:P:Si ratio by NH₄-N and PO₄-P supplementation then gave increased biomass production. In this case, the maximum cell density found was 450 000 cell mL^–1, dominated byChaetoceros spp.Author for correspondence     

Synthesis and biological activity of 17α-alkynylamide derivatives of estradiol

Seven estradiol (E₂) derivatives with an alkynylamide side chain at the 17α position were synthesized starting from ethynylestradiol (EE₂). The main chemical step was the coupling reaction of the acetylide ion of EE₂ with carbon dioxide, glutaric anhydride or bromoalkyl ortho ester. The synthesis of these compounds is fast (3–6 steps according to the compound) and is easily achieved with good yield. Five compounds with different side chain lenghts were evaluated for uterotrophic and antiuterotrophic activity in the CD-1 mouse. None of the tested compounds shows estrogenic activity in this sensitive in vitro system. At low doses (1 and 3 μg), a 14–57% inhibition of E₂-induced uterine growth was observed while no additional inhibition was observed at the 10, 20 and 30 μg doses. In human breast carcinoma cells in culture, all compounds show estrogenic activity at high concentrations while only compound 39 (N-buty,N-methyl-8-[3′,17′β-dihydroxy estra-1′,3′,5′(10′)-trien-17′α-yl]-7-octynamide) possesses antiproliferative or antiestrogenic effects. No significant correlation could be demonstrated between alkynylamide side chain length and estrogenic or antiestrogenic activity. Among the compounds tested, the derivative of EE₂ possessing a five-methylene (CH₂) side chain (compound 39) possesses the best antiestrogenic activity (44 ± 7% in the CD-1 mouse uterus assay at the 3μg dose and 57 ± 4% at 0.1 nM in human ZR-75-1 cancer cells in culture).     

Regional variation in canopy transpiration of Central European beech forests

Forest hydrologists have hypothesised that canopy transpiration (E_c) of European temperate forests occurs at rather similar rates in stands with different tree species and hydrologic regimes. We tested this hypothesis by synchronously measuring xylem sap flow in four mature stands of Fagus sylvatica along a precipitation gradient with the aim (1) of exploring the regional variability of annual canopy transpiration (E_c(t)) in this species, and (2) of analysing the relationship between precipitation (P) and E_c(t). E_c(t) rates of 216, 225, 272 and 303 mm year^–1 corresponded to precipitation averages of 520, 710, 801 and 1,040 mm year^–1 in the four stands. We explored the regional variability of E_c(t) in Central European colline to sub-montane beech stands in two meta-analyses based on (1) existing sap flow data on beech (n=5 observations), or (2) all canopy transpiration data on beech obtained by different techniques (sap flow, micrometeorological or soil water budget approaches, n=25). With a coefficient of variation (CV) of 20%, the regional variability of E_c(t) (213–421 mm year^–1) was smaller than the variation in corresponding precipitation (550–1,480 mm year^–1). The mean E_c(t) for beech was 289 (±58) mm year^–1 (n=25). A humped-shaped relationship between E_c(t) and P, with a broad transpiration maximum in the precipitation range from ca. 700 to 1,000 mm year^–1, was found which may indicate soil moisture limitation of transpiration for P –1, and reduced transpiration by increased cloudiness or leaf wetness for P>1,000 mm year^–1. Thus, the precipitation level significantly influences canopy transpiration of humid temperate forests; however, the size of the P influence on E_c(t) and, in part, the direction of its effect differ from forests in semi-arid or arid climates. European beech has the capacity to maintain high E_c rates in both humid and partly dry summer climates (P<550 mm year^–1).     

Chemolithoautotrophy in the marine,magnetotactic bacterial strains MV-1 and MV-2

Magnetite-producing magnetotactic bacteria collected from the oxic–anoxic transition zone of chemically stratified marine environments characterized by O₂/H₂S inverse double gradients, contained internal S-rich inclusions resembling elemental S globules, suggesting they oxidize reduced S compounds that could support autotrophy. Two strains of marine magnetotactic bacteria, MV-1 and MV-2, isolated from such sites grew in O₂-gradient media with H₂S or thiosulfate (S₂O₃^2–) as electron sources and O₂ as electron acceptor or anaerobically with S₂O₃^2– and N₂O as electron acceptor, with bicarbonate (HCO₃^–)/CO₂ as sole C source. Cells grown with H₂S contained S-rich inclusions. Cells oxidized S₂O₃^2– to sulfate (SO₄^2–). Both strains grew microaerobically with formate. Neither grew microaerobically with tetrathionate (S₄O₆^2–), methanol, or Fe²⁺ as FeS, or siderite (FeCO₃). Growth with S₂O₃^2– and radiolabeled ¹⁴C-HCO₃^– showed that cell C was derived from HCO₃^–/CO₂. Cell-free extracts showed ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) activity. Southern blot analyses indicated the presence of a form II RubisCO (cbbM) but no form I (cbbL) in both strains. cbbM and cbbQ, a putative post-translational activator of RubisCO, were identified in MV-1. MV-1 and MV-2 are thus chemolithoautotrophs that use the Calvin–Benson–Bassham pathway. cbbM was also identified in Magnetospirillum magnetotacticum. Thus, magnetotactic bacteria at the oxic–anoxic transition zone of chemically stratified aquatic environments are important in C cycling and primary productivity.     

Oxidation-reduction midpoint potentials of mitochondrial flavoproteins and their intramitochondrial localization

Spectrophotometric and fluorimetric substrate couple titrations and potentiometric spectrophotometric titrations were used to determine the oxidation-reduction potentials of components showing absorbance or fluorescence at the wavelengths attributable to the flavoproteins of mitochondria fractionated using digitonin together with sonication. A pure mitoplast fraction devoid of cytochrome b₅ contamination could be obtained using 230 µg digitonin/mg of mitochondrial protein. The digitonin-soluble fraction contained a species havingE _m 7 .4=–123 mV and probably represents the outer membrane flavoproteins. The inner membrane-matrix fraction, treated with ultrasound, provided evidence of a flavoprotein species with low redox potential (E _m 7 .4=–302 mV) in the matrix fraction. The –302 mV component is probably lipoamide dehydrogenase. A high redox potential species withE _m 7 .4=+19 mV in titrations with the succinate fumarate couple was located in the inner membrane vesicles and is probably identical with succinate dehydrogenase. The electron-transferring flavoprotein (ETF) was isolated from bovine heart mitochondria and itsE _m 7 .4=–74 mV determined. The component in the matrix fraction with an apparentE _m 7 .4=–56 mV probably represents ETF, and that in the inner membrane fraction with an apparentE _m 7 .4=–43 mV the NADH dehydrogenase flavoprotein. A component in an apparently low concentration withE _m 7 .4=+30 mV was detected in the inner membrane fraction. This probably represents the ETF-dehydrogenase flavoprotein. The origin of the flavoprotein fluorescence of mitochondria and intact tissues is discussed.     

Tissue estradiol is selectively elevated in receptor positive breast cancers while tumour estrone is reduced independent of receptor status

Previous studies have suggested elevated estrogen production in tumour-bearing breast quadrants as well as in breast cancers versus benign tissue. Using highly sensitive assays, we determined breast cancer tissue estrogen concentrations together with plasma and benign tissue estrogen concentrations in each quadrant obtained from mastectomy specimens (34 postmenopausal and 13 premenopausal women). We detected similar concentrations of each of the three major estrogens estradiol (E₂), estrone (E₁) and E₁S in tumour-bearing versus non-tumour-bearing quadrants. Considering malignant tumours, intratumour E₁ levels were reduced in cancer tissue obtained from pre- as well as postmenopausal women independent of tumour ER status (average ratio E₁ cancer: benign tissue of 0.2 and 0.3, respectively; p < 0.001 for both groups), suggesting intratumour aromatization to be of minor importance. The most striking finding was a significant (4.1–8.6-fold) increased E₂ concentration in ER positive tumours versus normal tissue (p < 0.05 and <0.001 for pre- and postmenopausal patients, respectively), contrasting low E₂ concentrations in ER− tumours (p < 0.01 and <0.001 comparing E₂ levels between ER+ and ER− tumours in pre- and postmenopausals, respectively). A possible explanation to our finding is increased ligand receptor binding capacity for E₂ in receptor positive tumours but alternative factors influencing intratumour estrogen disposition cannot be excluded.     

A channel that allows inwardly directed fluxes of anions in protoplasts derived from wheat roots

An anion channel that only allows outward current flow (anion influx) has been identified in protoplasts derived from wheat (Triticum aestivum L., Triticum turgidum L.) roots. The anion outward rectifier (anion OR) measured by patch-clamp of whole cells activated very quickly, usually reaching a steady-state level in less than 100 ms and was easily distinguished from the cation outward rectifier (cation OR) which activated more slowly during membrane depolarisation. The anion OR is permeable to NO ₃ ^– and Cl^–, moderately permeable to I^–, and relatively impermeable to H₂PO₄/^– and ClO₄/^–. An anomalous mole-fraction effect between ClO_4/ ^– and Cl^– was observed on the outward current, indicating that the channel is a multi-ion pore. The anion OR is gated by both voltage and external anion concentration such that it activates near to the equilibrium potential for the permeant anion. It activated at more negative membrane potentials when NO ₃ ^– was substituted for Cl^– in the external medium, indicating that the channel may function to allow NO ₃ ^– influx under luxuriant external NO ₃ ^– concentrations. For most experiments, K⁺ and Cl^– were the main cation and anion in solution, and under these conditions it appeared likely that the anion OR functioned in membrane-potential regulation by facilitating a Cl^– influx at membrane potentials more positive than the chloride reversal potential (E_Cl). If E_Cl was more negative than the K⁺ reversal potential (E_K) then the anion OR dominated but both the anion and cation ORs occurred together when the membrane potential difference (V_m) was positive of both E_Cl and E_K. The cation OR was inhibited by increasing external Cl^– concentrations, but the anion OR was not affected by external K⁺ or Na⁺ concentration. The anion-transport inhibitors, zinc and phenylglyoxal were ineffective in blocking the anion OR. 4,4-Di-isothiocyanostilbene-2, 2-disulfonic acid (DIDS) irreversibly blocked about 34% of the current when applied extracellularly at a concentration of 25 M, and about 69% at a concentration of 200 M. However, DIDS (200 M) also occasionally acted as an irreversible blocker of the cation OR. Perchlorate blocked irreversibly 75% of the current at an external concentration of 10 mM and did not block the cation OR. Whole-cell currents also indicated that the anion OR was insensitive to external pH (pH=5–7) and calcium concentration ([Ca²⁺]=0.1–10 mM). Increasing intracellular calcium concentration significantly increased the occurrence of the fast outward current in whole cells (P < 0.005, X² test). With approximately 10 nM calcium inside the cell the anion outward current was observed in 64% (n = 45) of cells and with 50 nM calcium inside the cell the anion current was observed in 88% (n = 69) of cells. Single-anion OR channels observed in outside-out patches had a conductance in 300 mM KCl (external) of about 4 pS. When voltage pulses were applied to outside-out patches the average currents were similar to those observed in whole cells. The significance of the anion OR as a likely route for Cl uptake in high salinities is discussed.Abbreviations Bath solution bathing the extracellular face of the membrane - DIDS (4,4-diisothiocyanostilbene-2,2-disulfonic acid) - E_x reversal potential for ion x - OR outward rectifier - Pip solution inside the pipette - TEACl (tetraethyl-ammonium chloride) - V_m membrane potential difference We thank the Australian Research Council for financial support, G.P. Findlay and A. Garrill for helpful discussions, and K. Morris and D. Mackenzie for expert technical assistance. M.S. was supported by an Australian Postgraduate Research Award.     

The red Mutations Impair the Regulation of Flavinogenesis and Metal Homeostasis in Yeast Pichia guilliermondii

A method of positive selection of mutants with impaired regulation of flavinogenesis and metal homeostasis in yeast Pichia guilliermondii was developed. This positive selection system was based on the isolation of pseudo-wild-type revertants (the Rib⁺ phenotype) in riboflavin-dependent rib1-86 mutant (the Rib^– phenotype) of yeast P. guilliermondii. Mutation rib1-86 blocks activity of the GTP cyclohydrolase II catalyzing the first step in riboflavin (RF) biosynthesis. Study of a collection of spontaneous Rib⁺ revertants allowed the identification of a considerably large number of genetic loci responsible for the suppression of rib1-86, which include both previously identified three loci (rib80, rib81, andhit1) and six new loci designated red1–red6 (reduction). A comparative analysis of the wild-type strain and red mutants revealed that these mutants had higher activity levels of GTP cyclohydrolase and RF-synthase, elevated levels of RF biosynthesis, enhanced Fe/Cu reductase activity and higher total iron content in cells and that they are characterized by enhanced sensitivity to transition metals (Fe(III), Cu(II), Cd(II), Co(II), Zn(II), Ag(I)) and to H₂O₂. The metal hypersensitivity of mutant cells can be prevented by an increased amount of extracellular iron ions. Mutations red1 and red6 synergistically interact with the locus rib81 in the course of RF biosynthesis. Obviously, each RED gene plays an important role in the regulation of both flavinogenesis and metal homeostasis in P. guilliermondii cells.     

Effect of experimental nitrogen load on methane and nitrous oxide fluxes on ombrotrophic boreal peatland

Methane (CH₄) and nitrous oxide (N₂O) dynamics were studied in a boreal Sphagnum fuscum pine bog receiving annually (from 1991 to 1996) 30 or 100 kg NH₄NO₃-N ha^–1. The gas emissions were measured during the last three growing seasons of the experiment. Nitrogen treatment did not affect the CH₄ fluxes in the microsites where S. fuscum and S. angustifolium dominated. However, addition of 100 kg NH₄NO₃-N ha^–1 yr^–1 increased the CH₄ emission from those microsites dominated by S. fuscum. This increase was associated with the increase in coverage of cotton grass (Eriophorum vaginatum) induced by the nitrogen treatment. The differences in the CH₄ emissions were not related to the CH₄ oxidation and production potentials in the peat profiles. The N₂O fluxes were negligible from all microsites. Only minor short-term increases occurred after the nitrogen addition.     

Influence of soil temperature on methane emission from rice paddy fields

Methane emission rates from an Italian rice paddy field showed diel and seasonal variations. The seasonal variations were not closely related to soil temperatures. However, the dieL changes of CH₄ fluxes were significantly correlated with the diel changes of the temperature in a particular soil depth. The soil depths with the best correlations between CH4 flux and temperature were shallow (1–5cm) in May and June, deep (10–15cm) in June and July, and again shallow (1–5 cm) in August. Apparent activation energies (E_a) calculated from these correlations using the Arrhenius model were relatively low (50–150 kJ mol^–1) in May and June, but increased to higher values (80–450 kJ mol^–1) in August. In the laboratory, CH₄ emission from two rice cultures incubated at temperatures between 20 and 38°C showed E . values of 41 and 53 kJ mol^–1) Methane production in anoxic paddy soil suspensions incubated between 7 and 43°C showed E values between 53 and 132 kJ mol^–1 with an average value of 85 kJ mol^–1) and in pure cultures of hydrogenotrophic methanogenic bacteria E _a values between 77 and 173 (average 126) kJ mol^–1. It is suggested that diel changes of soil properties other than temperature affect CH₄ emission rates, e.g. diel changes in root exudation or in efficiency of CH⁴ oxidation in the rhizosphere.     

Action of a cell-envelope proteinase (CEPIII-type) from Lactococcus lactis subsp. cremoris AM1 on bovine κ-casein

The specificity of the cell-envelope proteinase (CEP_III-type) from Lactococcus lactis subsp. cremoris AM₁ in its action on bovine -casein was studied. A 4-h digest (pH 6.2, 15°C) of -casein was made with the purified proteinase. The pH-4.6 soluble fraction, representing more than 95% of the whole hydrolysate, was ultrafiltered to obtain a high-molecular-mass (HMM) and a low-molecular-mass (LMM) fraction, which were separately further purified by electrophoretic and chromatographic techniques. Isolated HMM and LMM products were identified by amino acid analysis, end-group determination and mass spectrometry. On-line HPLC/mass spectrometry was also used for the separation of an LMM peptide mixture and the identification of its components. The HMM products formed were the fragments 1–160, 1–151, 1–95 and 1–79 of -casein, whereas the main LMM products found were the 161–169 and 152–160 fragments. The enzyme specificity was concluded to be primarily directed towards the C-terminal region of the substrate molecule by cleavage of the 160–161 and 151–152 peptide bonds. Two minor LMM products were identified as the fragments 96–104 and 103–106, indicating additional cleavage at positions 102–103, 104–105 and 106–107 of the sequence. Also several peptide bonds within the 161–169 sequence were found to be subject to secondary cleavage by the proteinase. From electrophoretic and identification data it is concluded that the lactococaal CEP_I, CEP_III and several mixed-type proteinases all act on the peptide bonds at positions 79–80 and 95–96. However, the C-terminal region of the -casein sequence is the exclusive target of the CEP_III-aand, to variable extents, of the mixed-type enzymes.     

Single measurement reliability and reproducibility of volitional and magnetically-evoked indices of neuromuscular performance in adults

This study documents intra-session and inter-day reproducibility (coefficient of variation [V%]) and single measurement reliability (intra-class correlations [R_I]; standard error of a single measurement [SEM%] [95% confidence limits]) of indices of neuromuscular performance elicited during peripheral nerve magnetic stimulation. Twelve adults (five men and seven women) completed 3 assessment sessions on 3 days, during which multiple assessments of knee flexor volitional and magnetically-evoked indices of electromechanical delay (EMD_V; EMD_E), rate of force development (RFD_V; RFD_E), peak force (PF_V; P_TF_E), and compound muscle action potential latency (LAT_E) and amplitude (AMP_E) were obtained. Results showed that magnetically-evoked indices of neuromuscular performance offered statistically equivalent levels of measurement reproducibility (V%: 4.3–31.2%) and reliability (R_I: 0.98–0.51) compared to volitional indices (V%: 3.7–25.2%; R_I: 0.98–0.64), which support the efficacy of both approaches to assessment and the indices PF_V, EMD_V, EMD_E and LAT_E offer the greatest practical utility for assessing neuromuscular performance.     

The Succinate Dehydrogenase from the Thermohalophilic Bacterium Rhodothermus marinus: Redox–Bohr Effect on Heme bL

The succinate dehydrogenase from the thermohalophilic bacterium Rhodothermus marinus is a member of the succinate:menaquinone oxidoreductases family. It is constituted by three subunits with apparent molecular masses of 70, 32, and 18 kDa. The optimum temperature for succinate dehydrogenase activity is 80°C, higher than the optimum growth temperature of R. marinus, 65°C. The enzyme shows a high affinity for both succinate (K _m = 0.165 mM) and fumarate (K _m = 0.10 mM). It contains the canonical iron–sulfur centers S1, S2, and S3, as well as two B-type hemes. In contrast to other succinate dehydrogenases, the S3 center has an unusually high reduction potential of +130 mV and is present in two different conformations, one of which presents an unusual EPR signal with g values at 2.035, 2.009, and 2.001. The apparent midpoint reduction potentials of the hemes, +75 and –65 mV at pH 7.5, are also higher than those reported for other enzymes. The heme with the lower potential (heme b_L) presents a considerable dependence of the reduction potential with pH (redox–Bohr effect), having a pK _a ^OX = 6.5 and a pK _a ^red = 8.7. This behavior is consistent with the proposal that in these enzymes menaquinone reduction occurs close to heme b_L, near to the periplasmic side of the membrane, and involving dissipation of the proton transmembrane gradient.     

Search for polythionates in cultures of Chromatium vinosum after sulfide incubation

Cultures of Chromatium vinosum, devoid of sulfur globules, were supplemented with sulfide and incubated under anoxic conditions in the light. The concentrations of sulfide, polysulfides, thiosulfate, polythionates and elemental sulfur (sulfur rings) were monitored for 3 days by ion-chromatography and reversed-phase HPLC. While sulfide disappeared rapidly, thiosulfate and elemental sulfur (S₆, S₇ S₈ rings) were formed. After sulfide depletion, the concentration of thiosulfate decreased fairly rapidly, but elemental sulfur was oxidized very slowly to sulfate. Neither polysulfides (S _x ^2– ), polythionates (S_nO ₆ ^2– , n=4–6), nor other polysulfur compounds could be detected, which is in accordance with the fact that sulfide-grown cells were able to oxidize polysulfide without lag. The nature of the intracellular sulfur globules is discussed.     

Optimal temperature and concentration profiles in a cascade of CSTR's performing Michaelis-Menten reactions with first order enzyme deactivation

A necessary condition is found for the intermediate temperatures and substrate concentrations in a series of CSTR's performing an enzyme-catalyzed reaction which leads to the minimum overall volume of the cascade for given initial and final temperatures and substrate concentrations. The reaction is assumed to occur in a single phase under steady state conditions. The common case of Michaelis-Menten kinetics coupled with first order deactivation of the enzyme is considered. This analysis shows that intermediate stream temperatures play as important a role as intermediate substrate concentrations when optimizing in the presence of nonisothermal conditions. The general procedure is applied to a practical example involving a series of two reactors with reasonable values for the relevant five operating parameters. These parameters are defined as dimensionless ratios involving activation energies (or enthalpy changes of reaction), preexponential factors, and initial temperature and substrate concentration. For negligible rate of deactivation, the qptimality condition corresponds to having the ratio of any two consecutive concentrations as a single-parameter increasing function of the previous ratio of consecutive concentrations.List of Symbols C _E,0 mol.m^–3 Initial concentration of active enzyme - C _E,i mol.m^–3 Concentration of active enzyme at the outlet of the i-th reactor - C _S,0 mol.m^–3 Initial concentration of substrate - C _S,i mol.m^–3 Concentration of substrate at the outlet of the i-th reactor - Da _i Damköhler number associated with the i-th reactor ((V _i.k_v,0.C_E,0)/(Q.C_S,0)) - Da _min Minimum value of the overall Damköhler number - Da _tot Overall Damköhler number - E _d J.mol^–1 Activation energy of the step of deactivation of the enzyme - E _m J.mol^–1 Standard enthalpy change of the step of binding of substrate to the enzyme - E _v J.mol^–1 Activation energy of the step of enzymatic transformation of substrate - i Integer variable - j Dummy integer variable - k Dummy integer variable - k _d,i s^–1 Kinetic constant associated with the deactivation of enzyme in the i-th reactor (k _d,o·exp{–E _d/(R.T _i}) - k _d,0 s^–1 Preexponential factor of the kinetic constant associated with the deactivation of the enzyme - K _m,i mol.m^–3 Equilibrium constant associated with the binding of substrate to the enzyme in the i-th reactor, (k _m,o·exp{–E _m}(R.T _i}) - K _m,0 mol.m^–3 Preexponential factor of the Michaelis-Menten constant associated with the binding of substrate to the enzyme - k _v,i s^–1 Kinetic constant associated with the transformation of the substrate by the enzyme in the i-th reactor (k _v,o·exp{–E _v/(R.T _i})) - k _v,0 s^–1 Preexponential factor of the kinetic constant associated with the transformation of the substrate by the enzyme - N Number of reactors in the series - Q m³.s^–1 Volumetric flow rate of reacting liquid through the reactor network - R J.K^–1.mol^–1 Ideal gas constant - T _i K Absolute temperature at the outlet of the i-th reactor - T ₀ K Initial absolute temperature - V _i m³ Volume of the i-th reactor - v _max mol.m^–3.s^–1 Maximum rate of reaction under saturation conditions of substrate - x _i Normalized concentration of substrate (C_S,i/C_{S, 0}) - x _i,opt Optimum value of the normalized concentration of substrate - y _i Dimensionless temperature (exp{–T ₀/T _i}) - y _i,opt Optimum value of the dimensionless temperature Greek Symbols Dimensionless preexponential factor associated with the Michaelis-Menten constant (K _m,0/C_s,0) - Dimensionless activation energy of the step of enzymatic transformation of substrate (E _v/R.T₀)) - Dimensionless standard enthalpy change of the step of binding of substrate to the enzyme (E _m/(R.T₀)) - Dimensionless activation energy of the step of deactivation of the enzyme (E _d/(R.T₀)) - Dimensionless deactivation preexponential factor ((k _d,0.C_S,0)/(k_v,0.C_E,0)     

Analog circuit of theAcetabularia membrane

Summary The high membrane potential ofAcetabularia (E _m=–170 mV) is due to an electrogenic pump in parallel with the passive diffusion system (E _d=–80 mV) which could be studied separately in the cold, when the pump is blocked. Electrical measurements under normal conditions show that the pump pathway consists of its electromotive forceE _p with two elementsP ₁ andP ₂ in series;P ₂ is shunted by a large capacitance (C _p=3 mF cm^–2). The nonlinear current-voltage relationship ofP ₁ (light- and temperature-sensitive) could be determined separately; it reflects the properties of a carrier-mediated electrogenic pump. The value ofE _p (–190 mV) indicates a stoichiometry of 21 between electrogenically transported charges and ATP. The electrical energy, normally stored inC _p, compares well with the metabolic energy, stored in the ATP pool. The nonlinear current-voltage relationship ofP ₂ (attributed to phosphorylating reactions) is also sensitive to light and temperature and is responsible for the region of negative conductance of the overall current-voltage relationship. The power of the pump (1 W cm^–2) amounts to some percent of the total energy turnover. The high Cl^– fluxes (1 nmol cm^–2 sec^–1) and the electrical properties of the plasmalemma are not as closely related as assumed previously. For kinetic reasons, a direct and specific Cl^– pathway between the vacuole and outside is postulated to exist.     

Oxygen utilization by Lactobacillus plantarum

Cell-free extracts of Lactobacillus plantarum contain non-proteinaceous compounds which mimic superoxide dismutase activity. Using the test system in which O ₂ ^– is generated by xanthine oxidase, superoxide dismutase activity is found in cell-free extracts, where proteins are removed by precipitation. This activity is strongly decreased after dialysis of cell-free extracts. Superoxide dismutase activity was also investigated by means of pulse radiolysis. Cell-free extracts of Escherichia coli were also investigated as a comparison, which were known to contain superoxide dismutase. With cell-free extracts of both L. plantarum and E. coli the decay of O ₂ ^– was markedly increased. However, the type of reaction of the O ₂ ^– decay was of first order in the presence of E. coli extracts due to superoxide dismutase(s), and of second order in the presence of L. plantarum extracts, indicating that O ₂ ^– elimination is not an enzymic reaction. Mn²⁺ phosphate(s) might be responsible for the observed elimination of O ₂ ^– . The production of O ₂ ^– is not detectable during NADH-, lactate- or pyruvate oxidase reactions in L. plantarum extracts.     

Functional studies of chloroplast glyceraldehyde-3-phosphate dehydrogenase subunits A and B expressed in Escherichia coli: formation of highly active A4 and B4 homotetramers and evidence that aggregation of the B4 complex is mediated by the B subunit carboxy terminus

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (phosphorylating, E.C. 1.2.1.13) (GAPDH) of higher plants exists as an A₂B₂ heterotetramer that catalyses the reductive step of the Calvin cycle. In dark chloroplasts the enzyme exhibits a molecular mass of 600 kDa, whereas in illuminated chloroplasts the molecular mass is altered in favor of the more active 150 kDa form. We have expressed in Escherichia coli proteins corresponding to the mature A and B subunits of spinach chloroplast GAPDH (GapA and GapB, respectively) in addition to a derivative of the B subunit lacking the GapB-specific C-terminal extension (CTE). One mg of each of the three proteins so expressed was purified to electrophoretic homogeneity with conventional methods. Spinach GapA purified from E. coli is shown to be a highly active homotetramer (50–70 U/mg) which does not associate under aggregating conditions in vitro to high-molecular-mass (HMM) forms of ca. 600 kDa. Since B₄ forms of the enzyme have not been described from any source, we were surprised to find that spinach GapB purified from E. coli was active (15–35 U/mg). Spinach GapB lacking the CTE purified from E. coli is more highly active (130 U/mg) than GapB with the CTE. Under aggregating conditions, GapB lacking the CTE is a tetramer that does not associate to HMM forms whereas GapB with the CTE occurs exclusively as an aggregated HMM form. The data indicate that intertetramer association of chloroplast GAPDH in vitro occurs through GapB-mediated protein-protein interaction.Abbreviations GAPDH glyceraldehyde-3-phosphate dehydrogenase - CTE carboxy-terminal extension - HMM high molecular mass - ATP adenosine triphosphate - 3PGA 3-phosphoglycerate - 1,3bisPGA 1,3-bisphosphoglycerate - HMM high-molecular mass