Abiotic metal stress enhances diosgenin yield in Dioscorea bulbifera L. cultures

Lower concentrations of CuSO₄ (25–75 M) in the MS medium supplemented with 0.1 mg l^–1 IAA+5.0 mg l^–1 Kn+500 mg l^–1 CH+10 mg l^–1 Cyst hyd enhanced the growth of regenerants of Dioscorea bulbifera L. CuSO₄ (75 M) induced an appreciable diosgenin yield in the regenerants compared to those obtained on media without Cu. The presence of Cu thus seems to stimulate diosgenin production. The regenerants also differentiated bulbils on lower concentrations of Cu. At CuSO₄ (100 M), however, cultures showed poor growth as well as a low diosgenin yield. Increased proline and protein contents were recorded in cultures grown on Cu-enriched media.     

Immobilization of Anabaena azollae from Azolla filiculoides in polyvinyl foam for ammonia production in a photobioreactor system

Anabaena azollae (AS-DS), isolated from Azolla filiculoides and grown in nitrogen-free medium, was immobilized in 5-mm-cube polyvinyl foam pieces and incorporated into a photobioreactor system for the production of NH₃. NH₃ was produced continuously and in significant amounts. Benlate (methyl-1-butyl-carbamoyl)-2-benzimidazole carbamate at 5 ppm and l-methionine-d,l-sulphoximine at 50 m stimulated NH₃ production continuously for a period of 1 week.S. Kannaiyan is with the Biotechnology Unit, Department of Agricultural Microbiology, Tamilnadu Agricultural University, Coimbatore-641003, Tamilnadu, India; K.K. Rao and D.O. Hall are with the Division of Life Sciences, King's College London, Campden Hill Road, London W8 7AH, UK.     

Binding of [3H]muscimol to calf cerebrocortical synaptic membranes and the effects of sulphur-containing convulsant and non-convulsant compounds

Endogenous and xenobiotic sulphur-containing convulsant and non-convulsant compounds containing structural moieties of, or bearing a structural resemblance to, GABA and homocysteine were tested in binding studies for their potency in displacing the GABA-mimetic [³H]muscimol from specific, high-affinity sites (K _d=3.6 nM;B _max=3.94 pmol/mg protein) on freeze-thawed, Triton-treated calf-brain synaptic membranes. The xenobiotic convulsants, 4-mercaptobutyric acid (MBA), 3-mercaptopropionic acid (3-MPA) and 2-mercaptopropionic acid (2-MPA) were found to be two-site competitive inhibitors exhibiting apparent inhibition affinity constants (K _i ^app ) of 5000 M, 3750 M, and 4800 M, respectively; while homocysteic acid (K _i ^app =4800 M) was shown to be a one-site partial competitive inhibitor. Intermediary metabolites of methionine: S-adenosyl-l-homocysteine,l-cysteine, the convulsantl-homocysteine, and its non-convulsant disulphide oxidation product, homocystine, were found to be one-site partial competitive inhibitors exhibitingK _i ^app values of 5750 M, 8350 M, 5000 M, and 510 M, respectively. The endogenous anticonvulsant neuroeffector, taurine, and the tripeptide, reduced glutathione (GSH) were shown to be, respectively, one-site (K _i=20 M) and two-site (K _i ^app =4300 M) competitive inhibitors of [³H]muscimol binding. These findings are discussed with regard to a previously proposed mechanism for the convulsant action of homocysteine.     

Nonidentity of the cDNA sequence of human breast cancer cell malic enzyme to that from the normal human cell

A cDNA coding for human breast cancer cell cytosolic NADP⁺-dependent malic enzyme was obtained. This cDNA is composed of a length of 2084 base pairs, with 1698 base pairs coding for 565 amino acid residues and a length of 386 base pairs representing a 3-noncoding region. Comparing this nucleotide sequence with that from the normal human tissue [Loeber, G., Dworkin, M. B., Infante, A., and Ahorn, H. (1994),FEBS Lett. 344, 181–186] reveals that three nucleotides in the open reading frame and the length of 3-noncoding region of the cDNA are different. One of the changes results in a substitution of serine at position 438 for proline, which, however, may not cause significant changes in the predicted secondary structure. A partial cDNA lacking the first 84 nucleotides in the open reading frame was successfully cloned and expressed functionally inEscherichia coli cells. ItsK _m value forl-malate (1.21±0.11 mM) is four times higher than that for the natural human breast cancer cell malic enzyme (0.29±0.04 mM) but similar to that for the full-length recombinant enzyme (1.06±0.07 mM). TheK _m values for Mn²⁺ and NADP⁺ (0.26±0.03 and 0.97±0.4M, respectively) are similar to those for the natural enzyme (0.12±0.02 and 1.9±0.3M, respectively) or the recombinant wild-type enzyme (0.56±0.04 and 0.44±0.02M, respectively). A recombinant pigeon liver malic enzyme without the first 13 amino acid residues was used for comparison. TheK _m values forl-malate and Mn²⁺ of the truncated enzyme (11.2±0.9 mM and 61.2±4.6M, respectively) are over 40 times larger than those for the natural pigeon liver malic enzyme (0.21±0.02 mM and 1.06±0.08M, respectively) or the recombinant wild-type enzyme (0.25±0.01 mM and 1.48±0.05M, respectively). We suggest that the N-terminus of malic enzyme may be required for the substrate binding during the catalytic cycle.     

Presence of an X AG − carrier in frog (Rana esculenta) red blood cells

Evidence is presented that the high levels of internal l-glutamic and l-aspartic acid in frog Rana esculenta red blood cells are due to the existence of a specific carrier for acidic amino acids of high affinity K _m = 3 m and low capacity (V_max) 0.4 mol l-Glu · Kg^–1 dry cell mass · 10 min^–1. It is Na+ dependent and the incorporation of l-glutamic acid can be inhibited by l and d-aspartate and l-cysteic acid, while d-glutamic does not inhibit. Moreover, this glutamic uptake shows a bell-shaped dependence on the external pH. All these properties show that this carrier belongs to the system X _AG ^– family. Besides the incorporation through this system, l-glutamic acid is also taken up through the ASC system, although, under physiological conditions, this transport is far less important, since it has relatively low affinity K _m 39 m but high capacity (V _max) 1.8 mol l-Glu · Kg^–1 dry cell mass · 10 min^–1.     

Ammonium photoproduction by free and immobilized cells of Chlamydomonas reinhardtii

Summary Free-living or immobilized Chlamydomonas reinhardtii cells photoproduce ammonium from nitrite in a medium containing 1 mM of l-methionine-d,l-sulphoximine (MSX). Ammonium is accumulated in the medium to 8 mM final concentration, which inhibits nitrite uptake by the MSX-treated cells and consequently the excretion of ammonium is blocked. However, if ammonium was removed from the medium and nitrite and MSX periodically restored, the photoproduction process could be maintained over 96 h, with a final ammonium concentration of about 18 mM for free-living cells and 28 mM for immobilized ones. The MSX-treated cells showed a photoproduction productivity of 1300 mol NH ₄ ⁺ · mg chlorophyll (Chl)^-1, with an average production rate of 14 mol NH ₄ ⁺ · mg Chl^-1 per hour, for calcium alginate-entrapped cells, while the corresponding data for free-living ones was 650 mol NH ₄ ⁺ · mg Chl^-1 and 6.7 mol NH ₄ ⁺ · mg Chl^-1 per hour, respectively. Immobilized cells showed a significant increase in the nitrite uptake rate, probably due to a change in membrane permeability as a consequence of cell-matrix interactions.     

Differences between magnesium- and manganese-activated pyruvate kinase fromThiobacillus versutus (A2)

Tan variants isolated fromBradyrhizobium (NRRL strains L-179, L181 and L-344 fromIndigofera hosts) enrichment cultures catabolized exogenousl-tryptophan (trp) to produce indolepyruvic (IPA), indoleacetic (IAA), and indolelactic (ILA) acids extracellularly. Of 184 moles, 20% of the trp was detectable by GC-MS as IPA (26 moles) and IAA (10.4 moles), and 50% was deaminated and oxidatively decarboxylated after 5 days of aerobic fermentation. Tan variants were obtained from bradyrhizobia capable of asymbiotic acetylene reduction and, like their parents, did not cross-nodulate soybean roots. Tan variants fromB. japonicum L-259 stimulated soybean growth and nodulation only when seedlings were leached and may supply absent growth components.     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

Photoproduction of ammonium by Chlamydomonas reinhardtii cells immobilized in barium alginate: a reactor feasibility study

Summary Chlamydomonas reinhardtii cells immobilized in Ba-alginate beads provide a stable and effective system for photoproducing ammonium from nitrite in a culture medium containing l-methionine-d,l-sulphoximine. The process was studied in either air-lift, fluidized or packed-bed reactors, the last one providing the most suitable system with a volumetric activity of 2700 mol NH _inf+ ^sup4 ·1^–1 per hour.     

Production of l-glutamate by immobilized protoplasts

Summary Protoplasts of Brevibacterium flavum cultured in a medium containing 50 g·l^-1 of biotin were prepared with lysozyme and immobilized in matrices of agar-acetylcellulose filters. The immobilized protoplasts were applied to l-glutamate production from glucose and urea in a batch system. The productivity of l-glutamate by the immobilized protoplasts was 2.5 times higher than that by immobilized whole cells under optimal conditions. Maximal productivity initially reached 1.5 mg·ml^-1. The immobilized protoplasts of B. flavum could be used six times for l-glutamate production with retention of about 70% of the initial productivity.     

The differential allosteric regulation of two chorismate-mutase isoenzymes of Nicotiana silvestris

The reaction catalyzed by chorismate mutase (EC 5.4.99.5) is a crucial step for biosynthesis of two aromatic amino acids as well as for the synthesis of phenylpropanoid compounds. The regulatory properties of two chorismate-mutase isoenzymes expressed in Nicotiana silvestris Speg. et Comes are consistent with their differential roles in pathway flow routes ending with l-phenylalanine and l-tyrosine on one hand (isoenzyme CM-1), and ending with secondary metabolites on the other hand (isoenzyme CM-2). Isoenzyme CM-1 was very sensitive to allosteric control by all three aromatic amino acids. At pH 6.1, l-tryptophan was a potent allosteric activator (K _a =1.5 M), while feedback inhibition was effected by l-tyrosine (K _i =15 M) or by l-phenylalanine (K_i=15 M). At pH 6.1, all three effectors acted competitively, influencing the apparent K _m for chorismate. All three allosteric effectors protected isoenzyme CM-1 at pH 6.1 from thermal inactivation at 52° C. l-Tryptophan abolished the weak positive cooperativity of substrate binding found with isoenzyme CM-1 only at low pH. At pH 7.2, the allosteric effects of l-tyrosine and l-tryptophan were only modestly different, in striking contrast to results obtained with l-phenylalanine. At pH 7.2 (i) the K _i for l-phenylalanine was elevated over 30-fold to 500 M, (ii) the kinetics of inhibition became non-competitive, and (iii) l-phenylalanine now failed to protect isoenzyme CM-1 against thermal inactivation. l-Phenylalanine may act at different binding sites depending upon the intracellular pH milieu. In-vitro data indicated that the relative ability of allosteric activation to dominate over allosteric inhibition increases markedly with both pH and temperature. The second isoenzyme, CM-2, was inhibited competitively by caffeic acid (K _i =0.2 mM). Aromatic amino acids failed to affect CM-2 activity over a broad range of pH and temperature. Inhibition curves obtained in the presence of caffeic acid were sigmoid, yielding an interaction coefficient (from Hill plots) of n=1.8.Abbreviation DAHP synthase 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase     

Purification and characterization of glutamine synthetase from the commerical mushroom Agaricus bisporus

Agaricus bisporus glutamine synthetase, a key enzyme in nitrogen metabolism, was purified to apparent homogeneity. The native enzyme appeared to be a GS-II type enzyme. It has a molecular weight of 325 kDa and consists of eight 46-kDa subunits. Its pI was found at 4.9. Optimal activity was found at 30°C. The enzyme had low thermostability. Stability declined rapidly at temperatures above 20°C. The enzyme exhibits a K _m for glutamate, ammonium, and ATP of 22mm, 0.16mm and 1.25mm respectively in the biosynthetic reaction, with optimal activity at pH 7. The enzyme is slightly inhibited by 10mm concentrations of l-alanine, l-histidine, l-tryptophan, anthranilic acid, and 5-AMP and was strongly inhibited by methionine sulfoximine and phosphinothricine. For the transferase reaction K _i-values were 890 m and 240 m for methionine sulfoximine and phosphinothricine respectively. For the biosynthetic reaction K _i was 17 m for both methionine sulfoximine and phosphinothricine.     

Molecular environment of the phencyclidine binding site in the nicotinic acetylcholine receptor membrane

Summary Phencyclidine is a highly specific noncompetitive inhibitor of the nicotinic acetylcholine receptor. In a novel approach to study this site, a spin-labeled analogue of phencyclindine. 4-phenyl-4-(1-piperidinyl)-2.2.6.6.-tetramethylpiperidinoxyl (PPT) was synthesized. The binding of PPT inhibits⁸⁶Rb flux (IC₅₀=6.6m), and [³H] phencyclidine binding to both resting and desensitized acetylcholine receptor (IC₅₀=17 m and 0.22 m, respectively). From an indirect Hill plot of the inhibition of [³H]phencyclidine binding by PPT. a Hill coefficient of approximately one was obtained in the presence of carbamylcholine and 0.8 in -bungarotoxin-treated preparations. Taken together, these results indicate that PPt mimics phencyclidine in its ability to bind to the noncompetitive inhibitor site and is functionally active in blocking ion flux across the acetylcholine receptor channel. Analysis of the electron spin resonance signal of the bound PPT suggests that the environment surrounding the probe within the ion channel is hydrophobic, with a hydrophobicity parameter of 1.09. A dielectric constant for the binding site was estimated to be in the range of 2–3 units.     

Characteristics of the ATP-ase activity of isolated neurons of rabbit

Summary Isolated and homogenised Deiters' neurons from the lateral vestibular nucleus of rabbit in a Krebs-Ringer solution containing no added Mg⁺⁺, 1.3 moles/ ml and 5 moles/ml Mg⁺⁺, broke down ATP at the maximal rate of 0.29+-0.20, 2.40+–0.20, and 5.95+–0.63 moles/cell/hr. In 1.3 mM Mg⁺⁺ solution the single cell homogenates took up phosphate at the mean rate of 2.6+–0.2 moles/cell/hr. If the rabbits were injected 1 hour before with 20 mg/kg body weight of 2-amino-1-propene-1,1,3, tricarbonitrile (triap), the breakdown of ATP in these latter media was 0.82+–0.44, 2,5+–0.60, and 6.7+– 1.1 moles/cell/hr, respectively, and the quantity of inorganic liberated did not decrease.     

Enzymatic synthesis of l-β-chloroalanine using amino acid dehydrogenase

l--Chloroalanine is a useful intermediate for the synthesis of several l-amino acids. Conditions for synthesizing optically pure l--chloroalanine from 3-chloropyruvate using alanine dehydrogenase (AlaDH), leucine dehydrogenase and phenylalanine dehydrogenase with a regeneration of NADH by formate dehydrogenase (FDH) were investigated. The enzymatic reaction was carried out at neutral pH because of a chemical instability of 3-chloropyruvate on the alkaline side. Commercially available AlaDH from Bacillus stearothermophilus IFO 12550 showed the highest activity for the production of l--chloroalanine at pH 7.5. The K _m and V _max values for 3-chloropyruvate of AlaDH were calculated to be 300 units/mg and 62.5 mm, respectively. Although 3-chloropyruvate had no inhibitory effect on AlaDH, it acted as a non-competitive inhibitor with FDH. 3-Chloropyruvate was added into the reaction mixture in a stepwise manner to avoid the inhibition. l--Chloroalanine was produced with high chemical (>90%) and optical yields (100% enantiometric excess) and at a high concentration (43 g/l).     

Methylobacterium rhodesianum MB 126 possesses two acetoacetyl-CoA reductases

Two constitutive acetoacetyl-CoA (AcAc-CoA) reductases were purified from Methylobacterium rhodesianum MB 126, an NADPH-linked d(-)--hydroxybutyryl-CoA forming reductase (enzyme A) and an NADH-and NADPH-linked l(+)--hydroxybutyryl-CoA forming reductase (enzyme B). Enzyme A and B give apparent K _m values of 15 M and 30 M for AcAc-CoA, 18 M for NADPH and 30 M for NADH, respectively. They are inhibited by AcAc-CoA at concentrations higher than 25 M and 50 M, respectively. The contribution of the two reductases to poly--hydroxybutyrate synthesis is discussed.     

Inhibition by excitatory sulphur amino acids of the high-affinityl-glutamate transporter in synaptosomes and in primary cultures of cortical astrocytes and cerebellar neurons

A detailed kinetic study of the inhibitory effects ofl- andd-enantiomers of cysteate, cysteine sulphinate, homocysteine sulphinate, homocysteate, and S-sulpho-cysteine on the neuronal, astroglial and synaptosomal high-affinity glutamate transport system was undertaken.d-[³H] Aspartate was used as the transport substrate. Kinetic characterisation of uptake in the absence of sulphur compounds confirmed the high-affinity nature of the transport systems, the Michaelis constant (K _m) ford-aspartate uptake being 6 M, 21 M and 84 M, respectively, in rat brain cortical synaptosomes and primary cultures of mouse cerebellar granule cells and cortical astrocytes. In those cases where significant effects could be demonstrated, the nature of the inhibition was competitive irrespective of the neuronal versus glial systems. The rank order of inhibition was essentially similar in synaptosomes, neurons and astrocytes. Potent inhibition (K _iK _m) of transport in each system was exhibited byl-cysteate, andl- andd-cysteine sulphinate whereas substantially weaker inhibitory effects (K _i>10–1000 times the appropriateK _m value) were exhibited by the remaining sulphur amino acids. In general, inhibition: (i) was markedly stereospecific in favor of thel-enantiomers (except for cysteine sulphinate) and (ii) was found to decrease with increasing chain length. Computer-assisted molecular modelling studies, in which volume contour maps of the sulphur compounds were superimposed on those ofd-aspartate andl-glutamate, demonstrated an order of inhibitory potency which was, qualitatively, in agreement with that obtained quantitatively by in vitro kinetic studies.Special issue dedicated to Dr. Elling Kvamme     

Purification and characterization of anl-amino-acid oxidase fromChlamydomonas reinhardtii

Anl-amino-acid oxidase (EC 1.4.3.1) that catalyzes the oxidative deamination of twelvel-amino acids has been purified 21-fold and with 14% yield to electrophoretic homogeneity fromChlamydomonas reinhardtii cells by ammonium-sulfate fractionation, gel filtration through Sephacryl and Superose, anion-exchange chromatography and preparative electrophoresis in polyacrylamide gels. The native enzyme is a protein of 470 kDa and consists of eight identical or similarsized subunits of 60 kDa each. Optimum pH and temperature were 8.2 and 55° C, respectively, with a Q₁₀ (45–55° C) of 1.7 and an activation energy of 45 kJ · mol^–1. Its absorption spectrum showed, in the visible region, maxima at 360 and 444 nm, characteristic of a flavoprotein with a calculated flavin content of 7.7 mol FAD per mol of native enzyme. ApparentK _m values of the twelvel-amino acids which can act as substrates ofl-amino-acid oxidase ranged between 31 M for phenylalanine and 176 M for methionine. The effect of several specific group reagents, chelating agents and bivalent cations on enzyme activity has also been studied.This work was supported by Grant 780-CO2-01 from CICYT, Spain. The skillful secretarial assistance of C. Santos and I. Molina is gratefully acknowledged.     

Some effects of tetrazolium salt on the metabolism of mitochondria isolated from Jerusalem artichoke tubers

Summary A series of tetrazolium salts were found to accept electrons more readily from succinate than malate even though the rate of oxygen uptake was similar with both substrates. This difference was explained by showing that all the tetrazolium salts tested caused a reduction in electron flow between NAD⁺ and Cyt.b. The tetrazolium salts were also found to be able to uncouple phosphorylation from electron transport. The monotetrazolium salts causing complete uncoupling around 100 moles/litre and the ditetrazolium salts causing complete uncoupling around 20 moles/litre.     

Chloride conductive and cotransport mechanisms in cultures of canine tracheal epithelial cells measured by an entrapped fluorescent indicator

Summary To study Cl conductive and cotransport mechanisms, primary cultures of canine tracheal cells were grown to confluency on thin glass cover slips and on porous filters. Transepithelial resistance was >100 ·cm², and short circuit current (I _sc=2–20 A/cm²), representing active secretion of Cl, increased >threefold with addition of 10 m isoproterenol to the serosal solution. Cells made transiently permeable in hypotonic solution were loaded with the Cl-sensitive fluorophore 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) (5mm, 4 min, 150 mOsm). The electrical properties of the cell monolayers were not altered by the loading procedure. Intracellular SPQ fluorescence was monitored continuously by epifluorescence microscopy (excitation 360±5 nm, emission>410 nm). SPQ leakage from the cells was <10% in 60 min at 37°C. Intracellular calibration of SPQ fluorescencevs. [Cl] (0–90mm) was carried out using high-K buffers containing the ionophores nigericin (5 m) and tributyltin (10 m); SPQ fluorescence was quenched with a Stern-Volmer constant of 13m ^–1. Intracellular Cl activity was 43±4mm. Cl flux was measured in response to addition and removal of 114mm Cl from the bathing solution. Addition of 10 m isoproterenol increased Cl efflux from 0.10 to 0.27mm/sec. The increase was inhibited by the Cl-channel blocker diphenylamine-2-carboxylic acid (1mm). In the absence of isoproterenol, removal of external Na or addition of 0.5mm furosemide, reduced Cl influx by >fourfold. In ouabain-treated monolayers, removal of external K in the presence of 5mm barium diminished Cl influx by >twofold, suggesting that Cl entry is in part K dependent. These results establish an accurate optical method for the realtime measurement of intracellular Cl activity in tracheal cells that does not require an electrically tight cell monolayer. The data demonstrate the presence of an isoproterenol-regulated Cl channel and a furosemide-sensitive cation-coupled transport mechanism.