Analysis of essential leucine residue for catalytic activity of novel thermostable chitosanase by site-directed mutagenesis

Bacterial chitosanases share weak amino acid sequence similarities at certain regions of each enzyme. These regions have been assumed to be important for catalytic activities of the enzyme. To verify this assumption, the functional importance of the conserved region in a novel thermostable chitosanase (TCH-2) from Bacillus coagulans CK108 was investigated. Each of the conserved amino acid residues (Leu64, Glu80, Glu94, Asp98, and Gly108) was changed to aspartate and glutamine or asparagine and glutamate by site-directed mutagenesis, respectively. Kinetic parameters for colloidal chitosan hydrolysis were determined with wild-type and 10 mutant chitosanases. The Leu64 Arg and Leu64 Gln mutations were essentially inactive and kinetic parameters such as V _max and k _cat were approximately 1/10⁷ of those of the wild-type enzyme. The Asp98 Asn mutation did not affect the K _m value significantly, but decreased k _cat to 15% of that of wild-type chitosanase. On the other hand, the Asp98 srarr; Glu mutation affected neither K _m nor k _cat. The observation that approximately 15% of activity remained after the substitution of Asp98 by Asn indicated that the carboxyl side chain of Asp98 is not absolutely required for catalytic activity. These results indicate that the Leu64 residue is directly involved in the catalytic activity of TCH-2.     

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     

New Substrates for Enteropeptidase: I. Biologically Active Hepta-, Octa-, and Nonapeptides

Enteropeptidase (enterokinase, EC 3.4.21.9) hydrolyzes peptide bonds formed by carboxyl groups of Lys or Arg residue if less than four negatively charged amino acid residues are in positions P ₂–P ₅ of its substrate. We determined the kinetic parameters of three substrates of this type: human angiotensin II (AT) (DR VYIHPF) and the Hb(2–8) (LTAEEK A) and Hb(1–9) (MLTAEEK AA) peptides of the cattle hemoglobin -chain. The K _m values for all the substrates (10^–3 M) were one order of magnitude higher than those of the typical synthetic substrates of enteropeptidase or chimeric proteins with the –DDDDK– full-size linker (K _m 10^–4 M). The k _cat values for AT and Hb(2–8) were also close and low (30 min^–1). The general hydrolysis efficiency of such substrates is no more than 1% of the corresponding value for the typical peptide and protein substrates of the enteropeptidase. However, the elongation of Hb(2–8) peptide by one amino acid residue from both its N- and C-termini results in a dramatic increase in the catalytic efficiency of the hydrolysis: the k _cat value for Hb(1–9) is 1510 min^–1, which means that it is hydrolyzed only three times less effective than the chimeric protein with the full-size linker.     

Arabidopsis ammonium transporters,AtAMT1;1 and AtAMT1;2, have different biochemical properties and functional roles

We have compared the biochemical properties of two different Arabidopsis ammonium transporters, AtAMT1;1 and AtAMT1;2, expressed in yeast, with the biophysical properties of ammonium transport in planta. Expression of the AtAMT1;1 gene in Arabidopsis roots increased approximately four-fold in response to nitrogen deprivation. This coincided with a similar increase in high-affinity ammonium uptake by these plants. The biophysical characteristics of this high-affinity system (K_m for ammonium and methylammonium of 8 M and 31 M, respectively) matched those of AtAMT1;1 expressed in yeast (K_m for methylammonium of 32 M and K_i for ammonium of 1–10 M). The same transport system was present, although less active, in nitrate-fed roots. Ammonium-fed plants exhibited the lowest rates of ammonium uptake and appeared to deploy a different transporter (K_m for ammonium of 46 M). Expression of AtAMT1;2 in roots was insensitive to changes in nitrogen nutrition. In contrast to AtAMT1;1, AtAMT1;2 expressed in yeast exhibited biphasic kinetics for methylammonium uptake: in addition to a high-affinity phase with a K_m of 36 M, a low-affinity phase with a K_m for methylammonium of 3.0 mM was measured. Despite the presence of a putative chloroplast transit peptide in AtAMT1;2, the protein was not imported into chloroplasts in vitro. The electrophysiological data for roots, together with the biochemical properties of AtAMT1;1 and Northern blot analysis indicate a pre-eminent role for AtAMT1;1 in ammonium uptake across the plasma membrane of nitrate-fed and nitrogen-deprived root cells.     

N 5,N 10-Methylenetetrahydromethanopterin reductase (coenzyme F420-dependent) and formylmethanofuran dehydrogenase from the hyperthermophile Archaeoglobus fulgidus

Methylene-H₄MPT reductase was found to be present in Archaeoglobus fulgidus in a specific activity of 1 U/mg. The reductase was purified 410-fold. The native enzyme showed an apparent molecular mass of approximately 200 kDa. Sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed the presence of only 1 polypeptide of apparent molecular mass 35 kDa. The ultraviolet/visible spectrum of the reductase was almost identical to that of albumin indicating the absence of a chromophoric prosthetic group. The reductase was dependent on reduced coenzyme F₄₂₀ as electron donor. Neither NADH, NADPH, nor reduced viologen dyes could substitute for the reduced deazaflavin. From reciprocal plots, which showed an intersecting patter, a K _m for methylene-H₄MPT of 16 M, a K _m for F₄₂₀H₂ of 4 M, and a V _max of 450 U/mg (K_cat=265 s^-1) were obtained. The enzyme was found to be rapidly inactivated when incubated at 80°C in 100 mM Tris/HCl pH 7. The rate of inactivation, however, decreased to essentially zero in the presence of either F₄₂₀ (0.2 mM), methylene-H₄MPT (0.2 mM), albumin (1 mg/ml), or KCl (0.5 M). The N-terminal amino acid sequence was determined and found to be similar to that of methylene-H₄MPT reductase (F₄₂₀-dependent) from the methanogens Methanobacterium thermoautotrophicum, Methanosarcina barkeri, and Methanopyrus kandleri. The purification and some properties of formylmethanofuran dehydrogenase from A. fulgidus are also described.Abbreviations H₄MPT tetrahydromethanopterin - CH₂=H₄MPT N ⁵,N ¹⁰-methylene-H₄MPT - CH₃–H₄MPT N ⁵-methyl-H₄MPT - CHH₄MPT methenyl-H₄MPT - F₄₂₀ coenzyme F₄₂₀ - MFR methanofuran - CHO-MFR formyl-MFR - 1 U 1 mol/min     

Purification and characterization of ferredoxin-NADP<Superscript>+</Superscript> reductase encoded by <Emphasis Type="Italic">Bacillus subtilis yumC</Emphasis>

From Bacillus subtilis cell extracts, ferredoxin-NADP⁺ reductase (FNR) was purified to homogeneity and found to be the yumC gene product by N-terminal amino acid sequencing. YumC is a 94-kDa homodimeric protein with one molecule of non-covalently bound FAD per subunit. In a diaphorase assay with 2,6-dichlorophenol-indophenol as electron acceptor, the affinity for NADPH was much higher than that for NADH, with K_m values of 0.57 M vs >200 M. K_cat values of YumC with NADPH were 22.7 s^–1 and 35.4 s^–1 in diaphorase and in a ferredoxin-dependent NADPH-cytochrome c reduction assay, respectively. The cell extracts contained another diaphorase-active enzyme, the yfkO gene product, but its affinity for ferredoxin was very low. The deduced YumC amino acid sequence has high identity to that of the recently identified Chlorobium tepidum FNR. A genomic database search indicated that there are more than 20 genes encoding proteins that share a high level of amino acid sequence identity with YumC and which have been annotated variously as NADH oxidase, thioredoxin reductase, thioredoxin reductase-like protein, etc. These genes are found notably in gram-positive bacteria, except Clostridia, and less frequently in archaea and proteobacteria. We propose that YumC and C. tepidum FNR constitute a new group of FNR that should be added to the already established plant-type, bacteria-type, and mitochondria-type FNR groups.     

Cadmium-copper antagonism in the activation of periplasmic nitrous oxide reductase of copper-deficient cells fromPseudomonas stutzeri

Summary Copper-deficient cells ofPseudomonas stutzeri strain ZoBell synthesize catalytically inactive nitrous oxide (N₂O) reductase which is activated by added Cu(II) in the absence of de novo protein synthesis. The apparentK _m for the activation process is 0.13 M. Activation is temperature-dependent and is inhibited by Cd(II)(K _i 1.27 M) and less strongly by Zn(II), Ni(II), and Co(II). The same metal ions at 20 M have little or no effect on N₂O reduction of intact cells. Apo-N₂O reductase of transposon Tn5-inducednos ^– mutants with defective Cu-chromophore biosynthesis is not reactivated by Cu(II). N₂O reductase of Cu-sufficient and Cu-deficient wild type, and ofnos ^– mutants is localized in the periplasm, the latter providing the likely site of metal incorporation into the apoenzyme.     

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.     

Purification and characterization of two cold-adapted extracellular tannin acyl hydrolases from an Antarctic strain <Emphasis Type="Italic">Verticillium</Emphasis> sp. P9

Two extracellular tannin acyl hydrolases (TAH I and TAH II) produced by an Antarctic filamentous fungus Verticillium sp. P9 were purified to homogeneity (7.9- and 10.5-fold with a yield of 1.6 and 0.9%, respectively) and characterized. TAH I and TAH II are multimeric (each consisting of approximately 40 and 46 kDa sub-units) glycoproteins containing 11 and 26% carbohydrates, respectively, and their molecular mass is approximately 155 kDa. TAH I and TAH II are optimally active at pH of 5.5 and 25 and 20°C, respectively. Both the enzymes were activated by Mg²⁺and Br⁻ ions and 0.5–2.0 M urea and inhibited by other metal ions (Zn²⁺, Cu²⁺, K⁺, Cd²⁺, Ag⁺, Fe³⁺, Mn²⁺, Co²⁺, Hg²⁺, Pb²⁺ and Sn²⁺), anions, Tween 20, Tween 60, Tween 80, Triton X-100, sodium dodecyl sulphate, β-mercaptoethanol, α-glutathione and 4-chloromercuribenzoate. Both tannases more efficiently hydrolyzed tannic acid than methyl gallate. E _a of these reactions and temperature dependence (at 0–30°C) of k _cat, k _cat/K _m, ΔG*, ΔH* and ΔS* for both the enzymes and substrates were determined. The k _cat and k _cat/K _m values (for both the substrates) were considerably higher for the combined preparation of TAH I and TAH II.     

Purification and characterization of β-xylosidase from <Emphasis Type="Italic">Streptomyces</Emphasis>sp. CH7 and its gene sequence analysis

A thermostable -xylosidase was extracted and purified from Streptomyces sp. CH7 mycelium. The apparent molecular weight of the native enzyme estimated by gel filtration was around 173 and 87 kDa for the two subunits estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Its optimal pH and temperature were 6.5 and 55 °C, respectively. The enzyme was stable at pH 6–9 and at 50 °C after 30 min. The K _m values for p-nitrophenyl---xylopyranoside and o-nitrophenyl---xylopyranoside were 0.56 and 0.94 mM with the V _m values of 26.3 and 6.6 U/mg protein, respectively. The enzyme was inhibited strongly by Hg²⁺, Fe²⁺, Cu²⁺ and Zn²⁺. It was inhibited by xylose competitively for p-nitrophenyl---xylopyranoside with the K _i value of 40 mM. Characterization of the nucleotide sequence of pCH7-1 carrying the -xylosidase gene from Streptomyces sp. CH7 revealed 3 open reading frames (ORF). The first truncated ORF, bxlI, encodes a putative ABC-type sugar transport system, permease component. The second ORF, bxl2, encodes -xylosidase, while the third truncated ORF, bxl3, encodes a putative oxidoreductase. The deduced 791 amino acid sequence of Bxl2 showed 84, 71 and 66% identity to those of Streptomyces coelicolor, Streptomyces lividansand Streptomyces thermoviolaceus, respectively. The calculated molecular mass of the deduced amino acid sequence revealed close similarity to that of the purified enzyme.     

Inducibility of Rat Liver Drug-Metabolizing Enzymes as an Index of Food-Screeing for Safety Assessment

D-Lactate dehydrogenase (D-LDH) from Pediococcus pentosaceus ATCC 25745 was found to produce D-3-phenyllactic acid from phenylpyruvate. The optimum pH and temperature for enzyme activity were pH 5.5 and 45 °C. The Michaelis-Menten constant (K _m), turnover number (k _cat), and catalytic efficiency (k _cat?K _m) values for the substrate phenylpyruvate were estimated to be 1.73 mmol/L, 173 s^?1, and 100 (mmol/L)^?1 s^?1 respectively.     

Influence of inorganic microelements on the production of camptothecin with suspension cultures of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

The effects of eight microelements (I^–, BO₃ ^3–, MoO₄ ^2–, Co²⁺, Cu²⁺, Mn²⁺, Fe²⁺, Zn²⁺) on the biosynthesis of camptothecin and the growth of suspension cultures of Camptotheca acuminata were studied. The increase of I^– to 25 M l^–1, Cu²⁺ to 1 M l^–1, Co²⁺ to 2 M l^–1 and MoO₄ ^2– to 10 M l^–1 in Murashige and Skoog (MS) medium resulted in 1.66, 2.84, 2.53 and 2.04 times higher of camptothecin yield than that in standard MS medium respectively. Combined treatment of I^– (25 M l^–1), Cu²⁺ (1 M l^–1), Co²⁺ (2 M l^–1) and MoO₄ ^2– (10 M l^–1) lead to improve cell dry weight, camptothecin content, and camptothecin yield to 30.56 g l^–1, 0.0299%, and 9.15 mg l^–1, respectively, which were 20.2, 208.9 and 273.8% increment respectively when compared with those of control.     

Competition between sulfate-reducing and methanogenic bacteria for H2 under resting and growing conditions

The basis for the outcome of competition between sulfidogens and methanogens for H₂ was examined by comparing the kinetic parameters of representatives of each group separately and in co-culture. Michaelis-Menten parameters (V _max and K _m) for four methanogens and five sulfate-reducing bacteria were determined from H₂-depletion data. Further, Monod growth parameters (_max, K _s, Y _H2) for Desulfovibrio sp. G11 and Methanospirillum hungatei JF-1 were similarly estimated. H₂ K _m values for the methanogenic bacteria ranged from 2.5 M (Methanospirillum PM1) to 13 M for Methanosarcina barkeri MS; Methanospirillum hungatei JF-1 and Methanobacterium PM2 had intermediate H₂ K _m estimates of 5 M. Average H₂ K _m estimates for the five sulfidogens was 1.2 M. No consistent difference among the V _max estimates for the above sulfidogens (mean=100 nmol H₂ min^-1 mg^-1 protein) and methanogens (mean=110 nmol H₂ min^-1 mg^-1 protein) was found. A two-term Michaelis-Menten equation accurately predicted the apparent H₂ K _m values and the fate of H₂ by resting co-cultures of sulfate-reducers and methanogens. Half-saturation coefficients (K _s) for H₂-limited growth of Desulfovibrio sp. G11 (2–4 M) and Methanospirillum JF-1 (6–7 M) were comparable to H₂ K _m estimates obtained for these organisms. Maximum specific growth rates for Desulfovibrio sp. G11 (0.05 h^-1) were similar to those of Methanospirillum JF-1 (0.05–0.06 h^-1); whereas G11 had an average yield coefficient 4 x that of JF-1. Calculated _max and V _max/K _m values for the methanogens and sulfidogens studied predict that the latter bacterial group will process more H₂ whether these organisms are in a growing or resting state, when the H₂ concentration is in the first-order region.     

Chorismate mutase of<Emphasis Type="Italic"> Thermus thermophilus</Emphasis> is a monofunctional AroH class enzyme inhibited by tyrosine

aroG, encoding the monofunctional chorismate mutase (TtCM) of the thermophilic gram-negative bacterium Thermus thermophilus, was cloned and its gene product characterized. TtCM was purified to homogeneity on an SDS polyacrylamide gel as a His-fusion protein with a deduced molecular mass of 15.8 kDa. The enzyme belongs to the rare group of AroH-type chorismate mutases which are mainly found in gram-positive bacteria of the Bacillus/Clostridia group and have recently also been described for gram-negative organisms. The native molecular mass is consistent with a pseudo-/ barrel enzyme that is organized as a trimer. Comparison of the enzymes structure with that of its mesophilic counterpart from Bacillus revealed an increase in hydrophilicity on the proteins surface, greater hydrophobicity in cavities within the protein, and greater restriction of conformational freedom, features that contribute to the thermal stability of this chorismate mutase. The kinetic data show Michaelis-Menten substrate saturation with a K _m of 290 M, and a k _cat/K _m value of 180 s^–1 mM^–1. TtCM was inhibited by tyrosine with a K _i =34 M, possibly in a competitive manner.     

The influence of temperature on glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and the regulation of these enzymes in a mesophilic and a thermophilic cyanobacterium

The activities and kinetics of the enzymes G6PDH (glucose-6-phosphate dehydrogenase) and 6PGDH (6-phosphogluconate dehydrogenase) from the mesophilic cyanobacterium Synechococcus 6307 and the thermophilic cyanobacterium Synechococcus 6716 are studied in relation to temperature. In Synechococcus 6307 the apparent K _m's are for G6PDH: 80M (substrate) and 20M (NADP⁺); for 6PGDH: 90M (substrate) and 25M (NADP⁺). In Synechococcus 6716 the apparent K _m's are for G6PDH: 550M (substrate) and 30M (NADP⁺); for 6PGDH: 40M (substrate) and 10M (NADP⁺). None of the K _m's is influenced by the growth temperature and only the K _m's of G6PDH for G6P are influenced by the assay temperature in both organisms. The idea that, in general, thermophilic enzymes possess a lower affinity for their substrates and co-enzymes than mesophilic enzymes is challenged.Although ATP, ribulose-1,5-bisphosphate, NADPH and pH can all influence the activities of G6PDH and 6PGDH to a certain extent (without any difference between the mesophilic and the thermophilic strain), they cannot be responsible for the total deactivation of the enzyme activities observed in the light, thus blocking the pentose phosphate pathway.Abbreviations G6PDH glucose-6-phosphate, dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase - G6P glucose-6-phosphate - 6PG 6-phosphogluconate - RUDP ribulose-1,5-bisphosphate - Tricine N-Tris (hydroxymethyl)-methylglycine     

Two N 5, N 10-methylenetetrahydromethanopterin dehydrogenases in the extreme thermophile Methanopyrus kandleri: characterization of the coenzyme F420-dependent enzyme

It was recently reported that the extreme thermophile Methanopyrus kandleri contains only a H₂-forming N ⁵, N ¹⁰-methylenetetrahydromethanopterin dehydrogenase which uses protons as electron acceptor. We describe here the presence in this Archaeon of a second N ⁵,N ¹⁰-methylenetetrahydromethanopterin dehydrogenase which is coenzyme F₄₂₀-dependent. This enzyme was purified and characterized. The enzyme was colourless, had an apparent molecular mass of 300 kDa, an isoelectric point of 3.7±0.2 and was composed of only one type of subunit of apparent molecular mass of 36 kDa. The enzyme activity increased to an optimum with increasing salt concentrations. Optimal salt concentrations were e.g. 2 M (NH₄)₂SO₄, 2 M Na₂HPO₄, 1.5 M K₂HPO₄, and 2 M NaCl. In the absence of salts the enzyme exhibited almost no activity. The salts affected mainly the V _max rather than the K _m of the enzyme. The catalytic mechanism of the dehydrogenase was determined to be of the ternary complex type, in agreement with the finding that the enzyme lacked a chromophoric prosthetic group. In the presence of M (NH₄)₂SO₄ the V _max was 4000 U/mg (k _cat=2400 s^-1) and the K _m for N ⁵,N ¹⁰-methylenetetrahydromethanopterin and for coenzyme F₄₂₀ were 80 M and 20 M, respectively. The enzyme was relatively heat-stable and lost no activity when incubated anaerobically in 50 mM K₂HPO₄ at 90°C for one hour. The N-terminal amino acid sequence was found to be similar to that of the F₄₂₀-dependent N ⁵, N ¹⁰-methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum, Methanosarcina barkeri, and Archaeoglobus fulgidus.Abbreviations H₄MPT tetrahydromethanopterin - F₄₂₀ coenzyme F₄₂₀ - CH₂=H₄MPT N ⁵,N ¹⁰-methylenetrahydromethanopterin - CHH₄MPT⁺ N ⁵,N ¹⁰-methenyltetrahydromethanopterin - methylene-H₄MPT dehydrogenase N ⁵,N ¹⁰-methylenetetrahydromethanopterin dehydrogenase - Mops N-morpholinopropane sulfonic acid - Tricine N-[Tris(hydroxymethyl)-methyl]glycine - 1 U = 1 mol/min     

Cytosolic calcium regulates a potassium current in corn (Zea mays) protoplasts

Summary The voltage- and time-dependent K⁺ current,I _K ⁺ _out , elicited by depolarization of corn protoplasts, was inhibited by the addition of calcium channel antagonists (nitrendipine, nifedipine, verapamil, methoxyverapamil, bepridil, but not La³⁺) to the extracellular medium. These results suggested that the influx of external Ca²⁺ was necessary for K⁺ current activation. The IC₅₀, concentration of inhibitor that caused 50% reduction of the current, for nitrendipine was 1 m at a test potential of +60 mV following a 20-min incubation period.In order to test whether intracellular Ca²⁺ actuated the K⁺ current, we altered either the Ca²⁺ buffering capacity or the free Ca²⁺ concentration of the intracellular medium (pipette filling solution). By these means,I _K ⁺ _out could be varied over a 10-fold range. Increasing the free Ca²⁺ concentration from 40 to 400nm also shifted the activation of the K⁺ current toward more negative potentials. Maintaining cytoplasmic Ca²⁺ at 500nm with 40nm EGTA resulted in a more rapid activation of the K⁺ current. Thus the normal rate of activation of this current may reflect changes in cytoplasmic Ca²⁺ on depolarization. Increasing intracellular Ca²⁺ to 500nm or 1 m also led to inactivation of the K⁺ current within a few minutes. It is concluded thatI _K ⁺ _out is regulated by cytosolic Ca²⁺, which is in turn controlled by Ca²⁺ influx through dihydropyridine-, and phenylalkylamine-sensitive channels.     

Characterization and regulation of NADP+-isocitrate dehydrogenase from Saccharopolyspora erythraea

NADP⁺-Isocitrate dehydrogenase (ICDH) activity was detected in cell-free extracts of Saccharopolyspora erythraea CA340, an erythromycin producer. Apparent K _m values for dl-isocitrate and NADP⁺ were 0.14 M and 0.026 M, respectively. ATP, ADP, GTP, citric acid, oxaloacetate, -ketoglutarate, glyoxalate and glyoxalate plus oxaloacetate, each at 1 mM concentration, caused 50, 20 10, 50, 25, 60, 20 and 50% inhibition of ICDH activity, respectively. Phosphoenolpyruvate, fructose 1,6-diphosphate and pyruvate had no effect. ICDH specific activity profile was growth-associated and activity with dextrose or fructose as sole carbon source, was twice of that obtained with lactose.     

Chemical composition of soil solutions extracted from New Zealand beech forests and West German beech and spruce forests

Concentrations of ions were measured in soil solutions from beech (Nothofagus) forests in remote areas of New Zealand and in solutions from beech (Fagus sylvatica) and Norway spruce (Picea abies) forests in North-East Bavaria, West Germany, to compare the chemistry of soil solutions which are unaffected by acid deposition (New Zealand) with those that are affected (West Germany). In New Zealand, soil solution SO₄ ^2– concentrations ranged between <2 and 58 mol L^–1, and NO₃ ^– concentrations ranged between <1 and 3 mol L^–1. In West Germany, SO₄ ^2– concentrations ranged between 80 and 700 mol L^–1, and NO₃ ^– concentrations at three of six sites ranged between 39 and 3750 mol L^–1, but was not detected at the remaining three sites. At all sites in New Zealand, and at sites where the soil base status was moderately high in West Germany, pH levels increased, and total Al (Al_t) and inorganic monomeric Al (Al_i) levels decreased rapidly with increasing soil depth. In contrast, at sites on soils of low base status in West Germany, pH levels increased only slightly, and Al levels did not decline with increasing soil depth.Under a high-elevation Norway spruce stand showing severe Mg deficiency and dieback symptoms in West Germany, soil solution Mg²⁺ levels ranged between 20 and 60 mol L^–, and were only half those under a healthy stand. Al_t and Al_i levels were substantially higher the healthy stand than under the unhealthy stand, indicating that Al toxicity was not the main cause of spruce decline.     

N 5,N 10-Methenyltetrahydromethanopterin cyclohydrolase from the extreme thermophile Methanopyrus kandleri: increase of catalytic efficiency (kcat/K M) and thermostability in the presence of salts

The activity of purified N ⁵,N ¹⁰-methenyltetrahydromethanopterin cyclohydrolase from Methanopyrus kandleri was found to increase up to 200-fold when potassium phosphate was added in high concentrations (1.5 M) to the assay. A 200-fold stimulation was also observed with sodium phosphate (1 M) and sodium sulfate (1 M) whereas stimulation by potassium sulfate (0.8 M), ammonium sulfate (1.5 M), potassium chloride (2.5 M), and sodium chloride (2 M) was maximal 100-fold. A detailed kinetic analysis of the effect of potassium phosphate revealed that this salt exerted its stimulatory effect by decreasing the K _m for N ⁵,N ¹⁰-methenyltetrahydromethanopterin from 2 mM to 40 M and by increasing the V _max from 2000 U/mg (k_cat=1385 s^-1) to 13300 U/mg (k_cat=9200 s^-1). Besides increasing the catalytic efficiency (k_cat/K _m) salts were found to protect the cyclohydrolase from heat inactivation. For maximal thermostability much lower concentrations (0.1 M) of salts were required than for maximal activity.Abbreviations H₄MPT tetrahydromethanopterin - N ⁵,N ¹⁰-methenyl-H₄MPT - CHO-H₄MPT N ⁵-formyl-H₄-MPT - CH₂=H₄MPT N ⁵,N ¹⁰-methylene-H₄MPT - CH₃–H₄-MPT N ⁵-methyl-H₄MPT - MOPS -N-morpholinopropane sulfonic acid - TRICINE N-[Tris(hydroxymethyl)-methyl]glycine - 1 U = 1 mol/min