Physiological comparison of d-cysteine desulfhydrase of Escherichia coli with 3-chloro-d-alanine dehydrochlorinase of Pseudomonas putida CR 1-1

d-Cysteine desulfhydrase of Escherichia coli W3110 trpED102/F trpED102 was physiologically characterized. It was found to be located in the cytosolic fraction, as 3-chloro-d-alanine dehydrochlorinase is. d-Cysteine desulfhydrase catalyzed not only the ,-elimination reaction of O-acetyl-d-serine to form pyruvate, acetic acid and ammonia, but also the -replacement reaction of O-acetyl-d-serine with sulfide to form d-cysteine. However, these reactions appeared not to proceed in vivo. No other activity of d-cysteine synthesis from O-acetyl-d-serine and sulfide was detected in a crude cell extract of E. coli which was immunotitrated with antibodies raised against the purified d-cysteine desulfhydrase. Although d-cysteine desulfhydrase catalyzes the degradation (,-elimination reaction) of 3-chloro-d-alanine, which is an effective antibacterial agent, E. coli W3110 trpED102/F trpED102 did not show resistance against 3-chloro-d-alanine. Therefore, d-cysteine desulfhydrase does not contribute to 3-chloro-d-alanine detoxification in vivo.     

Binding of chloride and a disulfonic stilbene transport inhibitor to red cell band 3

Summary The effect of chloride on 4,4-dibenzamido-2,2-disulfonic stilbene (DBDS) binding to band 3 in unsealed red cell ghost membranes was studied in buffer [NaCl (0 to 500mm) + Na citrate] at constant ionic strength (160 or 600mm). pH 7.4, 25°C. In the presence of chloride, DBDS binds to a single class of sites on band 3. At 160mm ionic strength, the dissociation constant of DBDS increases linearly with chloride concentration in the range [Cl]=450mm. The observed rate of DBDS binding to ghost membranes, as measured by fluorescence stopped-flow kinetic experiments, increases with chloride concentration at both 160 and 600mm ionic strength. The equilibrium and kinetic results have been incorporated into the following model of the DBDS-band 3 interaction: The equilibrium and rate constants of the model at 600mm ionic strength areK ₁=0.67±0.16 m,k ₂=1.6±0.7 sec^–1,k _–2=0.17±0.09 sec^–1,K ₁=6.3±1.7 m,k ₂=9±4 sec^–1 andk _–2=7±3 sec^–1. The apparent dissociation constants of chloride from band 3,K _Cl, are 40±4mm (160mm ionic strength) and 11±3mm (600mm ionic strength). Our results indicate that chloride and DBDS have distinct, interacting binding sites on band 3.     

The xylogalactan sulfate from Chondria macrocarpa (Ceramiales,Rhodophyta)

A structure is proposed for the complex xylogalactan sulfate from Chondria macrocarpa. The hot-water extract of C. macrocarpa was desulfated or alkali-treated and Smith degraded. Constituent sugars and their substitution patterns were identified using a modified Hakamori methylation procedure suited to sulfated polysaccharides and a double hydrolysis-reduction protocol that yielded derivatives from all of the sugar residues, including the labile 3,6-anhydrogalactosyl residues. The polymer has an agar-type backbone of alternating 3-linked \-d- and 4-linked -L-galactopyranosyl units. The d-residues are partially sulfated on O-2 (50%) and O-6 (20–30%). About 40% of the l-residues are present as the 3,6-anhydride and 25% as its precursor l-galactose 6-sulfate. A significant proportion of the remaining l-galactosyl residues have both a d-xylopyranosyl substituent on O-3 and a sulfate ester on O-6 and are stable to alkali.     

Development of medium components for the production of glucosyl-transferring enzyme by Aureobasidium

Aureobasidium sp. ATCC 20524 produced a glucosyl-transferring enzyme which produced panose (O--D-glucopyranosyl-(1»6)-O--D-glucopyranosyl-(1»4)-d-glucose) from maltose. Optimum production for the enzyme was with maltose at 2% (w/v) and yeast extract at 1.5% (w/v). Enzymatic activity reached 0.7×10³ U/g dry cells after 48 h.     

Pleconax Rafin. — eine bis heute unbeachtete Silenoideen-Gattung (Caryophyllaceae)

Zusammenfassung Die zwischen den Arten der SektionConoimorpha Otth (UntergattungConocalyx Willk.) der GattungSilene und den übrigen Arten derselben Gattung sowie aller übrigen Gattungen der TribusLychnideae A. Br. existierenden Unterschiede berechtigen zur Abtrennung dieser Sektion (Untergattung) als selbständige GattungPleconax Rafin. Nach bisherigen Untersuchungen gehören in diese Gattung folgende Arten und Unterarten:Pleconax ammophila (Boiss.)ourková mit subsp.ammophila und subsp.carpathae (Chowdhuri)ourková,P. amphorina (Pomel)ourková,P. conica (L.)ourková mit subsp.conica und subsp.conomaritima (D.Jord. et P.Pan.)ourková,P. coniflora (Nees)ourková,P. conoidea (L.)ourková,P. lydia (Boiss.)ourková,P. macrodonta (Boiss.)ourková,P. multinervia (Wats.)ourková,P. sartorii (Boiss. etHeldr.)ourková,P. subconica (Friv. emend. D.Jord. et P.Pan.)ourková mit subsp.subconica und subsp.grisebachii (David.)ourková sowieP. tempskyana (Freyn etSint.)ourková. Die angeführten nomenklatorischen Umkombinationen werden hier zum ersten Male veröffentlicht.     

Isolation and Functional Characterization of Siderophore-Producing Lead- and Cadmium-Resistant Pseudomonas putida KNP9

Heavy metals, being phytotoxic, cause growth inhibition and even plant death. Siderophore-producing bacterial strain KNP9 is growth promoting and has been isolated from Panki Power Plant, Kanpur, India. It simulated significant (p > 5%) root and shoot growth of mung bean to the extent of 16.48% and 28.80%, respectively in the presence of CdCl₂ (110 M). However, the increase in root and shoot growth was 20% and 19.5%, respectively, in the presence of (CH₃COO)₂Pb (660 M). Moreover, concentration of accumulated lead and cadmium in root and shoot was also reduced in the presence of this isolate ranging from 37.5 to 93.19%. A moderate reduction in chlorophyll content (39.14%) in the presence of 110 M CdCl₂ was rescued by bioinoculant KNP9. However, the 19.58% decrease in chlorophyll content in the case of lead acetate remained unchanged even in the presence of KNP9. Nevertheless, 16S ribosomal DNA (rDNA) sequencing identified KNP9 as a strain of Pseudomonas putida.     

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).     

Chromosome size and DNA values in sundews (Droseraceae)

Relative DNA content has been determined Feulgen cytophotometrically and autoradiographically for roottip nuclei of Drosophyllum lusitanicum L., (2n=12), Drosera rotundifolia L. (2x=20), D. intermedia Hayne (2x=20), D. linearis Goldie (2x= 20), D. binata Labill. (3x=32), D. capensis L. (4x= 40), D. spathulata Labill. (8x=80), all Droseraceae. Relative DNA values per diploid genome for Drosophyllum and diploid, triploid, and higher polyploid Drosera were approximately as 16421. These values are terms of a geometric series and are compatible with a multistranded (polyneme) interpretation of chromosome structure.     

Regulation of biosynthesis of aspartate family amino acids in the photosynthetic bacterium Rhodopseudomonas palustris

The four amino acids of the aspartate family (l-lysine, l-methionine, l-threonine, and l-isoleucine) are produced in bacteria by a branched biosynthetic pathway. Regulation of synthesis of early common intermediates and of carbon flow through distal branches of the pathway requires operation of a number of subtle feedback controls, which are integrated so as to ensure balanced synthesis of the several end products. Earlier studies with nonsulfur purple photosynthetic bacteria were instrumental in revealing the existence of alternative regulatory schemes, and in this communication we report on the control pattern of a representative of this physiological group not previously investigated, Rhodopseudomonas palustris. The results obtained from study of the properties of four key regulatory enzymes of the aspartate family pathway (-aspartokinase, homoserine dehydrogenase, homoserine kinase, and threonine deaminase) and of the effects of exogenous amino acids (i. e., the end products) on growth of the bacterium indicate that the control schema in Rps. palustris differs substantially from the schemes described for other Rhodopseudomonas species, but resembles the regulatory pattern observed in Rhodospirillum rubrum.Abbreviations A absorbancy - AK -aspartokinase - ASA aspartate -semialdehyde - DTT dithiothreitol - HS l-homoserine - HSDH homoserine dehydrogenase - HSK homoserine kinase - I l-isoleucine - KU Klett-Summerson photometer units - L l-lysine - M l-isoleucine - KU Klett-Summerson photometer units - L l-lysine - M l-methionine - ME -mercaptoethanol - PABA p-aminobenzoic acid - T l-threonine - TD threonine deaminase - RCV synthetic growth medium (see text) - YP agar medium containing 0.3% yeast extract, 0.3% peptone, and 1.5% agar - Y2T synthetic growth medium (see text)     

Interaction of free indole-3-acetic acid and its amino acid conjugates in tomato hypocotyl cultures

The interaction of free IAA and its amino acid conjugates on growth and development of cultured tomato hypocotyl tissue (Lycopersicon esculentum Mill. cv. Marglobe) was studied. In a nutrient medium containing 10 mol/L of benzyladenine, free IAA stimulated shoot and root development with little callus proliferation. In contrast, all IAA-amino acid conjugates tested supported mostly callus growth. Simultaneous application of free IAA and its conjugates resulted in the expression of mixed morphogenetic responses (i.e., both vigorous callus growth and organogenesis resulted). Growth kinetics and the effect of temporal exposure of the tissues to the bound and the free auxin suggest that some IAA-amino acid conjugates may specifically influence plant morphogenesis in ways that cannot be easily explained as simply a function of their slow hydrolysis to release free IAA.Abbreviations IAA indole-3-acetic acid - IAA-Ala N-(indol-3-ylacetyl)-l-alanine - IAA-Asp N-(indol-3-ylacetyl)-dl-aspartic acid - IAA-Lys N -(indol-3-ylacetyl)-l-lysine - IAA-Orn N -(indol-3-ylacetyl)-l-ornithine - IAA-Thr N-(indol-3-ylaetyl)-l-threonine     

Agaroids from New Zealand members of the Gracilariaceae (Gracilariales,Rhodophyta) — a novel dimethylated agar

Polysaccharide extracts from four New Zealand members of the Gracilariaceae have been characterized by ¹³C-NMR spectroscopy and GLC analysis of alditol acetate derivatives prepared using a new double hydrolysis-reduction procedure. All were based on variously substituted repeating disaccharide units of agarobiose and 20% of its precursor containing l-galactose-6-sulfate. Gracilaria truncata yielded a firm gelling agar with 67% methylation on the 6-position of the d-galactose residues. The other extracts belong to a new class of agar molecules having methylation on both the 6-position of the d-galactose units and the 2-position of the l-sugar units. The Curdiea coriacea polysaccharide displayed this double methylation almost completely ( 96 %); the alkali-modified polymer thus had only two free hydroxy-groups per disaccharide repeat unit, yet still gave a firm gel. The Curdiea flabellata and Melanthalia abscissa extracts had this double methylation pattern but to a lesser extent, and additional xylosyl branch units on up to 18% of the repeating disaccharide units.     

l-Ascorbic-acid biosynthesis in the euryhaline diatom Cyclotella cryptica

l-Ascorbic acid (AA) production in cells of Cyclotella cryptica Reimann, Lewin, Guillard (Bacillariophyceae) is enhanced when darkadapted cells are exposed to light.Heterotrophically grown cells incubated with d-[6-³H,6-¹⁴C]glucose and d-[1-³H,6-¹⁴C]glucose (2 h in dark followed by 15 h light) produced labeled AA with significantly different ratios of ³H and ¹⁴C. Comparisons of labeling patterns in AA and chitin-derived d-glucosamine support a path of conversion in Cyclotella from d-glucose to AA that inverts the carbon chain of the sugar. This process resembles similar conversions found in AA-synthesizing animals and species from two other algal classes.Abbreviations AA l-Ascorbic acid - glc d-glucose - glcN d-glucosamine     

Regulation of acetohydroxy acid synthase in Corynebacterium glutamicum during fermentation of α-ketobutyrate to l-isoleucine

Summary Corynebacterium glutamicum ATCC 13 032 produces 13 g/l l-isoleucine from 200 mM -ketobutyrate as a synthetic precursor. In fed batch cultures up to 19 g/l l-isoleucine is formed. For optimal conversion the addition of 0.3 mM l-valine plus 0.3 mM l-leucine to the fermentation medium is required. The affinity constants for the acetohydroxy acid synthase (AHAS) were determined. (This enzyme directs the flow of -ketobutyrate plus pyruvate towards l-isoleucine and that of two moles of pyruvate to l-valine and l-leucine, respectively.) For -ketobutyrate the K _m is 4.8×10^-3 M, and V _max 0.58 U/mg, for pyruvate the K _m is 8.4×10^-3 M, and V _max 0.37 U/mg. Due to these characteristics the presence of high -ketobutyrate concentrations apparently results in a l-valine, l-leucine deficiency. This in turn leads to a derepression of the AHAS synthesis from 0.03 U/mg to 0.29 U/mg and high l-isoleucine production is favoured. The derepression of the AHAS synthesis induced by the l-valine, l-leucine shortage was directly proven with a l-valine, l-leucine, l-isoleucine auxotrophic mutant where the starvation of each amino acid resulted in an increased AHAS level. This is in accordance with the fact that only one AHAS enzyme could be verified by chromatographic and electrophoretic separations as being responsible for the synthesis of all three branched-chain amino-acids.     

Evidence for two modes of Ca2+ entry following muscarinic stimulation of a human salivary epithelial cell line

Summary We have investigated muscarinic receptor-operated Ca²⁺ mobilization in a salivary epithelial cell line, HSG-PA, using an experimental approach which allows independent evaluation of intracellular Ca²⁺ release and extracellular Ca²⁺ entry. The carbachol (Cch) dose response of intracellular Ca²⁺ release indicates the involvement of a single, relatively low-affinity, muscarinic receptor site (K _0.510 or 30 m, depending on the method for [Ca²⁺] _i determination). However, similar data for Ca²⁺ entry indicate the involvement of two Cch sites, one consistent with that associated with Ca²⁺ release and a second higher affinity site withK _0.52.5 m. In addition, the Ca²⁺ entry response observed at lower concentrations of Cch (2.5 m) was completely inhibited by membrane depolarization induced with high K⁺ (>55mm) or gramicidin D (1 m), while membrane depolarization had little or no effect on Ca²⁺ entry induced by 100 m Cch. Another muscarinic agonist, oxotremorine-M (100 m; Oxo-M), like Cch, also induced an increase in the [Ca²⁺] _i of HSG-PA cells (from 72±2 to 104±5nm). This response was profoundly blocked (75%) by the inorganic Ca²⁺ channel blocker La³⁺ (25–50 m) suggesting that Oxo-M primarily mobilizes Ca²⁺ in these cells by increasing Ca²⁺ entry. Organic Ca²⁺ channel blockers (verapamil or diltiazem at 10 m, nifedipine at 1 m), had no effect on this response. The Oxo-M induced Ca²⁺ mobilization response, like that observed at lower doses of Cch, was markedly inhibited (70–90%) by membrane depolarization (high K⁺ or gramicidin D). At 100 m Cch the formation of inositol trisphosphate (IP₃) was increased 55% above basal levels. A low concentration of carbachol (1 m) elicited a smaller change in IP₃ formation (25%), similar to that seen with 100 m Oxo-M (20%). Taken together, these results suggest that there are two modes of muscarinic receptor-induced Ca²⁺ entry in HSG-PA cells. One is associated with IP₃ formation and intracellular Ca²⁺ release and is independent of membrane potential; the other is less dependent on IP₃ formation and intracellular Ca²⁺ release and is modulated by membrane potential. This latter pathway may exhibit voltage-dependent gating.     

Mapping of gal minus-mutants by transducing lambda dg carrying deletions of the gal-region

Summary To map numerousgal ^--mutants ofE. coli, advantage is taken of the fact that transducing dg's can carry different amounts of bacterial DNA of the host from which they originated (Adler andTempleton, 1963).A method is described with which a large number of transducing dg can be easily isolated, differing from each other with respect to the amount of bacterial DNA of thegal-region. By observing whethergal ⁺-colonies can arise as the result of recombination betweengal ^--mutants and dg's carrying deletions in thegal-region, so far 104 kinaseless mutants and 96 transferaseless mutants could be ordered into 26 groups. The mapping-tests were done by spotting the mutants with 52 HFT-lysates of dg's lacking more or less of the kinase- or the kinase- and transferase gene.     

Uptake of acetyl-l-carnitine in the brain

Analysis in mouse brain slices of the uptake of acetyl-l-[N-methyl-¹⁴C]carnitine with time showed it to be concentrative, and kinetic analysis gave aK _m of 1.92 mM and aV _max of 1.96 mol/min per ml, indicating the presence of a low-affinity carrier system. The uptake was energy-requiring and sodium-dependent, being inhibited in the presence of nitrogen (absence of O₂), sodium cyanide, low temperature (4°C), and ouabain, and in the absence of Na⁺. The uptake of acetyl-l-carnitine was not strictly substrate-specific; -butyrobetaine,l-carnitine,l-DABA, and GABA were potent inhibitors, hypotaurine andl-glutamate were moderate inhibitors, and glycine and -alanine were only weakly inhibitory. In vivo, acetyl-l-carnitine transport across the blood-brain barrier had a brain uptake index of 2.4±0.2, which was similar to that of GABA. These results indicate an affinity of acetyl-l-carnitine to the GABA transport system.     

Isolation and characterization of five serine proteases with trypsin-, chymotrypsin- and elastase-like characteristics from the gut of the lugworm Arenicola marina (L.) (Polychaeta)

Summary Five proteases were isolated from the digestive fluid of the lugworm, Arenicola marina L. The enzymes (molecular weight 24.0–24.6 kDa) were classified as serine proteases. Three enzymes showed a cleavage specificity corresponding to mammalian trypsin (E.C. 3.4.21.4). One protease possessed a chymotrypsin-like cleavage pattern (E.C. 3.4.21.1), and the fifth preferred cleavage behind short-chain amino acids like an elastase (E.C. 3.4.21.36). Detailed investigations revealed differences in molecular characteristics and cleavage patterns compared to mammalian proteases, especially in the chymotrypsin- and the elastase-like enzymes.Abbreviations APNE N-acetyl-d/l-Phe -naphthyl ester - BANA N-benzoyl-d/l-Arg -naphthylamide - BAPNA N-benzoyl-d/l-Arg-4-nitroanilide - BIGGANA N-benzoyl-l-Ile-l-Glu-Gly-l-Arg-4-nitroanilide - BLPNA N-benzoyl-d/l-Lys-4-nitroanilide - BTEE N-benzoyl-l-Tyr ethyl ester - enzyme T1/T2/T3 trypsin-like enzyme - enzyme ChT chymotrypsin-like enzyme - enzyme E elastase-like enzyme - GPANA N-glutaryl-l-Phe-4-nitroanilide - MUF 4-methylumbelliferryl - MW molecular weight - PMSF phenylmethylsulphonyl fluoride - SAAPPNA N-succinyl-l-Ala-l-Ala-l-Pro-l-Phe-4-nitroanilide - SBTI soybean trypsin inhibitor - SPPNA N-succinyl-l-Phe-4-nitroanilide - TAME N-tosyl-l-Arg methyl ester - TFA trifluoracetic acid - TLCK N-tosyl-l-Lys chloromethyl ketone - TPCK N-tosyl-l-Phe chloromethyl ketone - TRIS tris(hydroxymethyl)aminomethane     

A practical synthesis of N α -Fmoc protected l-threo-β-hydroxyaspartic acid derivatives for coupling via α- or β-carboxylic group

Abstract  

A simple and practical general synthetic protocol towards orthogonally protected tHyAsp derivatives fully compatible with Fmoc solid-phase peptide synthetic methodology is reported. Our approach includes enantioresolution of commercially available d,l-tHyAsp racemic mixture by co-crystallization with l-Lys, followed by ion exchange chromatography yielding enantiomerically pure l-tHyAsp and d-tHyAsp, and their selective orthogonal protection. In this way N ^α -Fmoc protected tHyAsp derivatives were prepared ready for couplings via either α- or β-carboxylic group onto the resins or the growing peptide chain. In addition, coupling of tHyAsp via β-carboxylic group onto amino resins allows preparation of peptides containing tHyAsn sequences, further increasing the synthetic utility of prepared tHyAsp derivatives.     

Synthesis of γ-aminobutyric acid by expressing <Emphasis Type="Italic">Lactobacillus brevis</Emphasis>-derived glutamate decarboxylase in the <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> strain ATCC 13032

Purpose of work  

Purpose of this work is to synthesize γ-aminobutyric acid by glutamate-producing species expressing Lactobacillus brevis-derived glutamate decarboxylase genes, i.e. recombinant Corynebacterium glutamicum strains, which directly convert endogenous l-glutamate precursor into γ-aminobutyric acid (GABA) through single-step fermentation.