Chemical composition of lipopolysaccharides from Legionella bozemanii and Legionella longbeachae

Lipopolysaccharides (LPS) from Legionella bozemanii serogroup 1 and Legionella longbeachae serogroup 1 were subjected to chemical analyses. The lipid A part of both LPSs contained 2,3-dideoxy-2,3-diamino-d-glucose as major constituents and d-glucosamine and glycerol as minor constituents of the sugar backbone structure. Both LPSs exhibited a very complex fatty acid composition. Twenty amide-linked 3-hydroxy fatty acids were detected in LPS of L. longbeachae, whereas seventeen were encountered in LPS of L. bozemanii. Both LPSs contained nine ester-linked nonhydroxy fatty acids and the unique long-chain fatty acids 27-oxo-octacosanoic acid, 29-oxotriacontanoic acid, heptacosane-1,27-dioic acid and nonacosane-1,29-dioic acid. SDS-PAGE showed that L. bozemanii produced smooth-form LPS, whereas L. longbeachae LPS was mainly of the R-type. Composition analyses were in accordance with these electrophoretic patterns. d-Quinovosamine and l-fucosamine constituted 80 mol% of the polysaccharide part of L. bozemanii LPS. Other sugars identified were d-glucosamine, d-mannose, d-glucose, l-rhamnose, d-glycero-d-manno-heptose, l-glycero-d-mannoheptose, 2-keto-3-deoxy-octonic acid and glycerol. The polysaccharide chain from LPS of L. longbeachae appeared to be shorter, but composed of the same sugars except l-fucosamine. Both LPSs contained glycerol phosphate and glucosamine phosphate and L. longbeachae LPS contained in addition glucose phosphate.Abbreviations EI Electron impact - GlcN3N 2,3-Diamino-2,3-dideoxy-d-glucose - HPAEC High pH anion-exchange chromatography - Kdo 2-Keto-3-deoxy-octonic acid - LPS Lipopolysaccharide - PCP Phenol/chloroform/petroleum ether solvent - PED Pulsed electrochemical detection - PS Polysaccharide - TFA Trifluoroacetyl - TMS Trimethylsilyl     

Characterization ofCampylobacter jejuni lipopolysaccharide

Lipopolysaccharides were isolated from dehydratedCampylobacter jejuni by combination of the phenol-chloroform-petroleum ether and phenol-water extraction techniques. Biochemical characterizations of lipopolysaccharide were performed on the two fractions of highest purity. Neutral sugar analyses detected galactose, glucose, trace amounts of mannose, and an unidentified deoxy-hexose. The primary amino sugars were galactosamine, glucosamine, and glucosamine-phosphate. Chemical analyses of other lipopolysaccharide components included phosphate, 3-deoxy-d-manno-octulosonic acid (KDO), and fatty acids. The predominant fatty acids were 3-hydroxytetradecanoic and hexadecanoic acids with lesser amounts of tetradecanoic acid. 3-Hydroxytetradecanoic acids were bound to lipid A by both amide and ester linkages.     

The structure of the lipid A component of Sphaerotilus natans

The lipopolysaccharide of Sphaerotilus natans afforded a ladder-like pattern of bands in sodium deoxycholate-polyacrylamide gel electrophoresis, indicating the presence of a S-form lipopolysaccharide. The chemical analysis showed neutral sugars (rhamnose, glucose, l-glycero-d-manno-heptose), 3-deoxy-octulosonic acid (Kdo), amino compounds (glucosamine, glucosamine phosphate, ethanolamine and ethanolamine phosphate), and phosphorus. The lipid A fraction contained saturated and unsaturated capric, lauric, and myristic acids, and 3-hydroxy capric acid (3-OH-10:0). Its chemical structure was consisting of a glucosamine disaccharide, glycosidically substituted by a phosphomonoester, and substituted at C-4 by a pyrophosphodiester esterified with ethanolamine. The amino groups of both glucosamines are acylated by 3-hydroxy capric acids and these in turn are substituted by saturated and unsaturated capric, lauric, and myristic acids. Hydroxyl groups of the backbone disaccharide at C-3 and C-3 were also esterified by 3-hydroxy capric acid, those at C-4 and C-6 were unsubstituted. The latter provides the attachment site for Kdo.Abbreviations Kdo 3-deoxy-d-manno-octulosonic acid - 3-OH-10:0 3-hydroxy capric acid - DOC-PAGE deoxycholate-polyacrylamide gel electrophoresis - GC-MS gas chromatography/mass spectrometry - LD-MS laser desorption mass spectrometry - LPS lipopolysaccharide - PS polysaccharide     

Cell wall and sheath constituents of the cyanobacterium Gloeobacter violaceus

Sheaths isolated from Gloeobacter violaceus were found to be composed of a major polysaccharide moiety (glucose, galactose, rhamnose, mannose, arabinose), a protein moiety, and negatively charged components (glucuronic acids, phosphate, sulfate). Outer membrane polypeptide patterns were dominated by two major peptidoglycan-associated proteins (M_r 62,000 and 53,000). Lipopolysaccharide constituents were glucosamine, 3-hydroxy fatty acids (3-OH-14:0, anteiso-3-OH-15:0, 3-OH-16:0, 3-OH-18:0), carbohydrates, and phosphate. A1-type peptidoglycan and non-peptidoglycan components (mannosamine, glucose, mannose, and glucosamine) indicated the presence of a peptidoglycan-polysaccharide complex in the cell walls of Gloeobacter violaceus.Abbreviations A₂pm diaminopimelic acid - ATCC American Type Culture Collection - CE cell envelope - CM cytoplasmic membrane - CW cell wall - dOcla 3-deoxy-d-manno-2-octulosonic acid - GalN galactosamine - GlcN glucosamine - GlcUA glucuronic acid - HF hydrofluoric acid - LPS lipopolysaccharide - ManN mannosamine - M relative molecular mass - MurN muramic acid - MurN-6-P muramic acid-6-phosphate - OMe O-methyl - PAGE polyacrylamide gel electrophoresis - PCC Pasteur Culture Collection - SDS sodium dodecyl sulfate - SH sheath     

Intracellular Proteases of Bacillus thuringiensis subsp. kurstaki and a Protease-Deficient Mutant Btk-q

The commencement of intracellular protease synthesis was studied by gelatin zymography in Bacillus thuringiensis (Btk) HD1, Btk HD73, and a protease-deficient mutant Btk-q derived from the former strain. By gelatin zymography, a 92-kDa protease was detected first at 3 h of sporulation, which continued until 48 h, whereas two other proteases of mol wt 78 and 69 kDa were detectable from 6 h onwards and continued until 48 h of growth in Btk HD1. Similar studies revealed the presence of two major intracellular proteases in Btk HD73 by gelatin zymography, which first appeared at 6 h of sporulation and continued until 48 h of growth. The quantitative azocasein assay confirmed that the total protease activity increases from 3 to 21 h, thereafter reaching a plateau up to 48 h of growth examined, in HD1 and HD73 strains. Btk-q, a protease-deficient mutant, showed traces of protease activity by azocasein analysis that could not be detected by gelatin zymography. The free amino acid pool content was also increased parallel to the way that the protease activity increased in all three strains. However, this increase was found to be low (16-fold) in Btk-q when compared with Btk HD1 and HD73 strains. The following amino acids were detected by paper chromatography in Btk HD1: DL-alanine, L-glutamic acid, L-aspartic acid, tyrosine, tryptophan/methionine/valine, arginine, leucine/norleucine/isoleucine, and glycine, whereas only DL-alanine, L-glutamic acid, and L-aspartic acid were in Btk-q at 24 and 48 h, when the protease activity was maximum. Received: 4 January 2002 / Accepted: 6 March 2002     

Control of l-amino acid oxidase in Neurospora crassa by different regulatory circuits

l-Amino acid oxidase is synthesized in Neurospora crassa in response to three different physiological stimuli: (i) starvation in phosphate buffer, (ii) mating, and (iii) nitrogen derepression in the presence of amino acids. During starvation in phosphate buffer, or after mating, l-amino acid oxidase synthesis occurred in parallel with that of tyrosinase. Exogenous sulfate repressed the formation of the two enzymes in starved cultures, but not in mated cultures. Sulfate repression was relieved by protein synthesis inhibitors, suggesting that the effect of sulfate required the synthesis of a metabolically unstable protein repressor. With amino acids as the sole nitrogen source only l-amino acid oxidase was produced. Under these conditions enzyme synthesis was repressed by ammonium and was insensitive to sulfate. Biochemical evidence suggested that the l-amino acid oxidase formed under the three different conditions was the same protein. Therefore, the expression of l-amino acid oxidase appeared to be under the control of least two regulatory circuits. One, also controlling tyrosinase, seems to respond to developmental signals related to sexual morphogenesis. The other, controlling other enzymes of the nitrogen catabolic system, is used by the organism to obtain nitrogen from alternative sources such as proteins and amino acids.     

Cell wall polymers of Actinomadura carminata INA 4281

The cell walls of Actinomadura carminata INA 4281 were found to contain peptidoglycan, teichoic acid, and nonpeptidoglycan amino acids. The peptidoglycan was of the A1 type and contained a small amount of ll-DAP in addition to m-DAP. The teichoic acid was an 1,3-poly(glycerol phosphate) chain composed of about eight glycerophosphate units, two of which had a 2-acetamido-2-deoxy--d-galactopyranosyl substituent and one, a 3-O-methyl--d-galactopyranosyl-(1 3)-2-acetamido-2-deoxy--d-galactopyranosyl residue at C2 of glycerol. The structure of the polymer was identified by chemical analysis and ¹³C-NMR spectroscopy. The teichoic acid contained 3-O-methyl-d-galactose (madurose) — the first ever finding of this compound within a teichoic acid. The nonpeptidoglycan amino acids made up some 30% of the cell wall's dry weight, about a quarter of the amino acids being removable with sodium dodecyl sulfate. Further treatment of the cell walls with LiCl and guanidine hydrochloride caused only a small loss of the amino acids and slight changes in their molar ratio.Abbreviations Gro glycerol - GroP monophosphate glycerol - GroP₂ diphosphate glycerol - Gro2P -monophosphate glycerol - P_TA phosphorus of teichoic acids - P_NA phosphorus of nucleic acids - TA teichoic acid     

Isolation and chemical characterization of lipopolysaccharides from four Mycoplana species (M. bullata,M. segnis,M. ramosa and M. dimorpha)

Lipopolysaccharides (LPS), isolated from four Mycoplana species, i.e. the type strains of M. bullata, M. segnis, M. ramosa and M. dimorpha, were characterized onto their chemical composition and their respective lipid A-types. Those of M. bullata and M. segnis showed on DOC-PAGE an R-type character and had lipid A's of the Lipid A_DAG-type which exclusively contained 2,3-diamino-2,3-dideoxy-d-glucose as lipid A sugar. LPS's of M. ramosa and M. dimorpha showed, although only weakly expressed, ladder-like patterns on DOC-PAGE indicating some S-type LPS's and lipid A of the d-glucosamine type (Lipid A_GlcN). M. bullata LPS contained mannose and glucose in major amounts and additionally l-glycero-d-mannoheptose, whereas M. segnis LPS was composed of rhamnose, mannose and glucose together with both, d-glycero-d-manno- and l-glycero-d-manno-heptoses in a molar ratio of 1:2. All LPS's contained 2-keto-3-deoxy-octonic acid (Kdo), phosphate and an unidentified acidic component X. In addition to X, M. segnis LPS contained glucuronic and galacturonic acids, whereas M. ramosa LPS contained only galacturonic acid. Acetic acid hydrolysis of the LPS resulted in splitting off lipid A moieties, very rich in 3-hydroxy fatty acids, in particular in 3-OH-12:0 (in Lipid A_DAG), or in 3-OH-14:0 (in Lipid A_GlcN). Analysis of the 3-acyloxyacyl residues revealed major amounts of amide-linked 3-OH(3-OH-13:0)12:0 in lipid A of M. bullata and 3-OH(12:0)12:0 in lipid A of M. segnis. The rare 4-oxo-myristic acid (4-oxo-14:0) was observed only in M. bullata LPS, where it is ester-linked. Amide linked diesters could not be traced in M. ramosa and M. dimorpha. All four lipid A's lacked erster-bound acyloxyacyl residues.Non-standard abbreviations DAG 2,3-diamino-2,3-dideoxy-d-glucose - Kdo 2-keto-3-deoxy-octonate - LPS lipopolysaccharide - PITC phenyl isothiocyanate - NANA N-acetyl neuraminic acid     

α-Chymotrypsin-Catalyzed Peptide Synthesis Using <Emphasis Type="Italic">N</Emphasis>-Protected <Emphasis Type="SmallCaps">D</Emphasis>-Amino Acid Carbamoylmethyl Esters as Acyl Donors

α-Chymotrypsin-catalyzed peptide synthesis was carried out between an N-protected D-amino acid ester and an L-amino acid amide (acyl donor, 10 mM; acyl acceptor, 50 mM; enzyme, 2 mg ml⁻¹; pH 8). By using a highly reactive carbamoylmethyl (Cam) ester as acyl donor, the D-amino acid was incorporated into the N-terminus of the resulting dipeptide amide. N-Protected dipeptide amides bearing D-amino acids such as D-Phe, D-Leu and D-Ala at their N-terminus were synthesized in high yields (up to 80%) in 1–3 h.     

The enzymatic malonylation of 1-aminocyclopropane-1-carboxylic acid in homogenates of mung-bean hypocotyls

Homogenates of hypocotyls of light-grown mung-bean (Vigna radiata (L.) Wilczek) seedlings catalyzed the formation of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC) from the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and malonyl-coenzyme A. Apparent K_m values for ACC and malonyl-CoA were found to be 0.17 mM and 0.25 mM, respectively. Free coenzyme A was an uncompetitive inhibitor with respect to malonyl-CoA (apparent K_i=0.3 mM). Only malonyl-CoA served as an effective acyl donor in the reaction. The d-enantiomers of unpolar amino acids inhibited the malonylation of ACC. Inhibition by d-phenylalanine was competitive with respect to ACC (apparent K_i=1.2 mM). d-Phenylalanine and d-alanine were malonylated by the preparation, and their malonylation was inhibited by ACC. When hypocotyl segments were administered ACC in the presence of certain unpolar d-amino acids, the malonylation of ACC was inhibited while the production of ethylene was enhanced. Thus, a close-relationship appears to exist between the malonylation of ACC and d-amino acids. The cis- as well as the trans-diastereoisomers of 2-methyl- or 2-ethyl-substituted ACC were potent inhibitors of the malonyltransferase. Treatment of hypocotyl segments with indole-3-acetic acid or CdCl₂ greatly increased their content of ACC and MACC, as well as their release of ethylene, but had little, or no, effect on their extractable ACC-malonylating activity.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - MACC 1-(malonylamino)-cyclopropane-1-carboxylic acid Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

The lipopolysaccharide of the phototrophic bacterium Ectothiorhodospira vacuolata

The lipopolysaccharide of Ectothiorhodospira vacuolata was obtained by the phenol-water procedure. It contained a 3-O-methyl-hexose, glucose, galacturonic and glucuronic acids. The finding of d-glycero-d-mannoheptose and 2-keto-3-deoxyoctonate (tentatively identified) suggested a core-structure. The lipid fraction of the lipopolysaccharide contained phosphate and both, 2,3-diamino-2,3-dideoxy-d-glucose and d-glucosamine. The major fatty acids were amine-bound 3-OH-10:0 and 3-OH-12:0 and esterbound 14:0 and 16:0 Sodium deoxycholate gel-electrophoresis, showing a single band only, indicated R-type character of the lipopolysaccharide of Ectothiorhodospira vacuolata.Abbreviations DOC sodium deoxycholate - GC/MS combined gasliquid chromatography - PAGE polyacrylamide gel-electrophoresis     

Racemic Resolution of some <Emphasis Type="SmallCaps">dl</Emphasis>-Amino Acids using <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis><Emphasis Type="SmallCaps">l</Emphasis>-Amino Acid Oxidase

The ability of Aspergillus fumigatus l-amino acid oxidase (l-aao) to cause the resolution of racemic mixtures of dl-amino acids was investigated with dl-alanine, dl-phenylalanine, dl-tyrosine, and dl-aspartic acid. A chiral column, Crownpak CR+ was used for the analysis of the amino acids. The enzyme was able to cause the resolution of the three dl-amino acids resulting in the production of optically pure d-alanine (100% resolution), d-phenylalanine (80.2%), and d-tyrosine (84.1%), respectively. The optically pure d-amino acids have many uses and thus can be exploited industrially. This is the first report of the use of A. fumigatus l-amino acid oxidase for racemic resolution of dl-amino acids.     

Sucrose phosphorylases catalyze transglycosylation reactions on carboxylic acid compounds

Two sucrose phosphorylases were employed for glycosylation of carboxylic acid compounds. Streptococcus mutans sucrose phosphorylase showed remarkable transglycosylating activity, especially under acidic conditions. Leuconostoc mesenteroides sucrose phosphorylase exhibited very weak transglycosylating activity. Three main products were detected from the reaction mixture using benzoic acid and sucrose as an acceptor and a donor molecule, respectively. These compounds were identified as 1-O-benzoyl α-d-glucopyranoside, 2-O-benzoyl α-d-glucopyranose, and 2-O-benzoyl β-d-glucopyranose by 1D-and 2D-NMR analyses of the isolated products and their acetylated products. Time-course analyses proved that 1-O-benzoyl α-d-glucopyranoside was initially produced by the transglycosylation reaction of the enzyme. 2-O-Benzoyl α-d-glucopyranose and 2-O-benzoyl β-d-glucopyranose were produced from 1-O-benzoyl α-d-glucopyranoside by intramolecular acyl migration reaction. S. mutans sucrose phosphorylase showed broad acceptor-specificity. This sucrose phosphorylase catalyzed transglycosylation to various carboxylic compounds such as short-chain fatty acids, hydroxy acids, dicarboxylic acids, and phenolic carboxylic acids. 1-O-Acetyl α-d-glucopyranoside was also enzymatically synthesized by transglucosylation reaction of the enzyme. The sensory test of acetic acid and the glucosides revealed that the sour taste of acetic acid glucosides was significantly lower than that of acetic acid.     

Chemical composition of the lipopolysaccharides of Ectothiorhodospira shaposhnikovii,Ectothiorhodospira mobilis,and Ectothiorhodospira halophila

Lipopolysaccharides were isolated from the moderate halophilic Ectothiorhodospira shaposhnikovii slight to and Ectothiorhodospira mobilis and from the extremely halophilic Ectothiorhodospira halophila by the hot phenol-water and purified by the phenol-chloroform-petroleum ether methods. The isolated lipopolysaccharides of all three species contained 3-deoxy-d-manno-octulosonic acid and d-glycero-d-mannoheptose indicating the existence of a core. They contained additionally glucose and uronic acids (E. shaposhnikovii and E. mobilis) or glucose, uronic acids and threonine (E. halophila). Sodium deoxycholate gel-electrophoresis of the three lipopolysaccharides, each showing only one major band, indicated R-type character of the lipopolysaccharides of the three Ectothiorhodospira species.The lipid A fractions of the lipopolysaccharides from E. shaposhnikovii and E. mobilis represented phosphorylated mixed lipid A types with both 2,3-diamino-2,3-dideoxy-d-glucose and d-glucosamine. The lipid A from E. halophila contained also phosphate and 2,3-diamino-2,3-dideoxy-d-glucose but only traces of d-glucosamine, which would indicated lipid A_DAG. The fatty acid spectra were characterized by amide-bound 3-OH-10:0 and 3-OH-12:0 (E. shaposhnikovii), 3-OH-10:0 (E. mobilis), or 3-OH-10:0,3-OH-14:0, and 3-oxo-14-0 (E. halophila). The predominant ester-bound fatty acids were 14:0 and 16:0 (E. shaposhnikovii and E. mobilis), or 12:0 and 14:1 (E. halophila).Abbreviations DAG 2,3-diamino-2,3-dideoxy-d-glucose - Kdo 3-deoxy-d-manno-octulosonic acid - GlcA glucuronic acid - GalA galacturonic acid - GC-MS combined gas liquid chromatographymass spectrometry - GlcN Glucosamine - DOC sodium deoxycholate - LPS lipopolysaccharide - PAGE polyacrylamide gel electrophoresis - PCP phenol-chloroform-petroleum ether     

Characterization of a novel lipopolysaccharide biosurfactant fromKlebsiella oxitoca

The chemical, physical, and emulsifying properties of BSF-1, which is an extracellular lipopolysaccharide biosurfactant produced byKlebsiella oxytoca strain BSF-1, were studied. BSF-1 was found to be composed mainly of carbohydrate and fatty acids. The average molecular weight was 1,700–2,000 kDa. The polysaccharide fraction containedl-rhamnose,d-galactose,d-glucose, andd-glucuronic acid at a molar ratio of 3∶1∶1∶1. The fatty acid content was 1.1% (w/w) and consisted mainly of palmitic acid (C16∶0), 3-hydroxylauric acid (3-OH-C12∶0), and lauric acid (C12∶0). In terms of thermal properties, BSF-1 was revealed to have inter- and intra-molecular hydrogen bonds. The hydrodynamic volume (intrinsic viscosity) of BSF-1 was 22.8 dL/g. BSF-1 could be maintained as a stable emulsion for 48 h through a low-level reduction in surface tension. The optimal emulsification temperature was 30°C. Emulsification by BSF-1 was efficient at both acidic and neutral pH values.     

Developmental regulation of aldoxime formation in seedlings and mature plants of Chinese cabbage (Brassica campestris ssp. pekinensis) and oilseed rape (Brassica napus): Glucosinolate and IAA biosynthetic enzymes

The first steps in the biosynthesis of glucosinolates and indole-3-acetic acid (IAA) in oilseed rape (Brassica napus L.) and Chinese cabbage (Brassica campestris ssp. pekinensis) involve the formation of aldoximes. In rape the formation of aldoximes from chain-extended amino acids, for aromatic and aliphatic glucosinolate biosynthesis, is catalysed by microsomal flavin-containing monooxygenases. The formation of indole-3-aldoxime from l-tryptophan, the potential precursor of both indole-3-acetic acid and indolyl-glucosinolates, is catalysed by several microsomal peroxidases. The biosynthesis of glucosinolates and indole-3-acetic acid was shown to be under developmental control in oilseed rape and Chinese cabbage. No monooxygenase activities were detected in cotyledons or old leaves of either species. The highest monooxygenase activities were found in young expanding leaves; as the leaves reached full expansion and matured the activities decreased rapidly. The indole-aldoxime-forming activity was found in all of the tissues analysed, but there was also a clear decrease in foliar activity with maturity in leaves of rape and Chinese cabbage. Partial characterisation of the Chinese cabbage monooxygenases showed that they have essentially identical properties to the previously characterised rape enzymes; they are not cytochrome P450-type enzymes, but resemble flavin-containing monooxygenases. No monooxygenase inhibitors were detected in microsomes prepared from either cotyledons or old leaves.Abbreviations DHMet dihomomethionine - FMO flavin-containing monooxygenase - HPhe homophenylalanine - IAA indole-3-acetic acid - l-Phe l-phenylalanine - l-Trp l-tryptophan - MO monooxygenase - IAALD indole-3-acetaldehyde - IAOX indole-3-aldoxime - THMet trihomomethionine     

Single-channel measurements of an <Emphasis Type="Italic">N</Emphasis>-acetylneuraminic acid-inducible outer membrane channel in <Emphasis Type="Italic">Escherichia coli</Emphasis>

NanC is an Escherichia coli outer membrane protein involved in sialic acid (Neu5Ac, i.e., N-acetylneuraminic acid) uptake. Expression of the NanC gene is induced and controlled by Neu5Ac. The transport mechanism of Neu5Ac is not known. The structure of NanC was recently solved (PDB code: 2WJQ) and includes a unique arrangement of positively charged (basic) side chains consistent with a role in acidic sugar transport. However, initial functional measurements of NanC failed to find its role in the transport of sialic acids, perhaps because of the ionic conditions used in the experiments. We show here that the ionic conditions generally preferred for measuring the function of outer-membrane porins are not appropriate for NanC. Single channels of NanC at pH 7.0 have: (1) conductance 100 pS to 800 pS in 100 mM KCl to 3 M KCl), (2) anion over cation selectivity (V _reversal = +16 mV in 250 mM KCl || 1 M KCl), and (3) two forms of voltage-dependent gating (channel closures above ±200 mV). Single-channel conductance decreases by 50% when HEPES concentration is increased from 100 μM to 100 mM in 250 mM KCl at pH 7.4, consistent with the two HEPES binding sites observed in the crystal structure. Studying alternative buffers, we find that phosphate interferes with the channel conductance. Single-channel conductance decreases by 19% when phosphate concentration is increased from 0 mM to 5 mM in 250 mM KCl at pH 8.0. Surprisingly, TRIS in the baths reacts with Ag|AgCl electrodes, producing artifacts even when the electrodes are on the far side of agar–KCl bridges. A suitable baseline solution for NanC is 250 mM KCl adjusted to pH 7.0 without buffer.     

Glutamate uptake system in the presynaptic vesicle: Glutamic acid analogs as inhibitors and alternate substrates

A variety of naturally occurring amino acids, their isomers, and synthetic analogs were tested for their ability to inhibit uptake of [³H]glutamate into presynaptic vesicles from bovine cerebral cortex. Strongest inhibition (K_i<1mM) was observed fortrans-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD) anderythro-4-methyl-L-glutamic acid (MGlu), while 4-methylene-L-glutamic acid (MeGlu) was only moderately inhibitory (Ki=3mM), indicating that the synaptic vesicle glutamate translocator has higher affinity forrans-ACPD and MGlu than for glutamate. A few other amino acids, e.g., 4-hydroxyglutamic acid, S-carboxyethyl cysteine, and 5-fluorotryptophan, were slightly inhibitory; alll- anddl-isomers of protein amino acids and longer chain acidic amino acids were without measurable inhibition. Potassium tetrathionate and S-sulfocysteine exhibited strong to moderate noncompetitive or irreversible inhibition. Inhibition by t-ACPD, MGlu, or MeGlu was competitive with glutamic acid. Each of these competitive inhibitors was also taken up by the vesicle preparation in an ATP-dependent manner, as indicated by their being recovered unchanged from filtered vesicles. Similar results were obtained with reconstituted vesicles, while glutamate uptake by partially purified rat synaptosomes was inhibited only by MGlu. These results indicate that the glutamate translocator of presynaptic vesicles has stringent structural requirements distinct from those of the plasma membrane translocator and the metabotropic type of postsynaptic glutamate receptor. They further suggest possible structural requirements of pharmacologically significant compounds that can substitute for glutamic acid in the presynaptic side of glutamatergic synapses, thus serving to moderate or control glutamate excitation and associated excitotoxic effects in these neurons.Special issue dedicated to Dr. Paul Greengard     

Bacterial assimilation of d- and l-2-chloropropionates and occurrence of a new dehalogenase

The occurrence of a new bacterial dehalogenase acting on both the optical isomers of 2-halogenated alkanoic acids was demonstrated. When the haloalkanoic acid-utilizing bacteria were screened in a medium containing dl-2-chloropropionate as a sole carbon source, two types of bacteria were isolated: (1) a few strains utilizing both d- and l-isomers of 2-chloropropionate and (2) strains utilizing only the l-isomer. A dehalogenating enzyme was obtained from the cells of Pseudomonas sp. which is able to utilize both isomers. The crude enzyme catalyzed the dehalogenation of d- and l-2-chloropropionates to yield l- and d-isomers of lactate, respectively. The enzyme showed the same pH optimum and heat inactivation rate for the d- and l-isomers. Apparent K _m values for d- and l-2-chloropropionates were 4.5 and 1.0 mM, respectively. The enzyme acted specifically on 2-haloalkanoic acids. Activity staining of disc-gels electrophoresed witg the crude enzyme preparation showed that the dehalogenation of d- and l-2-chloropropionates, monochloroacetate, dichloroacetate, 2,2-dichloropropionate, and dl-2-chlorobutyrate is due to a single protein.Abbreviations MCA monochloroacetic acid - DCA dichloroacetic acid - TCA trichloroacetic acid - 2 MCPA 2-monochloropropionic acid - 22 DCPA 2,2-dichloropropionic acid - 3 MCPA 3-monochloropropionic acid - 2 MCBA 2-monochlorobutyric acid - 3 MCBA 3-monochlorobutyric acid - 4 MCBA 4-monochlorobutyric acid     

Biochemical aspects on the germination of conidiospores of Aspergillus niger

Summary Biochemical events occurring in synchronously germinating spores of Aspergillus niger strain 1617 were investigated. The spores were found to require l-proline (or l-alanine), glucose and phosphate for the complete germination. The germination process in the above synthetic medium could be divided into three phases: endogenous swelling, exogenous swelling and sprouting. The first swelling phase was not influenced by the severe environmental factors so far tested, while the second phase was found to be affected by them, especially the CO₂ concentration. Rates of increase in cellular substances and in consumption of environmental substances changed markedly after germ tubes sprouted. The first cellular synthesis thus far detected was nucleic acid synthesis in the exogenous swelling phase. At the end of this phase accumulation of free amino acids, mainly glutamic acid and alanine, was observed. Protein synthesis then followed. A conspicuous increase in O₂-uptake commenced in parallel with the active synthesis of protein, when germ tubes began to sprout.During the course of germination a shift of metabolic pattern from that of the spore to the mycelium was indicated by the ratios of total nitrogen/dry weight, RNA/DNA, oxygen consumed/glucose consumed, and oxygen consumed/total nitrogen taken at various time intervals.Rosalie B. Hite Post-doctoral Fellow of the University of Texas.