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.     

D-Gluconate is an alternative growth substrate for cultivation of Schizosaccharomyces pombe mutants

Schizosaccharomyces pombe cells grow on d-gluconate as the sole carbon and energy source. d-Gluconate is taken up in symport with protons by a specific symporter, pH being the sole driving force. d-Gluconate uptake is independent of the sugar transporting system (e.g. for d-glucose) and of . The carrier is expressed constitutively, and its activity is not subject to glucose repression. Hence, d-gluconate is a suitable carbon and energy source for growth, when d-glucose or other hexoses have to be eliminated e.g. for selection of mutants deficient in hexose transport.Abbreviations 2-DG 2-deoxy-d-glucose - CCCP carbonylcyanide m-chlorophenylhydrazone - pH pH-gradient - electrical potential difference across the plasma membrane - SD standard deviation - SEM standard error of the mean - TPP⁺ tetraphenylphosphonium     

Formation of d(-)-1,2-propanediol and d(-)-lactate from glucose by Clostridium sphenoides under phosphate limitation

Clostridium sphenoides was grown on glucose in a phosphate-limited medium. Below 80 M phosphate two new products were formed in addition to ethanol, acetate, H₂ and CO₂: d(-)-1,2-propanediol and d(-)-lactate. These compounds were apparently synthesized via the methylglyoxal by-pass. The activity of the enzymes involvedmethylglyoxal synthase, methylglyoxal reductase, 1,2-propanediol dehydrogenase and glyoxalase-could be demonstrated in cell extracts of C. sphenoides. The formation of 1,2-propanediol from methylglyoxal proceeded via lactaldehyde. The enzyme methylgloxal synthase was inhibited by phosphate. Clostridium glycolicum, C. nexile, C. cellobioparum, C. oroticum and C. indolis did not produce propanediol under the condition of phosphate limitation. The latter two species, however, formed d(-)-lactate.Dedicated to Prof. Dr. G. Drews on the occasion of his 60th birthday     

Modulation of adenosine-induced cAMP accumulation via metabotropic glutamate receptors in chick optic tectum

Changes on cyclic adenosine monophosphate (cAMP) levels in response to adenosine and glutamate and the subtype of glutamate receptors involved in this interaction were studied in slices of optic tectum from 3-day-old chicks. cAMP accumulation mediated by adenosine (100 M) was abolished by 8-phenyltheophylline (15 uM). Glutamate and the glutamatergic agonists kainate or trans-d,l-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) did not evoke cAMP accumulation. Glutamate blocked the adenosine response in a dose-dependent manner. At 100 M, glutamate did not inhibit the effect of adenosine. The 1 mM and 10 mM doses of glutamate inhibited adenosine-induced cAMP accumulation by 55% and 100%, respectively. When glutamatergic antagonists were used, this inhibitory effect was not affected by 200 M 6,7-dihydroxy-2,3,dinitroquinoxaline (DNQX), an ionotropic antagonist, and was partially antagonized by 1 mM (rs)-alpha-methyl-4-carboxyphenylglycine [(rs)M-CPG], a metabotropic, antagonist, while 1 mMl-2-amino-3-phosphonopropionate (l-AP3) alone, another metabotropic antagonist, presented the same inhibitory effect of glutamate. Kainate (10 mM) and trans-ACPD (100 M and 1 mM) partially blocked the adenosine response. This study indicates the involvement of metabotropic glutamate receptors in adenylate cyclase inhibition induced by glutamate and its agonists trans-ACPD and kainate.Abbreviations ADO adenosine - DNQX 6,7-dihydroxy-2,3-dinitro-quinoxaline - KA kainate - l-AP3 l-2-amino-3-phosphonopropionate - mGluRs metabotropic glutamate receptors - P-THEO 8-phenyltheophylline - (rs)M-CPG (rs)-alpha-methyl-4-carboxyphenyl-glycine - trans-ACPD trans-d,l-1-aminocyclopentane-1,3-dicarboxyho acid     

Automethylation of protein (d -aspartyl/l -isoaspartyl) carboxyl methyltransferase, a response to enzyme aging

A question that is central to understanding the mechanisms of aging and cellular deterioration is whether enzymes involved in recognition and metabolism of spontaneously damaged proteins are themselves damaged, either becoming substrates for their own activity; or being unable to act upon themselves, initiating cascades of cellular damage. We show here byin vitro experiments that protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase (PCM) from bovine erythrocytes does methylate age-dependent amino acid damage in its own sequence. The subpopulation that is methylated, termed thePCM fraction, appears to be formed through age-dependent deamidation of an asparaginyl site to either anl-isoaspartyl ord-aspartyl site because (a) the stoichiometry of automethylation of purified PCM is less than 1%, a value typical of the substoichiometric methylation of many other aged protein substrates, (b)PCM is slightly more acidic than the bulk of PCM, and (c) the methyl esterified site inPCM has the characteristic base-lability of this type of methyl ester. Also, the methyl group is not incorporated into the enzyme as an active site intermediate because the incorporated methyl group is not chased onto substrate protein. The effect of enzyme dilution on the rate of the automethylation reaction is consistent with methylation occurring between protein molecules, showing that the pool of PCM is autocatalytic even though individual molecules may not be. The automethylation and possible self-repair of the PCM pool has implications for maintaining thein vivo efficiency of methylation-dependent protein repair.     

Organization and characterization of three genes involved in d-xylose catabolism in Lactobacillus pentosus

Summary A cluster of three genes involved in d-xylose catabolism (viz. xylose genes) in Lactobacillus pentosus has been cloned in Escherichia coli and characterized by nucleotide sequence analysis. The deduced gene products show considerable sequence similarity to a repressor protein involved in the regulation of expression of xylose genes in Bacillus subtilis (58%), to E. coli and B. subtilis d-xylose isomerase (68% and 77%, respectively), and to E. coli d-xylulose kinase (58%). The cloned genes represent functional xylose genes since they are able to complement the inability of a L. casei strain to ferment d-xylose. NMR analysis confirmed that ¹³C-xylose was converted into ¹³C-acetate in L. casei cells transformed with L. pentosus xylose genes but not in untransformed L. casei cells. Comparison with the aligned amino acid sequences of d-xylose isomerases of different bacteria suggests that L. pentosus d-xylose isomerase belongs to the same similarity group as B. subtilis and E. coli d-xylose isomerase and not to a second similarity group comprising d-xylose isomerases of Streptomyces violaceoniger, Ampullariella sp. and Actinoplanes. The organization of the L. pentosus xylose genes, 5-xylR (1167 bp, repressor) — xylA (1350 bp, D-xylose isomerase) — xylB (1506 bp, d-xylulose kinase) — 3 is similar to that in B. subtilis. In contrast to B. subtilis xylR, L. pentosus xylR is transcribed in the same direction as xylA and xylB.     

Purine glucosylating activity in cell suspension cultures ofSolanum tuberosum L.

Cell suspension cultures ofSolanum tuberosum L. cv. Adretta were established from leaf-derived calluses. In the search for purine glucosylating activity, the metabolism of 6-benzylaminopurine was studied. The main metabolite of BA was isolated and identified as 6-benzylaminopurine 7--d-glucopyranoside indicating the occurrence of purine glucosylating activity.Abbreviations BA 6-Benzylaminopurine - [3G]BA BA 3--d-glucopyranoside - [7G]BA BA 7--d-glucopyranoside - [9G]BA BA 9--d-glucopyranoside - RA Radioactivity - R _T Retention Time     

Adherence of a virulent strain of Listeria monocytogenes to the surface of a hepatocarcinoma cell line via lectin-substrate interaction

Listeria monocytogenes was examined for the presence of surface carbohydrates to ascertain whether surface sugars, if present, would interact with eucaryotic surface carbohydrate receptors. We found that a virulent, but not two avirulent strains had a surface -d-galactose residue as determined by agglutination with Griffonia simplicifolia (GS-I) and other lectins. The virulent strain bound to a human hepatocarcinoma cell line (HepG2), which has a well characterized receptor for -d-galactose. This interaction could be blocked by pretreatment of the HepG2 cells with either -d-galactose or neuraminidase, the latter of which will render the galactose receptor functionally inactive. We propose that the attachment of the virulent Listeria to eucaryotic cells occurs as a result of the interaction of the microbial -d-galactose with that of the eucaryotic galactose receptor. This surface carbohydrate may provide an explanation for the mechanism of attachment and penetration of virulent Listeria into host cells during infection. As such, this may allow for amplication of pathogenesis through intracellular multiplication in nonprofessional phagocytes prior to macrophage involvement.Abbreviations ATCC 19113 and ATCC 4428 Listeria monocytogenes, avirulent strains - EDG Listeria monocytogenes, virulent strain - GS-I Griffonia simplicifolia lectin - GepG2 Human, hepatocarcinoma cells - MES buffer 2(N-Morpholino)-ethane sulfonic acid - PBS buffer phosphate buffered saline This work was carried out and submitted in part by JL to the 47th Annual Westinghouse Science Talent Search while a senior at Union High School, Tulsa, OklahomaRecipient of a National Merit Scholarship Award. Presently attending Southwest Missouri State University, Springfield, Missouri     

Proliferation and metabolic significance of peroxisomes in Candida boidinii during growth on d-alanine or oleic acid as the sole carbon source

We have studied the induction of peroxisomes in the methylotrophic yeast Candida boidinii by d-alanine and oleic acid. The organism was able to utilize each of these compounds as the sole carbon source and grew with growth rates of =0.20 h^-1 (on d-alanine) or =0.43 h^-1 (on oleic acid). Growth was associated with the development of many peroxisomes in the cells. On d-alanine a cluster of tightly interwoven organelles was observed which made up 6.3% of the cytoplasmic volume and were characterized by the presence of d-amino acid oxidase and catalase. On oleic acid rounded to elongated peroxisomes were dominant which were scattered throughout the cytoplasm. These organelles contained increased levels of -oxidation enzymes; their relative volume fraction amounted 12.8% of the cytoplasmic volume.     

NovelN-linked oligo-mannose type oligosaccharides containing an α-d-galactofuranosyl linkage found in α-d-galactosidase fromAspergillus niger

Structures of oligosaccharides fromAspergillus niger -d-galactosidase [EC 3.2.1.22] were studied. Purified -d-galactosidase was treated withN-glycosidase F, and six kinds of oligosaccharides were isolated by gel chromatography and anion-exchange chromatography. The structures of the oligosaccharides were determined by¹H-NMR and compositional analysis to be Man₅GlcNAc₂, Man₆GlcNAc₂, Man₉GlcNAc₂, GlcMan₉GlcNAc₂, GalMan₄GlcNAc₂ and GalMan₅GlcNAc₂. From mild acid hydrolysis, methylation analysis and ROESY spectral analysis, it was ascertained that the galactosyl residue in two oligosaccharides was in the furanose form and was bound to mannose at the nonreducing end with an 1–2 linkage (GalfMan₄GlcNAc₂ and GalfMan₅GlcNAc₂).     

Induction of extracellular arabinases on monomeric substrates in Aspergillus niger

The induction of extracellular arabinases by pentose sugars and polyols generated by the metabolic pathway of l-arabinose and d-xylose catabolism in Aspergillus niger was investigated. Induction occurred with l-arabinose and l-arabitol but not with d-xylose or xylitol. l-arabitol in particular was found to be a good inducer for -l-arabinofuranosidase and endo-arabinase activities. Western blotting analysis showed both -l-arabinofuranosidase A and B to be present. No induction was observed using d-arabitol. Unlike the wild type A. niger N402 strain, the A. niger xylulose kinase negative mutant N572 also showed induction of -l-arabinofuranosidases A and B and endo-arabinase activity on d-xylose and xylitol. This is due to metabolic conversion of these compounds leading to the accumulation of both xylitol and l-arabitol in this mutant, the latter of which then acts as inducer. The induction of the two -l-arabinofuranosidases and endo-arabinase is under the control of two regulatory systems namely pathway specific induction and carbon catabolite repression. Under derepressing conditions in the wild type only -l-arabinofuranosidase B could be detected by Western blotting analysis. This indicates that -l-arabinofuranosidase B is of importance in the initiation of specific induction of the various arabinose activities in A. niger grown on arabinose containing structural polysaccharides.Abbreviations PNA p-nitrophenyl--l-arabinofuranoside     

d-Alanine dehydrogenase

Pseudomonas aeruginosa PA01 was found to utilise both thed- andl-isomers of -alanine and also -alanine as sole sources of carbon and energy for growth. Enzymological studies of wild-type cultures and comparison with mutants deficient in growth upon one or more isomers of alanine led to the following conclusions: (i) utilisation ofd-alanine involved its direct oxidation by an inducible, membrane-bound, cytochrome-linked dehydrogenase; (ii) utilisation ofl-alanine required its conversion to the directly oxidisabled-form by a soluble racemase; (iii) utilisation of -alanine, likel-alanine, involves both the racemase andd-alanine dehydrogenase enzymes, but in addition must involve other enzymes the identity, of which is still speculative; (iv)P. aeruginosa, likeEscherichia coli, appears to take upd-alanine andl-alanine by means of two specific permeases.Abbreviation DCPIP 2,6-dichlorophenol-indophenol     

Structural studies of neuropeptides composed ofl -Asp andd -Glu by13C-NMR spectroscopy

¹³C-NMR spectral data are presented for four neuropeptides composed ofl-Asp, Ac-l-Asp, andd-Glu, which contain and peptide linkages. The data for the various compounds are compared to related dipeptides, one of which is a known neuropeptide, in order to gain structural information about these compounds. Electron-nuclear relaxation rates were used to elucidate the metal-ion binding sites of these species.     

Variant forms ofα-2-l-fucosyltransferase in human submaxillary glands from blood group ABH “secretor” and “non-secretor” individuals

The properties of the -2-l-fucosyltransferases in submaxillary gland preparations from blood group ABH secrefors and non-secretors were compared. The level of activity in the non-secretor gland homogenates amounted to about 5% only of that found in the secretor gland preparations. The enzymes from the two sources differed in solubility properties, charge and affinities for donor and acceptor substrates. The enzyme from secretor glands showed a preference for acceptors with Type 1 [d-galactosyl(1–3)-N-acetyl-d-glucosamine] structures whereas the enzyme from non-secretor glands had a preference for Type 2 [d-galactosyl(1–4)-N-acetyl-d-glucosamine] structures.These results demonstrate that expression of the secretor gene (Se) is associated with a molecular form of the -2-l-fucosyltransferase that is different from the species present in the same tissue when theSe gene is not expressed.     

Catabolism of malate and related dicarboxylic acids in various Desulfovibrio strains and the involvement of an oxygen-labile NADPH dehydrogenase

Four out of five Desulfovibrio strains tested were able to oxidize l-malate to acetate in the presence of sulfate. Fumarate and succinate were also oxidized to acetate by these strains, but growth with the latter substrate was marginal. During growth on malate high NADP-dependent malic enzyme and NADPH DH activities were found in all strains. These activities were lower in lactate-or pyruvate-grown cells. An NADPH DH from D. gigas was partially purified. It was oxygen-labile, very sensitive to heavy metal ions and highly specific for NADPH. Growth yield studies indicated that energy conservation occurred during the transport of reducing equivalents from NADPH to the sulfate reduction pathway.Abbreviations DH dehydrogenase - DCPIP 2,6-dichlorophenolindophenol - MTT 3-(4,5-dimethylthiazol-2-yl)-2,4-diphenyltetrazolium bromide - HEPES 4-(2-hydroxyethyl-1-piperazine ethane sulfonic acid - PIPES piperazine-1,1-bis(2-ethane sulfonic acid) - MES 2-(N-morpholino)-ethane sulfonic acid - DTT dithiothreitol     

Effects of cytochalasins on developing frog embryos: protection with l-cysteine and α-d-glucosamine

Summary Cytochalasins A, B and H (CA, CB and CH) brought about cellular disaggregation and mortality in the developing embryos of the frog, Microhyla ornata. All three cytochalasins exhibited a dose-response relationship. CA was the most potent and its effects were significantly reduced by simultaneous additon of l-cysteine, a sulph-hydryl compound. -d-Glucosamine, a precursor of complex macromolecules important in cell adhesion, protected against the effects of CH. The effects of CB, however, were influenced neither by l-cysteine nor by -d-glucosamine. The results revealed differences in the mechanism of action of these three cytochalasins.     

β-Dl-Methylene-aspartate,an inhibitor of aspartate aminotransferase,potently inhibitsl-glutamate uptake into astrocytes

[³H]Glutamate uptake into astrocytes in primary culture was potently inhibited by the aspartate analoguesl- andd-aspartic acid,Dl-threo--hydroxy-aspartic acid,l-aspartic acid--hydroxymate (IC50's: 136, 259, 168, and 560 M, respectively) and by -Dl-methylene-aspartate, a suicide inhibitor of asparate aminotransferase (IC50: 524 M), and by the endogenous sulphur-containing amino acidl-cysteinesulfinic acid (IC50: 114 M). [³H]Glutamate uptake was not significantly affected by either N-methyl-d-aspartate orDl-homocysteine thiolactone. These results demonstrate that other excitatory amino acids including aspartate andl-cysteinesulfinic acid (but excludingl-homocysteic acid) interact with the glutamate transport system of astrocytes. Inhibition of glutamate uptake may significantly increase the level of neuronal excitability.     

Seaweed liquid fertilizer fromAscophyllum nodosum contains elicitors of plantd-glycanases

The structure of the major component contained in a liquid seaweed extract prepared fromAscophyllum nodosum (Phaeophyta, Fucales) was investigated. The extract was fractionated by gel permeation chromatography, and the various fractions were analysed by GLC, HPLC,¹³C NMR spectroscopy and mass spectrometry. All fractions were derivatives of the branched -d-(13) glucan known as laminaran. They were capable of elicitingd-glycanase activities (laminaranase, -amylase) inRubus fruticosus suspended-cell cultures.     

Purification and partial characterization of ad(−)-lactate dehydrogenase fromDesulfovibrio desulfuricans (ATCC 7757)

Summary A membrane-boundd(–)-lactate dehydrogenase (LDH), an important enzyme in carbon and energy metabolism in sulfate-reducing bacteria of the genusDesulfovibrio, was solubilized from the membrane fraction ofDesulfovibrio desulfuricans (ATCC 7757). The enzyme was purified 84-fold to a final specific activity of 525 nmol DCPIP-reduced/min/mg protein by ammonium sulfate precipitation, chloroform extraction, gel filtration with Sephadex G-150, and hydrophobic column chromatography withN-octylamine Sepharose 4B. The enzyme eluted off a Sephacryl S-300 column as a single peak with a molecular weight of 400 000±40 000 Da. Denaturing gel electrophoresis showed it to be composed of 5 protein bands. The oxidized and dithionite reduced spectra of LDH resembles the spectra ofc-type cytochromes found inDesulfovibrio species. The addition of lactate to LDH resulted in a partially reduced spectrum. The flavin/cytochromec/non-heme iron content per 400 000 Da LDH molecular weight was found to be 11.64.5. The LDH activity was specific ford(–)-lactate and had aK _m ford(–)-lactate of 4.3×10^–4 M. The pH optimum was between 6.5 and 8.5.     

Enzymic synthesis of the trisaccharide core region of the carbohydrate chain ofN-glycoprotein

Transmannosylation from mannotriose (Man1-4Man1-4Man) to the 4-position at the nonreducing end N-acetylglucosaminyl residue ofN,N-diacetylchitobiose was regioselectively induced through the use of -d-mannanase fromAspergillus niger. The enzyme formed the trisaccharide Man1-4GlcNAc1-4GlcNAc (3.7% of the enzyme-catalysed net decrease ofN,N-diacetylchitobiose) from mannotriose as a donor andN,N-diacetylchitobiose as an acceptor. Mannobiose (Man1-4Man) was also shown to be useful as a donor substrate for the desired trisaccharide synthesis.Abbreviations Man d-mannose - (M _n) (n=1–5) -linkedn-mer of mannose - GlcNAc₂ 2-acetamido-2-deoxy--d-glucopyranosyl-(1–4)-2-acetamido-2-deoxy-d-glucose