Showing papers on "Melibiose published in 1985"
TL;DR: In intact Escherichia coli cells, severe osmotic stress almost totally inhibited active transport of carbohydrate by all of the systems known to transport carbohydrates in E. coli: group translocation (glucose), binding protein mediated transport (maltose), proton symport (lactose), and sodium cotransport (melibiose).
84 citations
Journal Article•
TL;DR: The presence of a complex pattern of endogenous lectins and their biochemical characteristics may contribute to an understanding of intercellular interaction during the complex process of metastatic spread and may furthermore allow a new tool for diagnosis and a lectin-based therapy.
Abstract: Salt and detergent extracts of a malignant epithelial tumor, obtained by extraction of acetone powder, were fractionated on different sets of Sepharose columns covalently derivatized with lactose, asialofetuin, melibiose, mannan, fucose, and heparin. Successive elution by chelating reagent and specific sugar resulted in isolation of different Ca2+-dependent and Ca2+-independent endogenous carbohydrate-binding proteins, as analyzed by gel electrophoresis. It appears from the analysis that certain bands represent newly identified proteins capable of binding to lactose (at M 64,000), melibiose (at M , 28,000), and fucose (at M , 62,000 and 70,000). Other carbohydrate-binding proteins isolated from this human tumor have been identified in normal, especially embryonic, tissues of different nonhuman vertebrates. The carbohydrate-binding proteins are assayable as agglutinin with rabbit erythrocytes and show no detectable enzymatic activity. They can thus be defined as lectins. The presence of a complex pattern of endogenous lectins and their biochemical characteristics may contribute to an understanding of intercellular interaction during the complex process of metastatic spread and may furthermore allow a new tool for diagnosis and a lectin-based therapy.
72 citations
TL;DR: The results of this study indicate that the substrate recognition site of the lactose carrier is located within the plane of the lipid bilayer, and a tertiary structure model is proposed that suggests how certain transmembrane segments might be localized relative to one another.
Abstract: The wild-type lactose carrier of Escherichia coli has a poor ability to transport the disaccharide maltose. However, it is possible to select lactose carrier mutants that have an enhanced ability to transport maltose by growing E. coli cells on maltose minimal plates in the presence of isopropyl thiogalactoside (an inducer of the lac operon). We have utilized this approach to isolate 18 independent lactose permease mutants that transport maltose. The relevant DNA sequences have been determined, and all of the mutations were found to be single base pair changes either at triplet 177 or at triplet 236. The nucleotide changes replace alanine-177 with valine or threonine, or tyrosine-236 with phenylalanine, asparagine, serine, or histidine. Transport experiments indicate that all of the mutants have faster maltose transport compared with the wild-type strain. Position 177 mutants retain the ability to transport galactosides, such as lactose and melibiose, at rates similar to the rate of the wild-type strain. In contrast, the position 236 mutants are markedly defective in the ability to transport galactosides. With regard to secondary structure, alanine-177 and tyrosine-236 are located on adjacent hydrophobic segments of the lactose carrier that are predicted to span the membrane. Thus, the results of this study indicate that the substrate recognition site of the lactose carrier is located within the plane of the lipid bilayer. In addition, a tertiary structure model is proposed that suggests how certain transmembrane segments might be localized relative to one another.
72 citations
TL;DR: The isolation of structural mutants unable to grow on lactose was achieved by mutagenic treatment and these mutants showed no expression of the lactose transport system, the lmrT mutants, or the mainly expressed beta-galactosidase, lacZ mutants.
Abstract: Wild-type strains of the phytopathogenic enterobacterium Erwinia chrysanthemi are unable to use lactose as a carbon source for growth although they possess a beta-galactosidase activity. Lactose-fermenting derivatives from some wild types, however, can be obtained spontaneously at a frequency of about 5 X 10(-7). All Lac+ derivatives isolated had acquired a constitutive lactose transport system and most contained an inducible beta-galactosidase. The transport system, product of the lmrT gene, mediates uptake of lactose in the Lac+ derivatives and also appears to be able to mediate uptake of melibiose, raffinose, and galactose. Two genes encoding beta-galactosidase enzymes were detected in E. chrysanthemi strains. That mainly expressed in the wild-type strains was the lacZ product. The other, the lacB product, is very weakly expressed in these strains. These enzymes showed different affinities for the substrates o-nitrophenyl-beta-D-galactopyranoside and lactose and for the inhibitors isopropyl-beta-D-thiogalactopyranoside and galactose. The lmrT and lacZ genes of E. chrysanthemi, together with the lacI gene coding for the regulatory protein controlling lacZ expression, were cloned by using an RP4::miniMu vector. When these plasmids were transferred into Lac- Escherichia coli strains, their expression was similar to that in E. chrysanthemi. The cloning of the lmrT gene alone suggested that the lacZ or lacB gene is not linked to the lmrT gene on the E. chrysanthemi chromosome. One Lac+ E. chrysanthemi derivative showed a constitutive synthesis of the beta-galactosidase encoded by the lacB gene. This mutation was dominant toward the lacI lacZ cloned genes. Besides these mutations affecting the regulation of the lmrT or lacB gene, the isolation of structural mutants unable to grow on lactose was achieved by mutagenic treatment. These mutants showed no expression of the lactose transport system, the lmrT mutants, or the mainly expressed beta-galactosidase, lacZ mutants. The lacZ mutants retained a very low beta-galactosidase level, due to the lacB product, but this level was low enough to permit use of the lacZ mutants for the construction of gene fusions with the Escherichia coli lac genes.
65 citations
01 Jan 1985
TL;DR: This chapter discusses the salient features of lactose and melibiose permeases, and describes the topology of the proteins of the maltodextrin permease and catabolic enzyme system in Escherichia coli.
Abstract: This chapter discusses the salient features of lactose and melibiose permeases. Early experiments established that the melibiose permease in Salmonella typhimurium can couple substrate uptake to Na+ uptake with a stoichiometry of 1:1, and that sugar-cation symport can account for energization of the system. Some evidence suggests that the lactose and melibiose permeases exhibit common structural and functional attributes, and are, therefore, evolutionarily related. Both permeases function by cation co-transport, probably by a similar carrier-mediated mechanism. While the lactose permease functions exclusively by sugar-H + co-transport, the melibiose permease couples sugar uptake to the symport of H + , Li + , or Na + , depending of the conditions, the bacterial strain, and the sugar substrate under study. The chapter also discusses the genetics and biochemistry of the maltose regulon, encoding the maltose catabolic enzyme system. It describes the topology of the proteins of the maltodextrin permease and catabolic enzyme system in Escherichia coli . Maltoporin preferentially binds and transports hexoses of the gluco configuration and oligosaccharides in which the glucosyl residues are linked α-1,4. However, these in vitro specificity data are insufficient to account for relative in vivo transport rates.
62 citations
TL;DR: Neither alpha-galactosidase I nor enzyme II was able to remove galactose residues from intact guar gum, but both were capable of removing galactOSE residues from guar Gum which had been degraded into large fragments by mannanase.
Abstract: When Bacteroides ovatus is grown on guar gum, a galactomannan, it produces alpha-galactosidase I which is different from alpha-galactosidase II which it produces when grown on galactose, melibiose, raffinose, or stachyose. We have purified both of these enzymes to apparent homogeneity. Both enzymes appear to be trimers and have similar pH optima (5.9 to 6.4 for alpha-galactosidase I, 6.3 to 6.5 for alpha-galactosidase II). However, alpha-galactosidase I has a pI of 5.6 and a monomeric molecular weight of 85,000, whereas alpha-galactosidase II has a pI of 6.9 and a monomeric molecular weight of 80,500. alpha-Galactosidase I has a lower affinity for melibiose, raffinose, and stachyose (Km values of 20.8, 98.1, and 8.5 mM, respectively) than does alpha-galactosidase II (Km values of 2.3, 5.9, and 0.3 mM, respectively). Neither enzyme was able to remove galactose residues from intact guar gum, but both were capable of removing galactose residues from guar gum which had been degraded into large fragments by mannanase. The increase in specific activity of alpha-galactosidase which was associated with growth on guar gum was due to an increase in the specific activity of enzyme I. Low, constitutive levels of enzyme II also were produced. By contrast, enzyme II was the only alpha-galactosidase that was detectable in bacteria which had been grown on galactose, melibiose, raffinose, or stachyose.
55 citations
TL;DR: It is concluded that Proline 122 was replaced with serine in the melibiose carrier of all five mutants of Escherichia coli and caused the alteration in cation specificity (loss of coupling to H+) of the melibose carrier.
Abstract: The structural genes (melB) for the melibiose carrier of five mutants of Escherichia coli showing altered cation specificity for melibiose transport were cloned. The mutations were mapped in a 248-base-pair DNA fragment by a recombinational assay by using the mutants transformed with hybrid plasmids carrying various portions of the wild-type melB gene. The nucleotide sequences of the corresponding DNA fragments derived from mutated melB genes were determined, and the amino acid sequences of the carrier were deduced. Proline 122 was replaced with serine in the melibiose carrier of all five mutants (which were isolated independently). We conclude that this amino acid replacement caused the alteration in cation specificity (loss of coupling to H+) of the melibiose carrier.
49 citations
TL;DR: The results show strikingly that although the membrane potential primarily drives melibiose accumulation by both types of symport, it selectively affects the apparent affinity constant Kt of the H+-melibiose symport while it specifically changes the maximal rate of transport (Vmax) of the Na+.
38 citations
TL;DR: Measurements of specific activities of membrane fractions prepared from tissues incubated in labeled PCMBS show that the decrease concerns the 120,000 gravity pellet, but that very mild procedures must be chosen to prevent redistribution of label in the supernatant.
Abstract: Replacement of mannitol with sucrose decreases the binding of [(203)Hg]-p-chloromercuribenzenesulphonic acid (PCMBS) to Vicia faba leaf discs without epidermis. This decrease is optimal for 20 minutes on incubation, is concentration-dependent, and is also found with maltose and raffinose. In parallel experiments, the addition of sucrose, maltose, and raffinose during PCMBS pretreatment was shown to increase subsequent uptake of [U-(14)C]sucrose. In contrast, d- or l-glucose, 3-O-methylglucose, galactose, fructose, palatinose, turanose, or melibiose had no effect either on PCMBS binding or on [(14)C]sucrose uptake. The sucrose-induced decrease of PCMBS binding is retained after a cold and ionic shock. Measurements of specific activities of membrane fractions prepared from tissues incubated in labeled PCMBS show that the decrease concerns the 120,000 gravity pellet, but that very mild procedures must be chosen to prevent redistribution of label in the supernatant. Altogether, the data provide new support to the hypothesis that the active site of the sucrose carrier contains a group sensitive to PCMBS.
28 citations
TL;DR: Escherichia coli K-12 strains in the absence of the lactose carrier grew on the disaccharide melibiose as the sole source of carbon, and Li+-resistant mutants appeared after several days of incubation, which represent a new type of cation-coupling mutants of theMelibiose carrier.
Abstract: Escherichia coli K-12 strains in the absence of the lactose carrier grew on the disaccharide melibiose as the sole source of carbon. The presence of 0.1 mM Li+ in the medium strongly inhibited growth of such cells, and Li+-resistant mutants appeared after several days of incubation. These mutants showed altered cation coupling to melibiose transport via the melibiose carrier. Cotransport between H+ and melibiose was lost in the mutants, although Na+-melibiose cotransport was retained. We observed no Li+-melibiose cotransport. Therefore, these mutants represent a new type of cation-coupling mutants of the melibiose carrier.
26 citations
TL;DR: Strains of Leminorella gave positive tests for H2S production, acid production from L-arabinose and D-xylose, and tyrosine clearing; they were negative for indole production, Voges-Proskauer, urea hydrolysis, phenylalanine deaminase, motility, gelatin liquefaction, and arginine dihydrolase.
Abstract: Leminorella is proposed as a new genus for the group of Enterobacteriaceae formerly known as Enteric Group 57. Strains of Leminorella gave positive tests for H2S production, acid production from L-arabinose and D-xylose, and tyrosine clearing; they were negative for indole production, Voges-Proskauer, urea hydrolysis, phenylalanine deaminase, motility, gelatin liquefaction, lysine and ornithine decarboxylases, arginine dihydrolase, growth in KCN, and acid production from adonitol, D-arabitol, cellobiose, erythritol, D-galactose, myo-inositol, lactose, maltose, D-mannitol, D-mannose, melibiose, alpha-CH3-glucoside, raffinose, L-rhamnose, salicin, D-sorbitol, sucrose, and trehalose. By DNA hybridization, strains of Leminorella were only 3 to 16% related to other Enterobacteriaceae and were divided into three groups. Leminorella grimontii is proposed as the type species for the genus and strain CDC 1944-81, ATCC 33999, is designated as the type strain. There were four strains of L. grimontii from stool specimens and two from urine specimens. L. richardii is proposed as the name for the second species (type strain, CDC 0978-82, ATCC 33998). All four L. richardii strains were from stool specimens. L. grimontii can be distinguished from L. richardii because it produces gas from glucose (100%) and acid from dulcitol (83%) and is methyl red positive (100%). One strain, CDC 3346-72, was more related to L. grimontii by DNA hybridization than to L. richardii, but the lower relatedness to both of these species indicated that it may be a third species. Biochemically it could not be distinguished from L. grimontii. All Leminorella strains were resistant (no zone of inhibition) to ampicillin, carbenicillin, and cephalothin. Some of the Leminorella strains were sent to us for Salmonella serotyping, and two reacted weakly in Salmonella antisera. The clinical significance of Leminorella is unknown.
TL;DR: The raffinosaccharides and their possible metabolites can be rapidly and quantitatively extracted from soya bean with aqueous 80% methanol, and quantified by g.l.c. after trimethyl silylation or oximation and trimethylsilylation.
TL;DR: In this article, different conditions were studied for optimal solubilization and reconstitution of the [3H]-melibiose carrier of Escherichia coli, and the Km for melibiose counterflow was 0.42 mM.
Abstract: Different conditions were studied for optimal solubilization and reconstitution of the melibiose carrier of Escherichia coli. Several α-and β-galactosides, known to be substrates for the melibiose carrier, were found to inhibit [3H]-melibiose uptake by proteoliposomes. In the presence of 10 mM Na+ the Km for melibiose counterflow was 0.42 mM. Melibiose and raffinose were good substrates for counterflow, while thiomethyl-β-galactoside and p-nitrophenyl-α-galacto-side were accumulated very poorly. Although the latter two sugars are known to be substrates for the carrier, they showed a very rapid rate of passive diffusion across the liposome membrane. The proton ionophore carbonylcyanidechlorophenylhy-drazone had no effect on uptake, suggesting that a proton motive force is not essential for the counterflow phenomenon.
TL;DR: Isolation and characterization of a Lactobacillus species capable of proper acid production in a sausage environment is described andjugation was demonstrated through the transfer of plasmid pAMβ1, which encodes erythromycin resistance, from Streptococcus lactis 2301β to LactOBacillus sp.
TL;DR: The Escherichia coli K12 strain X71-54 carries the lac YUN allele, coding for a lactose/H+ carrier defective in the accumulation of a number of galactosides, which suggests that lactose- like and melibiose-like substrates interact with the carrier in slightly different ways.
Abstract: The Escherichia coli K12 strain X71-54 carries the lac YUN allele, coding for a lactose/H+ carrier defective in the accumulation of a number of galactosides [Wilson, Kusch & Kashket (1970) Biochem. Biophys. Res. Commun. 40, 1409-1414]. Previous studies proposed that the lower accumulation in the mutant be due to a faulty coupling of H+ and galactoside fluxes via the carrier. Immunochemical characterization of the carriers in membranes from mutant and parent strains with an antibody directed against the C-terminal decapeptide of the wild-type carrier leads to the conclusion that the mutant carrier is similar to the wild-type in terms of apparent Mr, C-terminal sequence, and level of incorporation into the membrane. The pH-dependence of galactoside transport was compared in the mutant and the parent. At pH 8.0-9.0, mutant and parent behave similarly with respect to the accumulation of beta-D-galactosyl 1-thio-beta-D-galactoside and to the ability to grow on the carrier substrate melibiose. At pH 6.0, both the maximal velocity for active transport and the level of accumulation of beta-D-galactosyl-1-thio-beta-D-galactoside are lower in the mutant. The mutant also is unable to grow on melibiose at pH 5.5. However, at pH 6.0 and low galactoside concentrations, the symport stoichiometry is 0.90 H+ per galactoside in the mutant as compared with 1.07 in the parent. These observations suggest that symport is normal in the mutant and that the lower rate of transport in the mutant is responsible for the phenotype. At higher galactoside concentrations, accumulation is determined not only thermodynamically but also kinetically, contrary to a simple interpretation of the chemiosmotic theory. Therefore lower rates of active transport can mimic the effect of uncoupling H+ and galactoside symport. Examination of countertransport in poisoned cells at pH 6.0 reveals that the rate constants for the reorientation of the loaded and unloaded carrier are altered in the mutant. The reorientation of the unloaded carrier is slower in the mutant. However, the reorientation of the galactoside-H+-carrier complex is slower for substrates like melibiose, but faster for substrates like lactose. These findings suggest that lactose-like and melibiose-like substrates interact with the carrier in slightly different ways.
TL;DR: The three-dimensional structure of Escherichia coli B/r porin (OmpF) was studied by chemical modification using activated sugars of different size and suggested that all the reactive groups modified by the pore-impermeant sugars are located on the surface of porin exposed on the outside of the outer membrane.
TL;DR: Examination of the effects of various carbon sources on the cell wall and cytoplasmic activities of acid invertase, trehalase, maltase, cellobiase, melibiase and lactase found they could not be correlated with the growth-promoting activity of their substrates.
Abstract: The capacity of various carbohydrates to support growth of Japanese morning-glory callus in the dark over a 14-day period was examined. Sucrose was the most effective compound, but glucose, fructose, trehalose, maltose, cellobiose, raffinose and soluble starch supported significant growth. The callus remained alive in the presence of inulin, mannitol, inositol, methyl-α-glucoside and glycerol; while cells grown on galactose, mannose, sorbose, xylose, arabinose, melibiose, lactose, dextran, carboxy-methyl cellulose, sorbitol, galactitol, ethylene glycol were necrotic. Examination of the effects of these various carbon sources on the cell wall and cytoplasmic activities of acid invertase, trehalase, maltase, cellobiase, melibiase and lactase could not be correlated with the growth-promoting activity of their substrates. Extracellular hydrolysis of sucrose, trehalose, maltose, cellobiose, lactose, raffinose, and inulin occurred as a consequence of the presence of cell wall hydrolases in the morning-glory callus, and hydrolytic products could be detected in the medium.
TL;DR: Fusion products were capable of utilizing melibiose and dextrin as carbon sources and could be employed as carbon source for the parental Saccharomyces diastaticus diploid strain, which was a petite mutant.
Abstract: One haploid and one diploid strain of Saccharomyces diastaticus carrying genes responsible for glucoamylase synthesis were fused with a brewing polyploid Saccharomyces uvarum lager strain. With the spheroplast fusion technique, the ability to use dextrin and starch was introduced in the brewing yeast. Spheroplasts of the strains to be used were obtained by enzymatic digestion of the cell walls. Fusion took place in polyethylene glycol; complete cells were then regenerated in hypertonic medium containing 3% agar at 37°C. In the first fusion experiment melibiose was used as carbon source; in the second fusion experiment glycerol was employed as carbon source, for the parental Saccharomyces diastaticus diploid strain was a petite mutant. Fusion products were capable of utilizing melibiose and dextrin as carbon sources.
TL;DR: It seems that the melibiose carrier recognizes H+ in addition to Na' and Li+ with different cation specificities for different substrates, suggesting H+-melibiose cotransport.
Abstract: A common mechanism for nutrient uptake by cells is cation-substrate cotransport. Representatives of this class of membrane carrier are found in all prokaryotic and eukaryotic cells that have been examined.' In bacteria the cation used for many such processes is the proton, while in animal cells it is exclusively the sodium ion that is used for cotransport. The melibiose carrier of Escherichia coli is unusual in its ability to use either H+ or Na+.' This is of interest from an evolutionary point of view since it may be considered an intermediate stage in the evolution from a proton cotransport system to that of Na+ (FIG. 1). The melibiose carrier of E . coli was discovered by Prestidge and Pardee in 1965.' Stock and Roseman4 then showed that the analogous carrier of Salmonella typhimurium functions with Na+ cotransport. This observation was subsequently confirmed and further studied in membrane vesicles of S. typhimurium.5*6 Cotransport with N a + was later demonstrated for the melibiose carrier of E . coli.' It therefore came as a surprise to find that melibiose addition to anaerobic cells of E. coli caused proton uptake' (suggesting H+-melibiose cotransport). A variety of explanations for these observations was explored,' including the possibility of a second carrier for melibiose and all proved inconsistent with the data. It now seems that the carrier recognizes H+ in addition to Na' and Li+ with different cation specificities for different substrates. Recent studies have given additional support for the view that the melibiose carrier can recognize several cations. Experiments have been carried out to show competitive inhibition between different cations for cotransport. Furthermore, mutants have been isolated with altered specificity for more than one cation. A significant advance has been the cloning and sequencing of the DNA for the melibiose transport geneg and the subsequent sequencing of the mutant W3133-28 for comparison.''
TL;DR: The emphasis of this study was put on the similarities and differences with lactose permease which is able to transport the same sugar to approximately the same extent.
TL;DR: GUPTA et al. as mentioned in this paper studied the kinetics and mechanism of oxidation of Maltose and Lactose by cuprammonium-sulphate in ammoniacal and buffered medium.
Abstract: Kinetics of oxidation of maltose and lactose by cuprammonium-sulphate in ammoniacal and buffered medium have been studied. The rate of reaction is independent of [Cu] and directly proportional to [Disaccharide] and square root of [NH3], On addition of NH4C1 the reaction rate decreases due to common ion elfect. A probable mechanism involving the formation of an intermediate enediol anion has been proposed and the rate of enolization is the respective rate of oxidation. The activation parameters have been also evaluated. Introduction Earlier, the kinetics of oxidation of some reducing sugars by coppersulphate in presence [1, 2] and absence [3, 4] of complexing agents have been studied by SINGH et al. in strong alkaline medium. The reaction path showed a short induction period and autocatalysis due to produced finer colloidal particles of Cu 2 0. A general mechanism of oxidation based on the formation of an intermediate 1,2 Enediol has been suggested b y them. Later MARSHALL and WATERS [5] confirmed the results of SINGH et al. while studying the kinetics of oxidation of D-glucose, acetoin and Benzoin by alkaline coppersulphate complexed with tartrate, citrate and picolinate etc. by elucidating the rate expression as: -d[Cu\"] /dr = 2 K J E ] [Cu], and the rate-determining step assuming to be cuprous chelate formation. There after WIBERG and NIGH [6] studied the kinetics of oxidation of a-hydroxyacetophenone by cupric acetate in buffered aqueous pyridine medium spectrophotometrically and pointed out that the reaction follow the rate law: v = tf,[Ketol] + tf2[Ketol] [Cu]. The first term corresponds to the independently determined rate of enolization and second term is the major one when [Cu] is greater than 0.01 M. Thus they agree with the findings of SINGH et al. at lower concentration of copper sulphate. The present paper deals with the kinetics and mechanism of oxidation of Maltose and Lactose by cuprammonium-sulphate in ammoniacal and buffered medium. The system is advantageous as compared to copperchelate oxidants as it becomes homogeneous during entire course of oxidation due to formation of complex Cu(NH3)2. ' k.. C. GUPTA M