Showing papers on "Melibiose published in 1990"

Proton-linked sugar transport systems in bacteria.

Abstract: The cell membranes of various bacteria contain proton-linked transport systems for D-xylose, L-arabinose, D-galactose, D-glucose, L-rhamnose, L-fucose, lactose, and melibiose. The melibiose transporter of E. coli is linked to both Na+ and H+ translocation. The substrate and inhibitor specificities of the monosaccharide transporters are described. By locating, cloning, and sequencing the genes encoding the sugar/H+ transporters in E. coli, the primary sequences of the transport proteins have been deduced. Those for xylose/H+, arabinose/H+, and galactose/H+ transport are homologous to each other. Furthermore, they are just as similar to the primary sequences of the following: glucose transport proteins found in a Cyanobacterium, yeast, alga, rat, mouse, and man; proteins for transport of galactose, lactose, or maltose in species of yeast; and to a developmentally regulated protein of Leishmania for which a function is not yet established. Some of these proteins catalyze facilitated diffusion of the sugar without cation transport. From the alignments of the homologous amino acid sequences, predictions of common structural features can be made: there are likely to be twelve membrane-spanning alpha-helices, possibly in two groups of six; there is a central hydrophilic region, probably comprised largely of alpha-helix; the highly conserved amino acid residues (40-50 out of 472-522 total) form discrete patterns or motifs throughout the proteins that are presumably critical for substrate recognition and the molecular mechanism of transport. Some of these features are found also in other transport proteins for citrate, tetracycline, lactose, or melibiose, the primary sequences of which are not similar to each other or to the homologous series of transporters. The glucose/Na+ transporter of rabbit and man is different in primary sequence to all the other sugar transporters characterized, but it is homologous to the proline/Na+ transporter of E. coli, and there is evidence for its structural similarity to glucose/H+ transporters in Plants. In vivo and in vitro mutagenesis of the lactose/H+ and melibiose/Na+ (H+) transporters of E. coli has identified individual amino acid residues alterations of which affect sugar and/or cation recognition and parameters of transport. Most of the bacterial transport proteins have been identified and the lactose/H+ transporter has been purified. The directions of future investigations are discussed.

Transfructosylation Catalyzed by β-Fructofuranosidase I from Arthrobacter sp. K-1

Abstract: Transfructosylation of the β-fructofuranosidase I from Arthrobacter sp. K-1 was investigated. This enzyme catalyzed both transfructosylation and hydrolytic action, when it was incubated with sucrose alone. But in the presence of a suitable acceptor such as d-xylose and lactose, the enzyme catalyzed mostly transfructosylation and transferred the fructose residue preferentially to the acceptor. The enzyme had broad acceptor specificities. d-Xylose, d-galactose, l-sorbose, d- and l-fucose, d- and l-arabinose, maltose, isomaltose, cellobiose, lactose, melibiose, xylobiose, maltotriose, methyl β-glucoside, and galactoside were efficient acceptors in the transfructosylation. On the other hand, d-ribose, l-rhamnose, d-mannose, 2-deoxy-d-glucose, d-galactosamine, d-galacturonic acid, and 1-kestose were not efficient acceptors. Various primary alcohols, polyhydric alcohols including some sugar alcohols, and some glycosides acted as acceptors, but secondary alcohols with one hydroxyl group such as 2-propanol and 2-...

The melibiose/Na+ symporter of Escherichia coli: kinetic and molecular properties.

Abstract: The role of the co-transported cation in the coupling mechanism of the melibiose permease of Escherichia coli has been investigated by analysing its sugar-binding activity, facilitated diffusion reactions and energy-dependent transport reactions catalysed by the carrier functioning either as an H+, Na+ or Li(+)-sugar symporter. The results suggest that the coupling cation not only acts as an activator for sugar-binding on the carrier but also regulates the rate of dissociation of the co-substrates in the cytoplasm by controlling the stability of the ternary complex cation-sugar-carrier facing the cell interior. Furthermore, there is some evidence that the membrane potential enhances the rate of symport activity by increasing the rate of dissociation of the co-substrates from the carrier in the cellular compartment. Identification of the melibiose permease as a membrane protein of 39 kDa by using a T7 RNA polymerase/promoter expression system is described. Site-directed mutagenesis has been used to replace individual carrier histidine residues by arginine to probe the functional contribution of each of the seven histidine residues to the symport mechanism. Only substitution of arginine for His94 greatly interferes with the carrier function. It is finally shown that mutations affecting the glutamate residue in position 361 inactivate translocation of the co-substrates but not their recognition by the permease.

Gal4 Mutations That Separate the Transcriptional Activation and Gal80-Interactive Functions of the Yeast Gal4 Protein

Abstract: The carboxy-terminal 28 amino acids of the Saccharomyces cerevisiae transcriptional activator protein GAL4 execute two functions--transcriptional activation and interaction with the negative regulatory protein, GAL80. Here we demonstrate that these two functions are separable by single amino acid changes within this region. We determined the sequences of four GAL4C-mutations, and characterized the abilities of the encoded GAL4C proteins to activate transcription of the galactose/melibiose regulon in the presence of GAL80 and superrepressible GAL80S alleles. One of the GAL4C mutations can be compensated by a specific GAL80S mutation, resulting in a wild-type phenotype. These results support the idea that while the GAL4 activation function tolerates at least minor alterations in the GAL4 carboxyl terminus, the GAL80-interactive function is highly sequence-specific and sensitive even to single amino acid alterations. They also argue that the GAL80S mutations affect the affinity of GAL80 for GAL4, and not the ability of GAL80 to bind inducer.

Characterization of a gram-positive bacterium from the proventriculus of budgerigars (Melopsittacus undulatus).

Abstract: The cellular, cultural, and biochemical characteristics of eight isolates of a large gram-positive bacillus that are commonly observed as apparently normal flora in the proventriculus of budgerigars (Melopsittacus undulatus) were determined. The bacterium was highly pleomorphic and changed markedly in both diameter and length when subcultured on agar media. The bacterium was facultative anaerobic and capnophilic, hemolytic on blood agar, and formed flat colonies with irregular edges after incubation for several days. All isolates grew on sodium azide agar but did not grow on MacConkey agar. The isolates were catalase-negative and oxidase-negative and did not reduce nitrate. All isolates failed to utilize arginine, lysine, ornithine or tryptophane but produced acid from glucose, galactose, levulose, maltose, melibiose, starch, and sucrose. All isolates produced acetoin from glucose and hydrolyzed esculin. The eight isolates could not be identified to either genus or species level based on the descriptions of currently classified organisms in the division Firmicutes as described in Bergey's Manual of Systematic Bacteriology.

β-Galactosidase of Pediococcus species: induction, purification and partial characterization

Abstract: Six strains of Pediococcus pentosaceus and two of P. acidilactici had intracellular β-galactosidase (β-gal) activity when grown in the presence of lactose; all but two strains of P. pentosaceus and one of P. acidilactici had such activity when grown in the presence of glucose. Synthesis of β-gal by P. pentosaceus ATCC 25745 was inducible with lactose, galactose, melibiose, lactobionic acid and possibly cellobiose but not with glucose, sucrose, maltose, glycerol, fructose or mannose. Lactose, galactose and possibly maltose, melibiose and lactobionic acid but not glucose, sucrose, glycerol, cellobiose, fructose or mannose induced β-gal synthesis by P. acidilactici ATCC 25740. Synthesis of β-gal was partially inhibited in P. pentosaceus ATCC 25745 and P. acidilactici ATCC 25740 by glucose added to the medium during growth in the presence of galactose or lactose. Isopropyl β-d-thiogalactopyranoside failed to induce synthesis of β-gal by either strain during growth on glucose. β-Gal from P. pentosaceus ATCC 25745 had a molecular weight of 66,000 and activity optima of pH 6.5 and 45° C. Activity of the enzyme was stimulated by reducing agents, Mg2+, Mn2+, Zn2+ and Co2+ but not by Ca2+, and was markedly inhibited by ethylenediaminetetraacetate (EDTA), HgCl2, 1,10-phenanthroline, and an oxidizing agent. The K mvalues of the enzyme for o-nitrophenol-β-d-galactopyranoside and lactose were 3.07 and 7.0 mM, respectively, suggesting its low affinity for lactose.

Carbohydrate binding activities of Bradyrhizobium japonicum. I. Saccharide-specific inhibition of homotypic and heterotypic adhesion.

Abstract: Bradyrhizobium japonicum (R110d) exhibited four saccharide-specific binding activities: (a) adsorption to Sepharose beads containing covalently coupled lactose; (b) homotypic agglutination through one pole of the cell (star formation); (c) heterotypic adhesion to the cultured soybean cell line, SB-1; and (d) attachment to roots of soybean plants. Each of these binding activities can be inhibited by the addition of galactose or lactose, but not by derivatives such as N-acetyl-D-galactosamine or melibiose. Treatment of wild-type bacteria with N-methyl-N'-nitro-N-nitrosoguanidine followed by selection on the basis of reduced binding to SB-1 cells, resulted in two specific mutants, designated N4 and N6. Compared to wild type, these two mutants also exhibited decreased binding activity in: (a) adsorption to lactose-Sepharose beads; (b) homotypic star formation; and (c) heterotypic attachment to roots of soybeans plants. These results suggest that all four of the saccharide-inhibitable binding activities of Bradyrhizobium japonicum may be mediated by the same mechanism(s) or molecular component(s).

Isolation and functional reconstitution of soluble melibiose permease from Escherichia coli.

Abstract: By use of techniques described recently for lac permease [Roepe, P.D., & Kaback, H.R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6087], the melibiose permease from Escherichia coli, another polytopic integral plasma membrane protein, has been purified in a metastable soluble form after overexpression of the melB gene via the T7 RNA polymerase system. As demonstrated with lac permease, soluble melibiose permease is dissociated from the membrane with 5.0 M urea and appears to remain soluble in phosphate buffer at neutral pH after removal of urea by dialysis, although the protein aggregates in a time- and concentration-dependent fashion. Moreover, soluble melibiose permease behaves as a monomer during purification by size exclusion chromatography in the presence of urea. Circular dichroism of purified soluble melibiose permease reveals that the protein is highly helical in potassium phosphate buffer and that secondary structure is disrupted in 5.0 M urea. Finally, purified melibiose permease can be reconstituted into proteoliposomes, and the preparations catalyze membrane potential driven H+/melibiose or Na+/methyl 1-thio-beta,D-galactopyranoside symport. The results provide further support for the notion that hydrophobic transmembrane proteins may be able to assume a nondenatured conformation in aqueous solution and extend the implication that the approach described may represent a general method for rapid isolation and reconstitution of this class of membrane proteins.

Binding-protein-dependent lactose transport in Agrobacterium radiobacter.

Abstract: Agrobacterium radiobacter NCIB 11883 was grown in lactose-limited continuous culture at a dilution rate of 0.045/h. Washed cells transported [14C]lactose and [methyl-14C]beta-D-thiogalactoside, a nonmetabolisable analog of lactose, at similar rates and with similar affinities (Km for transport, less than 1 microM). Transport was inhibited to various extents by the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone, by unlabeled beta-galactosides and D-galactose, and by osmotic shock. The accumulation ratio for methyl-beta-D-thiogalactoside was greater than or equal to 4,100. An abundant protein (molecular weight, 41,000) was purified from osmotic-shock fluid and shown by equilibrium dialysis to bind lactose and methyl-beta-D-thiogalactoside, the former with very high affinity (binding constant, 0.14 microM). The N-terminal amino acid sequence of this lactose-binding protein exhibited some homology with several other sugar-binding proteins from bacteria. Antiserum raised against the lactose-binding protein did not cross-react with two glucose-binding proteins from A. radiobacter or with extracts of other bacteria grown under lactose limitation. Lactose transport and beta-galactosidase were induced in batch cultures by lactose, melibiose [O-alpha-D-galactoside-(1----6)alpha-D-glucose], and isopropyl-beta-D-thiogalactoside and were subject to catabolite repression by glucose, galactose, and succinate which was not alleviated by cyclic AMP. We conclude that lactose is transported into A. radiobacter via a binding protein-dependent active transport system (in contrast to the H+ symport and phosphotransferase systems found in other bacteria) and that the expression of this transport system is closely linked to that of beta-galactosidase.

Melibiose is hydrolyzed exocellularly by an inducible exo-alpha-galactosidase in Azotobacter vinelandii.

Abstract: Azotobacter vinelandii hydrolyzed melibiose exocellularly, leading to an accumulation of free glucose and galactose in the medium. This enzyme could also be induced by galactose, raffinose, and stachyose. The alpha-galactosidase activity could be detected quantitatively by using p-nitrophenyl-alpha-galactopyranoside as a substrate for intact cells. Chloramphenicol totally inhibited the induction of this enzyme. However, benzyl alcohol inhibited the secretion of this enzyme but did not inhibit the biosynthesis of the enzyme.

Alpha‐galactosidase of germinating seeds of cassia sericea sw.

Abstract: Germinating seeds of Cassia sericea Sw contain two molecular forms of α-galactosidase which were partially purified and characterized Both enzyme forms had an optimum pH of 50 and an optimum temperature of 50 °C Km values for the substrate p-nitrophenyl-α-D-galactoside (PNPG) were 091 mM and 105 mM for the two forms, and substrate inhibition was observed at high concentrations of PNPG The two forms of the enzyme had molecular weights of 46,000 and 33,000 by gel filtration The enzyme forms were inhibited completely by Ag+, Cu2+ and Hg2+ ions and by p-chloromercuribenzoate showing that thiol groups are probably involved in catalysis Both α-galactosidases hydrolyzed melibiose and raffinose, although hydrolysis of stachyose could not be detected The enzyme may find potential use in the food industry to hydrolyze flatulence-causing sugars in processed foods and in production of sucrose from high-raffinose sugar beets That the source (C sericea seeds) of the enzyme is inexpensive provides an added advantage over use of cultivated legumes as a source of α-galactosidase

Successive binding of raf repressor to adjacent raf operator sites in vitro.

Abstract: The raf repressor negatively regulates the transcription of the raf operon which encodes functions required for the uptake and hydrolysis of raffinose in Escherichia coli. Overexpression of the repressor gene under lac promoter control led to the formation of inclusion bodies. These were partially purified by centrifugation, solubilized in 0.1 % SDS and reactivated by dilution. DNase I protection and gel retardation experiments demonstrated the specific binding of raf repressor to DNA fragments that contained the previously identified raf operator, an element comprising two 18 by palindromic nucleotide sequences that flank the −35 raf promoter box. By using DNA fragments with one, two, or four copies of the 18 by palindrome, these experiments revealed concentration dependent, successive occupation of all available binding sites by raf repressor. Melibiose released the repressor from the operator complexes, whereas raffinose and other α-galactosides did not, indicating that melibiose is the actual inducer in vivo. We suggest that successive occupation by repressor of two strategically located operator sites is a specific type of stepwise down-regulation of gene expression in response to repressor concentration.

Transgalactosylation products from melibose by the α-galactosidase of Absidia corymbifera

Abstract: α-Galactosidases of Absidia and Mortiella sp. have been used to decompose raffinose in industrial beet sugar production. In this paper is described isolation and characterization of three major transgalactosylation products from melibiose

Binding and hemagglutinating properties of the B subunit(s) of heat-labile enterotoxin isolated from human enterotoxigenic Escherichia coli.

Abstract: The binding and hemagglutinating activities of the B subunit(s) of the heat-labile enterotoxin (LTh-B) isolated from human enterotoxigenic Escherichia coli were investigated. The binding of 125I-labeled LTh-B to neuraminidase-treated human type B erythrocytes was most effectively inhibited by ganglioside GM1. A number of mono-, di- and polysaccharides, as well as several glycoproteins were at least 500 times less potent inhibitors. However, hemagglutination was effectively inhibited by galactose, melibiose and hog A + H but not by ganglioside GM1. Preincubation of the LTh-B with ganglioside GM1 gave much stronger hemagglutination than LTh-B alone. These results suggest that the predominant binding substance for LTh-B on neuraminidase-treated human type B erythrocytes is ganglioside GM1, but indicate that the interaction of LTh-B with ganglioside GM1 is different in hemagglutination.

Isolation and hemagglutinating activities of bovine immunoglobulins reactive with melibiose.

Abstract: Calcium-dependent and -independent bovine immunoglobulins (IgM and IgG) reactive with melibiose were isolated by affinity chromatography on melibiose-coupled Sepharose 4B. Hemagglutination and hemagglutination inhibition were carried out to study their carbohydrate specificities. Human and animal erythrocytes were agglutinated by these bovine IgM, whereas those were not by bovine IgG at the highest concentrations used. Neuraminidase- and pronase-treated erythrocytes were more strongly agglutinated by these IgM than untreated ones. On the other hand, melibiose-reactive human immunoglobulins isolated from AB serum showed strong hemagglutinating activities to rabbit erythrocytes. Hemagglutination of neuraminidase-treated human type B erythrocytes by calcium-independent bovine IgM reactive with melibiose was effectively inhibited by galactose, methyl alpha-D-galactopyranoside and melibiose, whereas that was not by methyl beta-D-galactopyranoside, lactose, and other substances at the highest concentrations used. Similar results were also obtained in hemagglutination inhibition with untreated rabbit erythrocytes and calcium-independent human immunoglobulins (IgM and IgG) reactive with melibiose. However, hemagglutination of pronase-treated human type A erythrocytes by calcium-dependent bovine IgM reactive with melibiose was not at all inhibited by these substances at the highest concentrations used. From these results, bovine serum is found to also contain antibodies with a specificity for alpha-linked galactosyl residues as found for human AB serum reported previously.

Comparative studies of carbohydrate-binding proteins from Xenopus laevis skin and eggs. Sugar-binding specificities and affinity purification.

Abstract: Salt and detergent extracts of acetone-dried powder of Xenopus laevis skin and eggs were fractionated on sugar-Sepharose columns, to which lactose, melibiose, galactose, rhamnose and mannose had been covalently linked, by successive elution with chelating reagent and specific sugars, resulting in separation of the different Ca2+ -dependent and Ca2+ -independent carbohydrate-binding proteins. The skin of X. laevis contains a salt-extractable Ca2+ -dependent lactose-binding lectin of 30 kilodalton (kDa) and the eggs a similar lectin of 43kDa, but they both lack Ca2+ -dependent galactose-binding lectins, The 30 kDa lactose-binding lectin which agglutinates human A erythrocytes was isolated by successive affinity chromatography on two linked sugar-Sepharose columns, i.e., a galactose-Sepharose-lactose-Sepharose (GL) column system. Since the 30 kDa lectin was not recovered in the Ca2+ -dependent lactose-binding protein fraction from the GL column system under the dithiothreitol (DTT)-free conditions, it was concluded that the lectin requires the presence of DTT and calcium for binding to the lactose-Sepharose column.

Demonstration of Streptococcus sobrinus in human oral cavity

Abstract: From 133 saliva samples of humans we isolated 32 strains (24%) of Streptococcus sobrinus on an BYCSB selective medium. The very hard colonies of S. sobrinus on this medium were characterized by a distinct chalky white halo of water insoluble glucan ("glucan-halo") produced in large amounts from the sucrose by extracellular glucosyltransferase which has diffused out into the isolation medium. All of the isolated S. sobrinus strains fermented ordinarily mannitol and sorbitol, but not raffinose and melibiose. The occurrence of this special subspecies within the S. mutans-group may play a more important role in the etiology of dental caries of caries-active persons in the future.

Digestive physiology and food utilization of the larvae of Earias vittella (Lepidoptera: Noctuidae) from its malvaceous host plant.

Abstract: The hydrogen-ion concentration of the contents of fore-, mid- and hindgut of the larva ofEarias vittella (F.) lays within a pH range of 8-8 and 9-6 These caterpillars possessed the ability to break down starch, raffmose, maltose, melibiose, sucrose and proteins. Synthesis of certain higher oligosaccharides indicating transglycosidic activity was also noticed during hydrolysis of raffinose, melibiose and sucrose inin vitro experiments. The activity of carbohydrases detected in the larval midgut was variably influenced by the hydrogen-ion concentration of the medium. Further, they differed from one another in their relative strength. Amylase was the most powerful and a-galactosidase was the weakest of all. An in vivo examination into the fate of fed starch, maltose, cellobiose and lactose within the gut revealed the physiological competency of the caterpillar to utilise only the first two carbohydrates (the former partly and the latter completely) through swiftly operating processes of digestion and absorption.