Showing papers on "Melibiose published in 1983"

Cooperative Binding of the Sugar Substrates and Allosteric Regulatory Protein (Enzyme IIIGlc of the Phosphotransferase System) to the Lactose and Melibiose Permeases in Escherichia coli and Salmonella typhimurium

Abstract: An Escherichia coli strain which overproduces the lactose permease was used to investigate the mechanism of allosteric regulation of this permease and those specific for melibiose, glycerol, and maltose by the phosphoenolpyruvate-sugar phosphotransferase system (PTS). Thio-β-digalactoside, a high affinity substrate of the lactose permease, released the glycerol and maltose permeases from inhibition by methyl-α-d-glucoside. Resumption of glycerol uptake occurred immediately upon addition of the galactoside. The effect was not observed in a strain which lacked or contained normal levels of the lactose permease, but growth of wild-type E. coli in the presence of isopropyl-β-thiogalactoside plus cyclic AMP resulted in enhanced synthesis of the lactose permease so that galactosides relieved inhibition of glycerol uptake. Thiodigalactoside also relieved the inhibition of glycerol uptake caused by the presence of other PTS substrates such as fructose, mannitol, glucose, 2-deoxyglucose, and 5-thioglucose. Inhibition of adenylate cyclase activity by methyl-α-glucoside was also relieved by thiodigalactoside in E. coli T52RT provided that the lactose permease protein was induced to high levels. Cooperative binding of sugar and enzyme IIIGlc to the melibiose permease in Salmonella typhimurium was demonstrated, but no cooperativity was noted with the glycerol and maltose permeases. These results are consistent with a mechanism of PTS-mediated regulation of the lactose and melibiose permeases involving a fixed number of allosteric regulatory proteins (enzyme IIIGlc) which may be titrated by the increased number of substrate-activated permease proteins. This work suggests that the cooperativity in the binding of sugar substrate and enzyme IIIGlc to the permease, demonstrated previously in in vitro experiments, has mechanistic significance in vivo. It substantiates the conclusion that PTS-mediated regulation of non-PTS permease activities involves direct allosteric interaction between the permeases and enzyme IIIGlc, the postulated regulatory protein of the PTS.

Characterization of β-galactosidase–lactose-permease chimaeras of Escherichia coli

Abstract: Escherichia coli strains have been isolated in which 3, 39 or 805 5'-end codons of lacZ, the gene for the cytoplasmic enzyme beta-galactosidase are fused to codon 9 of lacY, the gene for lactose permease. Lactose-permease-deficient cells, carrying the lacZ-Y fusions on F' lac pro episomes, are phenotypically positive on eosin/methylene blue/lactose or on melibiose plates, demonstrating that the beta-galactosidase--lactose-permease chimaeras transport lactose and melibiose in vivo. The apparent affinity for beta-D-galactopypanosyl 1-thio-beta-D-galactopyranoside (GalSGal) in cells is similar to that of the wild-type gene product. The maximum velocity of active GalSGal transport is reduced in all three fusion strains. Both lactose and p-nitrophenyl alpha-D-galactopyranoside inhibit GalSGal uptake. As demonstrated by immunoblot experiments the chimaeras cross-react with polyclonal antibodies directed against native lactose permease and they are present in the cell envelope fraction of homogenates. Their apparent molecular weights upon electrophoresis in NaDodSO4/polyacrylamide gels correspond to those expected from their respective primary sequences, taking into account the migration properties of wild-type lactose permease. It is proposed that substitution of eight N-terminal lactose permease residues by N-terminal beta-galactosidase residues neither prevents membrane incorporation of permease nor completely impairs the ability to transport galactosides actively. Alternative interpretations of the experimental results are discussed.

Purification and characterization of β-D-galactosidase and α-D-mannosidase from Papaya (Carica papaya) seeds

Abstract: β-D-Galactosidase was purified 115-fold from a saline extract of papaya seeds by fractionation with ammonium sulfate, DEAE-Sephadex chromatography and gel-filtration on Sephadex G-75, G-150, and G-100. The purified β-D-galactosidase (MW, 56,000 daltons) had an isoelectric point (pI) at pH 8.4 and the optimal pH for its activity was 3.5 to 4.5. The enzyme activity was inhibited by Cu2+,Ag+,Hg2+,Pb2+,NaAsO2 and р-chloromercuribenzoate at concentrations of 1x10-3 M. Among the various mono- and oligosaccharides tested, D-galactose, D-galacturonic acid, D-galactono-γ-lactone and melibiose significantly inhibited the enzyme activities at concentrations of 2xl0-3 to 1X10-2M. The purified enzyme hydrolyzed β-nitrophenyl β-D-galactoside (Km = 1.0X10-3M), methyl β-D-galactoside (Km=1.6x10-2M), aminoethyl β-D-galactoside (Km =3.3X10-2M) and lactose (Km = 9.1X10-2M). β-(l→3)-Linked galactotetraosyl-eryth itol and asialo-glycopeptide isolated from fetuin were also hydrolyzed to the extent of 78 and 75%, 4respectively,...

Isolation and characterization of the serotype g carbohydrate moiety from an enzyme lysate of Streptococcus mutans 6715 cell walls.

Abstract: The serotype-specific carbohydrate moiety of Streptococcus mutans was isolated by mild degradation of purified cell walls with a cell-wall lytic enzyme. Cell walls of serotype g S. mutans strain 6715 were digested with M1 enzyme, an endo-N-acetylmuramidase purified from culture supernatants of Streptomyces globisporus strain 1829. The enzyme lysate of the cell walls was applied to a CM Sephadex C-25 column to remove the M1 enzyme from the cell wall lysate and then subjected to Sephadex G-100 column chromatography. Carbohydrate antigens with serotype g specificity, designated M1g, and a peptidoglycan--polysaccharide complex lacking serotype specificity (M1PG) were separated. Purified serotype g antigen was also obtained by autoclaving the S. mutans 6715 whole cells in saline at 120 C for 30 min. The extract was applied to a DEAE Sephadex A-25 column to remove nucleic acids and teichoic acids. The unbound peak fraction was concentrated and re-chromatographed on a Bio-Gel P-100 column. The void volume fraction contained serotype g carbohydrate and was designated RRg antigen. M1g and RRg antigens formed a band of identity with anti-serotype g serum by immunodiffusion. These antigens were composed mainly of galactose, glucose, and rhamnose at an approximate weight ratio of 8 : 4: 1, while constituent sugars of M1PG consisted of rhamnose and glucose, with no detectable galactose. M1g also contained peptidoglycan residues other than threonine, an interpeptide bridge component of the native cell wall peptidoglycan. Marked inhibition of the quantitative precipitin reaction between M1g and anti-serotype g serum was obtained with melibiose and galactose, which suggests that the immunodeterminant of the serotype g carbohydrate is an alpha-linked galactose-glucose terminal linkage.

Association of bacterial carbohydrate-specific cold agglutinin antibody production with immunization by group C, group B type III, and Streptococcus pneumoniae type XIV streptococcal vaccines.

Abstract: Rabbits immunized with group B type III, group C, and Streptococcus pneumoniae type XIV streptococcal vaccines developed autoantibodies reactive with autologous and isologous erythrocytes and human O-positive erythrocytes at reduced temperatures. The cold agglutinin antibodies were present in both the immunoglobulin M (IgM) and IgG fractions of group C streptococcal antiserum and in the IgM fraction of group B type III and S. pneumoniae type XIV antisera. BALB/c, CF1, and local strains of mice immunized with group B type III and S. pneumoniae type XIV streptococcal vaccines also produced a cold agglutinin antibody reactive with rabbit and human erythrocytes. The cold agglutinin antibodies were reactive with saccharide compounds representative of the determinants present on the individual bacterial carbohydrate structures, individual vaccine preparations, and isolated polysaccharides. The group C antibodies in rabbits were reactive with sugar ligands in the following order: N-acetylgalactosamine greater than melibiose greater than lactose greater than galactose greater than glucose. Group B type III and S. pneumoniae type XIV cold agglutinin antibodies in rabbit antisera, however, displayed reactivities different from group C antibodies and from each other. Group B type III antibodies reacted with galactose greater than lactose greater than N-acetylgalactosamine greater than glucose greater than rhamnose; S. pneumoniae type XIV antibodies reacted with lactose greater than melibiose greater than galactose greater than glucose greater than N-acetylgalactosamine. The same relative ligand specificity was observed for the cold agglutinin antibodies in S. pneumoniae type XIV mouse antisera. The cold agglutinin antibodies in group B type III and S. pneumoniae type XIV antiserum reacted with erythrocytes at higher temperatures (up to 31 degrees C) than did group C antibodies (up to 14 degrees C). In addition, S. pneumoniae type XIV antibodies did not discriminate between I- or i-bearing human erythrocytes to a significant extent. The results obtained provide substantial evidence that autoreactive cold agglutinin antibodies produced by immunization with these vaccines represent subpopulations of bacterial carbohydrate-specific antibodies that cross-react with mammalian carbohydrate structures.

Saccharomyces uvarum inulyticus var. nov., a new high-concentration ethanol tolerant yeast from rice wine

Abstract: A new alcohol tolerant yeast which had the ability to ferment glucose, galactose, maltose, trehalose, melizitose, α-methyl-d-glucoside, melibiose and raffinose (complete) was isolated from fermented rice wine. This new isolate which was morphologically, culturally and physiologically similar to type species Saccharomyces uvarum Beijerinck was considered as a variety of the species, hence, designated as Saccharomyces uvarum inulyticus var. nov., because of its extraordinary ability to ferment inulin into ethanol and hyper-tolerance to high concentration of ethanol at a wide range of temperature. The new variant produced 21.6%, 20%, 18.6%, and 9.8% v/v ethanol at 15, 20, 30, and 40° C, respectively, when fermentation was carried out by the stepwise addition of sucrose to the synthetic medium. The addition of fungal mycelia into the synthetic medium increased the fermentation rate and ethanol concentration up to 22.4%, 21.8%, 20.1%, and 11.6% v/v at 15, 20, 30, and 40° C, respectively. The new variant was found to grow well at 40° C and did not require any vitamin or growth factor, such as biotin, calcium pantothenate, inositol, niacin, p-aminobenzoic acid, pyridoxine hydrochloride, riboflavin and thiamine hydrochloride for normal growth.

Partial isolation and characterisation of a hemagglutinating factor from avocado seed.

Abstract: Extracts of ground avocado seeds (Fuerte and Hass varieties), prepared in different buffer solutions (pH 2.0–12.0), show hemagglutinating activity towards A, B, AB, and H (0) human erythrocytes. The extract showing the highest titer of aggulination was extracted at pH 10.5. The crude extract also causes hemolysis of fresh washed erythrocytes. The hemagglutinating factor is not inhibited by most of the simple sugars tested, e.g., D-glucose, D-mannose, D-galactose, and glucose-amine. The only sugars which show some inhibitory effect are N-Acetyl-neuraminic acid, melibiose, and stachiose. Basic amino acids, e.g., lysine and arginine also inhibit its activity. However the most potent inhibitors of the agglutinin are proteins and glycoproteins such as bovine serum albumin, collagen, thyroglobulin, ovalbumin, mucin, and fetuin.

Preparation of red sake using red yeast

Abstract: PURPOSE:To prepare a new-type sake containing >=15% alcohol, by carrying out the alcoholic fermentation using a red yeast having specific characteristics and capable of producing non-carotinoidal and non-pulcherrimin pigment. CONSTITUTION:The objective sake is prepared by the alcoholic fermentation using a red yeast having strong fermenting power and capable of producing a non-carotinoidal and non-pulcherrimin red pigment and having the following four properties. (1) In the cultivation in an MY liquid medium, it gives little clouding in the liquid, proliferates mostly at the bottom, generates carbon dioxide gas and scarcely forms rings and thin films by long-term cultivation: (2) In the cultivation on a YM agar medium, it gives a pink or red colony after 3-4 days at 25-28 deg.C: (3) There is no formation of ascospore and ballistospore: and (4) It ferments glucose, maltose, saccharose and raffinose vigorously and trehalose slowly, and there is no reproducibility in the fermentation of galactose and melibiose.