PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “Galactosides”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed1976

Studies on the mechanism of phosphorylation and transport of beta-galactosides by the lactose phosphotransferase system of Staphylococcus aureus. Kinetic investigations using tosyl galactosides as reversible dead-end inhibitors.

Tosyl galactosides, previously shown to be potent reversible dead-end inhibitors of the membrane-bound Enzyme IIlac of the lactose phosphotransferase system of Staphylococcus aureus, were used for an investigation of the kinetic mechanism of the sugar phosphorylation/transport reaction catalyzed by this enzyme: phospho-Factor IIIlac&sugar Enzyme IIlac lead to Factor IIIlac&sugar phosphate. Inhibition of Enzyme IIlac was studied in three different systems. Washed membranes, and washed membranes in the presence of 0.1% Triton X-100 were used for phosphorylation experiments, and whole cells were used for transport studies. When washed membranes were used to supply Enzyme IIlac, inhibition of phosphorylation by tosyl galactoside was linear non-competitive against both the sugar and phospho-Factor IIIlac substrates, with an apparent Ki of about 0.5 mM. This Ki decreased with increasing Factor IIIlac concentration. In the presence of 0.1% Triton X-100, the phosphorylation reaction was stimulated; under these conditions the inhibition became strictly competitive against sugar, and completely uncompetitive against phospho-Factor IIIlac. Apparently washed membranes can catalyze phosphorylation both via a reaction sequence in which sugar binds first and via one in which phospho-Factor IIIlac binds first, but in the presence of 0.1% Triton the reaction does not occur by the former sequence. The inability of bound phospho-Factor IIIlac to hinder the binding of tosyl galactosides suggests that the initial binding sites of the two substrates of Enzyme IIlac are separated by at least the distance of the tosyl moiety. Radioactive methyl 6-O-(p-toluenesulfonyl) beta-galactoside was not converted into a phosphorylated product in the reaction mixtures, i.e. it is a true dead-end inhibitor. Inhibition of beta-galactoside transport into whole cells by tosyl galactosides was competitive, with an apparent Ki of 5-10 mM, an order of magnitude higher than the Ki for inhibition of phosphorylation by membrane preparations. This result suggest that a significant level of unphosphorylated phospho-Factor IIIlac is present inside the cells, or that cellular levels of this compound are considerably lower than those used for in vitro sugar phosphorylation assays. Radioactive tosyl galactoside inhibitor was not transported into whole cells.

Binding Sites↗
PubMed1980

Inhibition of lactose transport in E. coli by N-ethylmaleimide-beta-galactoside, N-ethylsuccinimide-beta-galactoside and N-ethylmaleimide.

N-ethylmaleimide (NEM) inhibits lactose uptake in E. coli by reacting with the M protein component of the lac permease system. In an attempt to estimate the distance between the NEM reactive site and the substrate binding site, we have synthesized a beta-galactoside with NEM as the aglycon moiety (NEM-gal). NEM-gal was a more effective inhibitor of lactose transport than was NEM. Part of the inhibition by NEM-gal was caused by competition with lactose for the substrate binding site. To estimate this part of the inhibition, we synthesized the saturated and thus the unreactive N-ethylsuccinimide (NES) analog of NEM-gal. Nes-gal was a competitive inhibitor of lactose uptake. The remainder of the inhibition by NEM-gal followed first-order kinetics with the same rate constant as NEM. In addition, the protective effect of thiodigalactoside against the inhibition of transport by NEM was also observed against irreversible inhibition by NEM-gal. We suggest that the reactivity of NEM was unaltered by bringing it near the beta-galactoside binding site by way of covalent attachment to galactose. We conclude that the distance between the NEM reactive site and the position of the glycosidic oxygen of beta-galactosides bound to the lactose site is greater than 8 A.

Biological Transport↗
PubMed1975

Inhibition by 6-O-tosyl galactosides of beta-galactoside phosphorylation and transport by the lactose phosphotransferase system of Staphylococcus aureus.

The effect of various galactose derivatives, substituted at C-6, on the phosphoenolpyruvate:beta-galactoside phosphotransferase system of Staphylococcus aureus was studied. Cells were grown by an improved procedure, which resulted in a 5- to 10-fold increase in cell yield. The four protein components of the system were separated. A membrane fraction containing negligible levels of the soluble components was prepared by alternate cycles of sonic treatment and differential centrifugation. The in vitro system reconstituted from these fractions was used to test the ability of the galactose derivatives to inhibit the phosphorylation of lactose analogs, under conditions where the membrane-bound component, Enzyme IIlac, was rate limiting. Derivaites in which the hydroxyl group of C-6 was missing, or replaced by a fluoro, O-methyl, or carboxyl group had no affinity for Enzyme IIlac, as judged by their inability to inhibit phosphorylation. Surprisingly, derivatives containing arylsulfonyl groups at C-6 were potent inhibitors; the O-tosyl compound has an apparent affinity five times that of galactose. The arylsulfonyl substitution in an absolute requirement; neither O-benzyl or O-methanesulfonyl derivatives were inhibitory. The specificity of the inhibition by tosyl derivatives parallels that of unsubstituted substrates; tosyl galactosides of the beta configuration were inhibitory, but those of the alpha configuration were not. The tosyl derivatives also strongly inhibited the uptake of lactose analogs into whole cells; the requirement for the arylsulfonyl moiety was again observed. The chemical analogy between the tosyl galactosides and possible intermediates in the transport-phosphorylation step catalyzed by Enzyme IIlac provides a possible explanation for the unexpected properties of these derivatives.

Bacterial Proteins↗
PubMed1996

Evaluation of cyclohexenoesculetin-beta-D-galactoside and 8-hydroxyquinoline-beta-D-galactoside as substrates for the detection of beta-galactosidase.

We describe the synthesis of two new substrates for the detection of beta-galactosidase and evaluate their performance in comparison with that of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). Of 171 Enterobacteriaceae strains that were able to hydrolyze X-Gal, 166 (97.1%) also hydrolyzed cyclohexenoesculetin-beta-D-galactoside whereas only 96 (56.1%) showed evidence of hydrolysis of 8-hydroxyquinoline-beta-D-galactoside. No false-positive results were observed with either substrate.

Enterobacteriaceae↗
PubMed1992

Evidence that Caenorhabditis elegans 32-kDa beta-galactoside-binding protein is homologous to vertebrate beta-galactoside-binding lectins. cDNA cloning and deduced amino acid sequence.

We have cloned a full-length cDNA for a beta-galactoside-binding protein with a relative molecular mass of 32 kDa (32-kDa GBP), recently purified from a nematode, Caenorhabditis elegans (Hirabayashi, J., Satoh, M., Ohyama, Y., and Kasai, K. (1992) J. Biochem. 111, 553-555). The clone contained a single open reading frame encoding 279 amino acids, including the initiator methionine. Significant sequence homology to metal-independent beta-galactoside-binding lectins (25-30% identities), which had previously been found only in vertebrates, was observed. Moreover, the nematode 32-kDa GBP proved to have a unique polypeptide architecture; that is, it is composed of two tandemly repeated homologous domains, each consisting of about 140 amino acids. The internal homology was about 32%. Thus, this protein is constructed with a duplicated fundamental unit which is similar to the subunit of vertebrate 14-kDa lectins. In spite of the extreme phylogenic distance between nematodes and vertebrates (divergence greater than 6 x 10(8) years ago), both of the two repeated domains of the nematode 32-kDa GBP retained most of the amino acid residues conserved in vertebrate lectins. This means that members of the metal-independent animal lectin family are distributed much more widely than had been believed: from nematodes to vertebrates. The implication is that proteins belonging to this family have fundamental roles which are not restricted to vertebrates but are common to almost all animals.

Amino Acid Sequence↗
PubMed1976

Properties of the entry and exit reactions of the beta-methyl galactoside transport system in Escherichia coli.

The Km, Vmax, and Ki of the entry reaction were determined for three substrates of the beta-methyl galactoside transport system: D-galactose, D-glycerol-beta-D-galactoside, and beta-methyl-D-galactoside. Although the data for D-galactose and D-glycerol-beta-D-galactoside followed simple Michaelis-Menten kinetics, the results for beta-methyl-D-galactoside deviated from Michaelis-Menten kinetics in that the Ki for beta-methyl-D-galactoside inhibition of both of the other two substrates was 10-fold greater than the Km for beta-methyl-D-galactoside entry. Furthermore, two partial mgl- strains retain 56% of the parental level of the beta-methyl-D-galactoside entry reaction, but only 12% of the parental level of transport of the other two substrates. The exit reaction of beta-methyl-D-galactoside was shown to be first order. It was stimulated sixfold when the cells were provided with an energy source. This stimulation required adenosine 5'-triphosphate or a related compound. The exit reaction was not altered by mutations in any of the three cistrons which inactivate the beta-methyl-D-galactoside entry reaction, was not increased by growth in the presence of inducers of the entry reaction, and was not repressed by growth on glucose. The striking differences between the entry and exit reactions suggest that they either use different carriers or that none of the three cistrons which are currently known to code for components of the beta-methyl galactoside transport system code for its membrane carrier.

Adenosine Triphosphate↗
PubMed1973

Dansyl-galactoside, a fluorescent probe of active transport in bacterial membrane vesicles.

A fluorescent galactoside, 2-(N-dansyl)-aminoethyl beta-D-thiogalactoside (dansyl-galactoside), competitively inhibits lactose transport by membrane vesicles of Escherichia coli, but is not actively transported. An increase in dansyl-galactoside fluorescence is observed upon addition of D-lactate. The fluorescence increase is not observed in membrane vesicles lacking the beta-galactoside transport system, and is blocked or rapidly reversed by addition of beta-galactosides, sulfhydryl reagents, inhibitors of D-lactate oxidation, or uncoupling agents. The fluorescence increase exhibits an emission maximum at 500 nm and excitation maxima at 345 nm and at 292 nm. The latter excitation maximum is absent unless D-lactate is added, indicating that the bound dansyl-galactoside molecules are excited by energy transfer from the membrane proteins. Titration of vesicles with dansyl-galactoside in the presence of D-lactate demonstrates that the beta-galactoside carrier protein represents about 3.3% of the total membrane protein. The data indicate that D-lactate oxidation leads to binding of the fluorescent galactoside to the beta-galactoside carrier protein in such a manner that the dansyl group is transferred to a hydrophobic environment within the membrane.

Binding Sites↗
PubMed1985

Initiation of chondroitin sulphate synthesis by beta-D-galactosides. Substrates for galactosyltransferase II.

beta-Galactosides were found to initiate chondroitin sulphate chain synthesis in chick-embryo cartilage in vitro and thereby relieve inhibition by cycloheximide of [3H]-acetate incorporation into chondroitin sulphate. beta-Galactosides with an apolar aglycan group such as phenyl O-beta-galactoside were active, whereas those with a charged or polar aglycan group such as pyridine 3-O-beta-galactoside or those with sulphur instead of oxygen in the glycosidic linkage (phenyl beta-thiogalactoside) were not. beta-Galactosides also serve as substrates for microsomal galactosyltransferase activity from chick-embryo cartilage. Phenyl O-beta-galactoside and pyridine 3-O-beta-galactoside were effective substrates for this enzyme, but phenyl S-beta-thiogalactoside and pyridine 2-S-beta-thiogalactoside were only slightly active. This galactosyltransferase was shown to be a separate enzyme from galactosyltransferase I, which catalyses transfer of galactose from UDP-galactose to beta-xylosides. It is proposed that the enzyme catalysing this reaction is galactosyltransferase II, responsible for transfer of the second galactose residue of the chondroitin sulphate linkage oligosaccharide. No transfer of glucuronic acid from UDP-glucuronic acid to beta-galactosides, catalysed by the microsomal preparation could be detected.

Animals↗
PubMed1982

Discrimination between activity of (alpha 2-3)-sialyltransferase and (alpha 2-6)-sialyltransferase in human platelets using p-nitrophenyl-beta-D-galactoside as acceptor.

Exogenous asialo-glycoproteins and endogenous acceptors are both sialylated by incubating cytidine 5'-monophosphate N-[14C]acetylneuraminic acid (CMP [14C]NeuAc) with a lysate of human platelets but their respective incorporation levels vary with the divalent cation concentration. P-Nitrophenyl-beta-D-galactoside has also been demonstrated to be an acceptor of sialyl residues, and two different sialyl derivatives are synthesized according to the concentration of divalent cations. P-Nitrophenyl-beta-D-[6-3H]galactoside has been prepared by reduction with tritiated borohydride of the compound previously oxidized by galactose oxidase. Using this labelled p-nitrophenyl-beta-D-galactoside as acceptor and unlabelled CMP-NeuAc as donor, the two sialyl derivatives have been identified by methylation analysis as alpha-sialosyl-(2-3)-p-nitrophenyl-beta-D-galactoside and alpha-sialosyl-(2-6)-p-nitrophenyl-beta-D-galactoside. In addition to their different responses to divalent cation requirements, the sialyltransferase activities responsible for the synthesis of the two sialylgalactoside isomers have been clearly distinguished by their temperature and pH optimal values. They also exhibit different susceptibilities to dithioerythritol and different stabilities. These results demonstrate the presence in human platelets of two sialyltransferases: a CMP-NeuAc: galactoside (alpha 2-3)-sialyltransferase and a CMP-NeuAc: galactoside (alpha 2-6)-sialyltransferase.

Blood Platelets↗
PubMed1995

Synthesis of cluster galactosides with high affinity for the hepatic asialoglycoprotein receptor.

High-affinity ligands for the asialoglycoprotein receptor, which is uniquely localized on the parenchymal liver cell and recognizes oligoantennary galactosides, might be utilized as homing device to specifically target drugs or genes to parenchymal liver cells. In the present study, the synthesis of galactose-terminated triantennary glycosides, provided with various spacers between the beta-galactopyranosyl moieties and the branching point of the dendrite, is described. N-[Tris[[(methylthio)methoxy]methyl]methyl]-N alpha-[1-(6- methyladipy)]glycinamide (3b) was glycosylated with monogalactosyl derivatives, containing propanediol or ethylene glycol units as hydrophilic spacer moieties, to yield the corresponding cluster galactosides. To determine the affinity of the cluster galactosides for the asialoglycoprotein receptor, we have performed competition studies of [125I]ASOR binding, a specific ligand for the asialoglycoprotein receptor, to isolated parenchymal cells. The affinity for the asialoglycoprotein receptor significantly increased with increasing spacer length. N-[[[Tris-O-(beta-D-galactopyranosyl)-3,6,9-trioxaunde- canoxy]methoxy]methyl]-N-alpha-[1-(6-methyladipyl)]glycinami de (4e), a cluster galactoside provided with a 20 A spacer, possessed an at least 2000-fold higher affinity for the receptor than N-[[tris-O-(beta-D-galactopyranosyl)methyl]methyl]-N alpha-[1-(6- methyladipyl)]glycinamide (4a), a cluster galactoside lacking the spacer. It is concluded that vicinal galactosyl moieties within a cluster galactoside are more optimal recognized by the galactose binding sites of the asialoglycoprotein receptor upon proper spacing. The most potent galactoside, TG(20A), may constitute an attractive targeting device for the specific delivery of drugs and/or genes to the parenchymal liver cell.

Animals↗
PubMed1987

Galactoside-proton symport in a lacYUN mutant of Escherichia coli investigated by analysis of transport progress curves.

The kinetics of galactoside-proton symport catalysed by a wild-type strain and one carrying a mutation, previously reported to cause uncoupling of the symport reaction, have been examined. The mutation does not affect the stoichiometry during the initial period of uptake, when the internal concentration of galactoside is low, but it does result in much greater competition from the galactoside as it is accumulated. Simple methods for the analysis of the uptake progress curves have been developed and used to estimate the initial rate of uptake and affinity for internal galactoside. The maximum rate of uptake is decreased by a factor of 2 at most whereas the affinity for internal galactoside is increased up to 50-fold by the mutation. The pH-dependence of the galactoside efflux reaction is changed in a manner which suggests that the defect is in the interaction between proton-binding and galactoside-binding sites rather than in the structure of either site.

Binding Sites↗
PubMed1973

Studies on beta-galactoside transport in a Proteus mirabilis merodiploid carrying an Escherichia coli lactose operon.

Merodiploid derivatives bearing an F-linked lac operon (i(+), o(+), z(+), y(+), a(+)) from Escherichia coli were prepared from a Proteus mirabilis strain unable to utilize lactose and from a lac deletion strain of E. coli. A suitable growth medium was found in which the episomal element in the P. mirabilis derivative was sufficiently stable to allow induction of the episome-borne lac operon and thus to permit a comparison of the activities and properties of E. coli lac products in the intracellular environments of P. mirabilis and E. coli. In both derivatives the episomal lac operon was shown to be repressed in the absence of inducer. Kinetics of induction with gratuitous inducer (isopropyl-1-thio-beta-d-galactoside) were similar for both beta-galactosidase activity (beta-d-galactoside galactohydrolase, EC 3.4.1.23) and beta-galactoside transport activity in both derivatives, although the ratio of galactoside transport to beta-galactosidase activity was approximately 1.6-fold higher in the E. coli derivative. Comparison of beta-galactosidase and M-protein (lac y gene product)-specific activities indicated coordinate expression of the induced lac operon in both derivatives. Quantitatively, the maximal beta-galactosidase specific activity was two or three times higher for the E. coli derivative. A significant sodium azide inhibition (65% inhibition by 10 mM sodium azide) of lactose permease-mediated transport of o-nitrophenyl-beta-galactoside from an outside region of high concentration to an inside region of very low concentration ("downhill transport") was observed for the P. mirabilis derivative. Identical conditions for the E. coli derivative yielded only about 15% inhibition. Active transport of thiomethyl-beta-galactoside was similar for both derivatives, the major difference being that active transport was more sensitive to azide poisoning in the P. mirabilis derivative. Preliminary examination of the thiomethyl-beta-galactoside derivatives following active transport did not demonstrate the accumulation of a phosphorylated product in either strain but did reveal an unidentified derivative present in the P. mirabilis merodiploid extract which was not detectable in the E. coli merodiploid.

Azides↗