Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Melibiose”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

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

At least 37 records · Page 2Linked to original sources
PubMed1990

Isolation and hemagglutinating activities of bovine immunoglobulins reactive with melibiose.

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.

Animals↗
PubMed1980

H + -substrate cotransport by the melibiose membrane carrier in Escherichia coli.

Proton entry into anaerobic Escherichia coli in response to the addition of HCl was measured by monitoring pH changes in the external solution. Preincubation of cells in a Na+ -free medium containing melibiose or methyl-alpha-galactoside (alpha MG) stimulated the rate of H+ entry in response to the acid pulse. This melibiose- or alpha MG-dependent proton pathway appeared to be identical to the melibiose carrier, since the channel was only observed in melibiose-induced cells. Furthermore, this membrane pathway for protons showed the same temperature sensitivity as the melibiose carrier (active at 30 degrees but inactive at 37 degrees). These observations are consistent with the idea that the melibiose transport system provides a pathway for protons in the presence of appropriate substrates, but that the pathway is closed to protons in the absence of the sugar. Such observations indicate that there is an obligatory coupling between H+ flux and melibiose or alpha MG flux through the carrier when Na+ is omitted from the incubation medium.

Biological Transport↗
PubMed1997

The construction of a cysteine-less melibiose carrier from E. coli.

The melibiose carrier of E. coli is a cation-sugar cotransport system. This membrane protein contains four cysteine residues and the transport function is inhibited by sulfhydryl reagents. In order to investigate the importance of the cysteines, we have constructed a set of four melibiose transporters each of which has one cysteine replaced with serine or valine. The sensitivity of this set of carriers to N-ethylmaleimide was tested and Cys364 was identified as the target of the reagent. In addition, we constructed a melibiose transporter in which all 4 cysteines were replaced with either serine (Cys110, Cys310, and Cys364) or valine (Cys235) and we found that, as expected, the resulting cysteine-less transporter was resistant to the action of N-ethylmaleimide. The cysteine-less melibiose carrier had no significant decrease in ability to accumulate melibiose with cotransported sodium ions or protons. Thus, none of the 4 cysteines are necessary for the function of the melibiose carrier.

Amino Acid Substitution↗
PubMed2001

A cryptic melibiose transporter gene possessing a frameshift from Citrobacter freundii.

Wild-type Citrobacter freundii cannot grow on melibiose as a sole source of carbon. The melibiose transporter gene melB was cloned from a C. freundii mutant M4 that could utilize melibiose as a sole carbon source. Although the cloned melB gene is closely similar to the melB genes of other bacteria, it is cryptic because of a frameshift mutation. Site-directed mutagenesis was used to construct a functional melB gene by deleting one nucleotide, resulting in the production of an active melibiose transporter. The active MelB transporter could utilize Na(+) and H(+) as coupling cations to melibiose transport. The amino acid sequence of the C. freundii MelB was found to be most similar to those of Salmonella typhimurium and Escherichia coli MelB. These facts are consistent with the phylogenetic relationship of bacteria and the cation coupling properties of the melibiose transporters.

Amino Acid Sequence↗
PubMed1981

Role of inducer exclusion in preferential utilization of glucose over melibiose in diauxic growth of Escherichia coli.

The role of inducer exclusion in diauxic growth of Escherichia coli on glucose and melibiose was investigated. The amounts of glucose and melibiose in the culture medium were determined during the diauxie. Glucose was consumed during the first growth cycle of the diauxie, and melibiose was consumed during the second cycle. The addition of adenosine 3',5'-cyclic monophosphate to the culture medium released both transient and catabolite repressions on the melibiose operon by glucose. Biphasic growth without a transient lag phase was observed in the presence of adenosine 3',5'-cyclic monophosphate. Preferential utilization of glucose over melibiose was observed even under such conditions. Thus, it is clear that inducer exclusion alone is sufficient to ensure the preferential utilization of glucose over melibiose. Similar results were obtained from a glucose-lactose diauxie. Inducer exclusion itself was not affected by adenosine 3',5'-cyclic monophosphate.

Biological Transport↗
PubMed1997

Lactose carrier mutants of Escherichia coli with changes in sugar recognition (lactose versus melibiose).

The purpose of this research was to identify amino acid residues that mediate substrate recognition in the lactose carrier of Escherichia coli. The lactose carrier transports the alpha-galactoside sugar melibiose as well as the beta-galactoside sugar lactose. Mutants from cells containing the lac genes on an F factor were selected by the ability to grow on succinate in the presence of the toxic galactoside beta-thio-o-nitrophenylgalactoside. Mutants that grew on melibiose minimal plates but failed to grow on lactose minimal plates were picked. In sugar transport assays, mutant cells showed the striking result of having low levels of lactose downhill transport but high levels of melibiose downhill transport. Accumulation (uphill) of melibiose was completely defective in all of the mutants. Kinetic analysis of melibiose transport in the mutants showed either no change or a greater than normal apparent affinity for melibiose. PCR was used to amplify the lacY DNA of each mutant, which was then sequenced by the Sanger method. The following six mutations were found in the lacY structural genes of individual mutants: Tyr-26-->Asp, Phe-27-->Tyr, Phe-29-->Leu, Asp-240-->Val, Leu-321-->Gln, and His-322-->Tyr. We conclude from these experiments that Tyr-26, Phe-27, Phe-29 (helix 1), Asp-240 (helix 7), Leu-321, and His-322 (helix 10) either directly or indirectly mediate sugar recognition in the lactose carrier of E. coli.

Amino Acids↗
PubMed1999

Arg-52 in the melibiose carrier of Escherichia coli is important for cation-coupled sugar transport and participates in an intrahelical salt bridge.

Arg-52 of the Escherichia coli melibiose carrier was replaced by Ser (R52S), Gln (R52Q), or Val (R52V). While the level of carrier in the membrane for each mutant remained similar to that for the wild type, analysis of melibiose transport showed an uncoupling of proton cotransport and a drastic reduction in Na(+)-coupled transport. Second-site revertants were selected on MacConkey plates containing melibiose, and substitutions were found at nine distinct locations in the carrier. Eight revertant substitutions were isolated from the R52S strain: Asp-19-->Gly, Asp-55-->Asn, Pro-60-->Gln, Trp-116-->Arg, Asn-244-->Ser, Ser-247-->Arg, Asn-248-->Lys, and Ile-352-->Val. Two revertants were also isolated from the R52V strain: Trp-116-->Arg and Thr-338-->Arg revertants. The R52Q strain yielded an Asp-55-->Asn substitution and a first-site revertant, Lys-52 (R52K). The R52K strain had transport properties similar to those of the wild type. Analysis of melibiose accumulation showed that proton-driven accumulation was still defective in the second-site revertant strains, and only the Trp-116-->Arg, Ser-247-->Arg, and Asn-248-->Lys revertants regained significant Na(+)-coupled accumulation. In general, downhill melibiose transport in the presence of Na(+) was better in the revertant strains than in the parental mutants. Three revertant strains, Asp-19-->Gly, Asp-55-->Asn, and Thr-338-->Arg strains, required a high Na(+) concentration (100 mM) for maximal activity. Kinetic measurements showed that the N248K and W116R revertants lowered the K(m) for melibiose, while other revertants restored transport velocity. We suggest that the insertion of positive charges on membrane helices is compensating for the loss of Arg-52 and that helix II is close to helix IV and VII. We also suggest that Arg-52 is salt bridged to Asp-55 (helix II) and Asp-19 (helix I).

Amino Acid Sequence↗
PubMed2006

Changes in secondary structures and acidic side chains of melibiose permease upon cosubstrates binding.

Infrared difference spectroscopy analysis of the purified melibiose permease of Escherichia coli reconstituted into liposomes was carried out as a function of the presence of the two symporter substrates (Na(+), melibiose) in either H(2)O or in D(2)O media. Essentially, the data first show that addition of Na(+) induces appearance of peaks assigned to changes in the environment and/or orientation of alpha-helical domains of purified melibiose permease. Likewise, melibiose addition in the presence of Na(+) produces peaks corresponding to additional changes of alpha-helix environment or tilt. In addition to these changes, a pair of peaks (1599 (+) cm(-1)/1576 (-) cm(-1)) appearing in the Na(+)-induced difference spectrum is assigned to the antisymmetric stretching of COO(-) groups, since they show practically no shift upon H/D exchange. It is proposed that these acidic groups participate in Na(+) co-ordination. A corresponding pair of peaks, again fairly insensitive to H/D substitution (1591 (-) cm(-1)/1567 (+) cm(-1)), appear in the melibiose-induced difference spectra, and may again be assigned to COO(-) groups. The latter carboxyl groups may correspond to part or all of the acidic residues interacting with Lys or Arg in the resting state that become free upon melibiose binding.

Amino Acid Motifs↗
PubMed1984

Physical and genetic characterization of the melibiose operon and identification of the gene products in Escherichia coli.

We have identified hybrid plasmids carrying the melibiose operon of Escherichia coli in a colony bank of Clarke and Carbon (Tsuchiya, T., Ottina, K., Moriyama, Y., Newman, M., and Wilson, T. H. (1982) J. Biol. Chem. 257, 5125-5128). Using one of the plasmids as a starting material, the DNA fragments containing the melibiose operon were recloned in a vector pBR322. Restriction maps were prepared, and several DNA segments were subcloned into pBR322. Genetic complementation tests and recombination analyses using those plasmids and melA- and melB- mutants as well as biochemical analyses of mel mutants transformed with those plasmids enabled us to determine the physical location of promoter, melA, and melB on the DNA segment. The size of the melAB region was about 3,000 base pairs. Gene products were identified using maxicells harboring plasmids carrying the melibiose operon. The apparent molecular weight of the alpha-galactosidase (coded by melA) was about 50,000 and that of the melibiose carrier (coded by melB) was about 31,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The melibiose carrier was also identified as a 30,000-dalton protein in reconstituted proteoliposomes which possessed melibiose transport activity.

Disaccharides↗
PubMed1994

Replacement of alanine 58 by asparagine enables the melibiose carrier of Klebsiella pneumoniae to couple sugar transport to Na+.

The melibiose carrier of Klebsiella pneumoniae couples sugar transport to H+ and Li+, while that of Escherichia coli uses Na+ besides the other two cation species (Hama and Wilson, 1992). We have shown that the K. pneumoniae melibiose carrier is capable of recognizing Na+ when the amino-terminal 81 residues are replaced by the corresponding region of the E. coli melibiose carrier (Hama and Wilson, 1993). In this amino-terminal region there are 5 residues that are not conserved between the two carriers. In this study, we changed each of the 5 residues of the K. pneumoniae carrier to the one in the E. coli carrier. The substitutions are Ile-36-->Val, Val-43-->Leu, Leu-54-->Trp, Ala-58-->Asn, and Cys-68-->Ala. With four of the five mutants, Ile-36-->Val, Val-43-->Leu, Leu-54-->Trp, and Cys-68-->Ala, sugar accumulation was not affected by Na+. In striking contrast, melibiose and methyl-1-thio-beta-D-galactopyranoside accumulation was greatly stimulated by Na+ with the Ala-58-->Asn mutant. Furthermore, Na+ uptake coupled to downhill melibiose transport was observed with the Ala-58-->Asn mutant. These results indicate that the Ala-58-->Asn substitution enables the K. pneumoniae melibiose carrier to couple sugar transport to Na+. It is clear that the Asn-58 residue (Asn-54 in the E. coli carrier) is involved in Na+ recognition.

Base Sequence↗
PubMed2001

Cysteine substitutions for individual residues in helix VI of the melibiose carrier of Escherichia coli.

The melibiose carrier of Escherichia coli is a cytoplasmic membrane protein that mediates the cotransport of galactosides with H(+), Na(+), or Li(+). In this study we used cysteine-scanning mutagenesis to try to gain information about the position of transmembrane helix VI in the three-dimensional structure of the melibiose carrier. We constructed 23 individual cysteine substitutions in helix VI and an adjacent loop of the carrier. The resulting melibiose carriers retained 22-100% of their ability to transport melibiose. We tested the effect of the hydrophilic sulfhydryl reagent p-chloromercuri-benzenesulfonic acid (PCMBS) on the cysteine-substitution mutants and we found that there was no inhibition of melibiose transport in any of the mutants. We suggest that helix VI is imbedded in phospholipid and does not face the aqueous channel through which melibiose passes.

Amino Acid Sequence↗
PubMed1992

Resistance of the melibiose carrier to inhibition by the phosphotransferase system due to substitutions of amino acid residues in the carrier of Salmonella typhimurium.

The melibiose carrier of Salmonella typhimurium is under the control of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). We isolated mutants of the melibiose carrier that showed resistance to inhibition via the PTS. Growth of the mutants on melibiose was not inhibited by 2-deoxyglucose, a non-metabolizable substrate of the PTS, although growth of the parent strain was inhibited. Transport activity of the melibiose carrier in the mutants was fairly resistant to inhibition by 2-deoxyglucose, although the activity in the parent was sensitive to inhibition. We cloned the mutated melB gene that encodes the melibiose carrier, determined the nucleotide sequences, and identified replaced nucleotides. The mutations resulted in substitutions of Asp-438 with Tyr, Arg-441 with Ser, or Ile-445 with Asn. All of these residues are in the COOH-terminal region of the carrier. The secondary structure of this region is predicted to be an alpha-helix, and the mutated residues were on the same side of the helix. This region showed sequence similarity to a region of the MalK protein, in which substitution of amino acid residues also resulted in PTS-resistant mutants. Thus the COOH-terminal portion of the melibiose carrier is important for the interaction of dephosphorylated IIIGlc, which is an entity causing reversible inactivation of the carrier.

Amino Acid Sequence↗
PubMed1985

Effect of membrane potential on the kinetic parameters of the Na+ or H+ melibiose symport in Escherichia coli membrane vesicles.

Comparison of the transport properties of the melibiose permease of E. coli acting as a H+-symport or a Na+-symport has been performed by measuring initial rates of [3H]-melibiose transport or its accumulation in isolated membrane vesicles. 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+-melibiose symport. It is suggested that modification(s) of some partial reaction constants of a given transport cycle might lead to important changes in the kinetic properties of this transport system.

Biological Transport, Active↗
PubMed1998

Evidence for a close association between helix IV and helix XI in the melibiose carrier of Escherichia coli.

The melibiose carrier of Escherichia coli is a cation-sugar cotransport protein. Asp124 in membrane-spanning helix IV of the carrier protein was replaced with Ser, Ile or Phe by site-directed mutagenesis of a plasmid containing the melB gene. Each of these mutants failed to show membrane transport of melibiose and melibiose-positive revertants could be isolated on melibiose MacConkey indicator plates. D124F showed only one type of revertant (D124C) and D124I showed only revertants to the normal (D124). Second site revertants were not found with either of these mutants. S124, however, showed two types of second site revertants: D124S/V375A and D124S/V375G. The revertant D124S/V375A had lost melibiose/proton cotransport but showed 25% of normal melibiose (20 mM) uptake in the presence of 10 mM sodium ion at 37 degrees C. The value for the parental strain D124S was 2%. The second revertant D124S/V375G showed greater activity than S124 in the presence of 100 mM NaCl at both 20 degrees C and 37 degrees C. It was concluded that in the normal carrier protein Asp124 in helix IV is probably close to Val375 in helix XI. Since Lys377 is close to Val375, it is possible that Asp124 may interact with Lys377 to form a salt bridge.

Base Sequence↗
PubMed2005

Substrate-induced conformational changes of melibiose permease from Escherichia coli studied by infrared difference spectroscopy.

Fourier transform infrared difference spectroscopy has been used to obtain information about substrate-induced structural changes of the melibiose permease (MelB) from Escherichia coli reconstituted into liposomes. Binding of the cosubstrate Na(+) gives rise to several peaks in the amide I and II regions of the difference spectrum Na(+).MelB minus H(+).MelB, that denote the presence of conformational changes in all types of secondary structures (alpha-helices, beta-sheets, loops). In addition, peaks around 1400 and at 1740-1720 cm(-1) are indicative of changes in protonation/deprotonation or in environment of carboxylic groups. Binding of the cosubstrate Li(+) produces a difference spectrum that is also indicative of conformational changes, but that is at variance as compared to that induced by Na(+) binding. To analyze the following transport steps, the melibiose permease with either H(+), Na(+), or Li(+) bound was incubated with melibiose. The difference spectra obtained by subtracting the spectrum cation.MelB from the respective complex cation.melibiose.MelB were roughly similar among them, but different from those induced by cation binding, and more intense. Therefore, major conformational changes that are induced during melibiose binding/substrate translocation, like those denoted by intense peaks at 1668 and 1645 cm(-)(1), are similar for the three cotransporting cations. Changes in the protonation state and/or in the environment of given carboxylic residues were also induced by melibiose-MelB interaction in the presence of cations.

Binding Sites↗
PubMed2004

Sugar binding induced charge translocation in the melibiose permease from Escherichia coli.

Electrogenic events associated with the activity of the melibiose permease (MelB), a transporter from Escherichia coli, were investigated. Proteoliposomes containing purified MelB were adsorbed to a solid supported lipid membrane, activated by a substrate concentration jump, and transient currents were measured. When the transporter was preincubated with Na(+) at saturating concentrations, a charge translocation in the protein upon melibiose binding could still be observed. This result demonstrates that binding of the uncharged substrate melibiose triggers a charge displacement in the protein. Further analysis showed that the charge displacement is neither related to extra Na(+) binding to the transporter, nor to the displacement of already bound Na(+) within the transporter. The electrogenic melibiose binding process is explained by a conformational change with concomitant displacement of charged amino acid side chains and/or a reorientation of helix dipoles. A kinetic model is suggested, in which Na(+) and melibiose binding are distinct electrogenic processes associated with approximately the same charge displacement. These binding reactions are fast in the presence of the respective cosubstrate (k > 50 s(-1)).

Binding Sites↗
PubMed1997

Proteolytic mapping and substrate protection of the Escherichia coli melibiose permease.

The topology and substrate-induced conformational change(s) of the Na+ (Li+ or H+)-melibiose cotransporter (MelB) of Escherichia coli were investigated by limited protease digestion. To facilitate these analyses, MelB was epitope-tagged both at its carboxyl-terminus and at its amino-terminus. Limited digestion with different proteases indicates that the cytoplasmic loops connecting transmembrane domains 4-5, 6-7, and 10-11 together with the carboxyl-terminus of MelB are exposed in the cytoplasm. In contrast, periplasmic loops are highly resistant to all the proteases examined, including nonspecific proteases such as proteinase K and thermolysin. The effect of Na+ or Li+ and/or melibiose on the rate of protease digestion of the cytoplasmic loops was also analyzed. The rate of protease digestion of loop 4-5 is specifically reduced, by approximately 3-fold, by the presence of Na+ or Li+. These results suggest that loop 4-5 is near or part of the cation binding site. Moreover, the presence of both melibiose and either Na+ or Li+ further reduced the rate of protease digestion of this loop 4-5 by up to 9-fold, although no protection from protease digestion was observed when melibiose was added alone. The increase in resistance to proteases observed in the presence of the cation alone or the cation plus melibiose suggests that the interaction of the two cosubstrate with MelB results in change(s) of MelB conformation.

Amino Acid Sequence↗
PubMedDate not supplied

Cybernetic model for the growth of Saccharomyces cerevisiae on melibiose.

Cybernetic modeling has traditionally been used in the modeling of microbial growth on multiple substrates. In this paper, cybernetic modeling has been applied to serve as a model for growth on substrates such as melibiose, which are disaccharides and enzymatically degrade to a monosaccharide mixture in the fermentation broth. The enzyme alpha-galactosidase has been shown to be strongly induced in the presence of galactose and severely repressed by glucose. In the present model, the relative concentration of alpha-galactosidase has been linked to that of the key enzyme for galactose metabolism. The enzymatic degradation process is placed under the control of the cybernetic variables. The maximum rate of melibiose degradation vm and the Monod parameters for growth of Saccharomyces cerevisiae on pure glucose and galactose were estimated by batch growth experiments. S. cerevisiae growth on melibiose and a mixture of melibiose and glucose under a variety of preculturing conditions was simulated. Depending on the rate of enzymatic degradation (i.e., the value of vm), the cell mass profile for microbial growth on a disaccharide can resemble profiles for growth on a single substrate (melibiose) or can resemble diauxie growth. Experiments indicate that the model is able to accurately predict the cell mass profiles for yeast growth.

Culture Media↗