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.

SourceResearch connectionFrom yearThrough year

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

At least 73 records · Page 4Linked to original sources

PubMed1998

Mutants of Citrobacter freundii that transport and utilize melibiose.

We have isolated mutants of Citrobacter freundii that can grow on melibiose. Inducible alpha-galactosidase activity and melibiose transport activity were detected in the mutant cells but not in the wild-type cells. We detected a DNA region which hybridized with melB (the gene for the melibiose transporter) DNA of Escherichia coli in the chromosomal DNA of wild-type C. freundii. Protons, but not sodium ions, were found to be the coupling cations for melibiose (and methyl-beta-D-thiogalactoside) transport in the mutant cells.

Biological Transport↗

PubMed1990

Sensitivity of efflux-driven carrier turnover to external pH in mutants of the Escherichia coli lactose carrier that have tyrosine or phenylalanine substituted for histidine-322. A comparison of lactose and melibiose.

Two Escherichia coli lactose carrier mutants (tyrosine or phenylalanine substituted for histidine 322) were studied under conditions of net efflux or equilibrium exchange. Net lactose efflux by either mutant was 10-20-fold slower than by the parent and was sensitive to extracellular pH (5.6-8.0). The presence of extracellular lactose (equilibrium exchange) failed to accelerate loss of [14C]lactose, indicating that the step(s) rate limiting for exchange were also rate limiting for net lactose efflux. Net melibiose efflux by the Phe-322 mutant was comparable to the normal carrier, while that by the Tyr-322 mutant was 5-fold faster (pH 7.0). Melibiose efflux by either mutant was sensitive to pH (5.6-8.0). Melibiose in the extracellular medium significantly accelerated loss of [3H]melibiose from either mutant, showing that slow exchange is a sugar-specific phenomenon and not an intrinsic property of these mutants. The sugar-specific effect of these mutations could mean that the defect in these mutants is not on the path of the proton, although alternative explanations cannot as yet be eliminated. The modest effect of these mutations on the transport rate indicates that His-322 contributes a far smaller free energy increment to catalyzing of H+/galactoside cotransport than active site histidines contribute to catalyzing peptide bond hydrolysis in serine proteases. We interpret this to mean that in chemical terms the function of these catalytic histidine residues differ considerably.

Disaccharides↗

PubMed1982

Altered cation coupling to melibiose transport in mutants of Escherichia coli.

The alpha-galactoside transport system of Escherichia coli utilizes either H+ of Na+ as a coupling cation for melibiose transport. Mutants were isolated which showed altered cation recognition for melibiose transport. The transport carrier of the mutants has lost the ability to accept H+ but can utilize either Na/ or Li+ for co-transport with melibiose. Such mutants might be predicted if the melibiose carrier were viewed as a descendant of an evolutionary transition between the H+-substrate co-transport systems of primitive cells and the Na+-substrate systems of more complex organisms. A second mutation was found in these mutants which involves the Na+ (Li+)-H+ exchange carrier. The maximal rate of Li+-H+ exchange in one of the mutants was 11 times higher than the parent and the Km for Li+ by this carrier was one-sixth that of the parent.

Biological Transport, Active↗

PubMed1993

Mutagenesis of acidic residues in putative membrane-spanning segments of the melibiose permease of Escherichia coli. II. Effect on cationic selectivity and coupling properties.

Individual substitution of Cys or Asn for Asp-31, Asp-51, Asp-55, or Asp-120, distributed in different membrane spanning segments of the NH2-terminal domain of melibiose (mel) permease partially or completely inactivates Na(+)-linked sugar transport and stimulation of sugar binding on mel permease by Na+ ions (Pourcher, T., Zani, M.-L., and Leblanc, G. (1993) J. Biol. Chem. 268, 3209-3215). To investigate further the effect of these substitutions on the cationic selectivity and coupling properties of mel permease, H(+)-melibiose coupled transport, coupling between H+ and melibiose movements, sugar counterflow, and zero-trans sugar efflux by the mutant permeases were analyzed. The results provide additional evidence indicating that manipulation of some of these Asp in the membrane-spanning segments of mel permease alters its cationic selectivity properties. The results also indicate that the individual mutations diversely affect mel permease-coupling properties. For example, only permease with Asn in place of Asp-31 or Cys in place of Asp-51 retains the capacity to actively transport melibiose. On the other hand, replacing Asp-55 by Cys produces uncoupling of cosubstrate flows by the carrier but does not hamper sugar translocation. These and other features of the mutant permeases are used to discuss the relative participation of Asp-31, Asp-51, Asp-55, or Asp-120 to the mel symport mechanism and to its ionic selectivity and also the existence of a possible gating mechanism that may contribute the obligatory coupling of cosubstrate flows by the symporter.

Amino Acid Sequence↗

PubMed1997

Sequence of a melibiose transporter gene of Enterobacter cloacae.

We cloned a fragment of the chromosomal DNA of Enterobacter cloacae, which enabled a melibiose-negative Escherichia coli mutant lacking melB to grow on melibiose as the sole source of carbon. Transformed cells harboring the hybrid plasmid carrying the cloned DNA showed melibiose transport activity. The nucleotide sequence of the DNA region was determined. One complete open reading frame (ORF) and a part of another ORF were found in the region, and the amino acid sequences were deduced. The complete ORF was found to encode a melibiose transporter which consisted of 425 amino acid residues. Hydropathy analysis revealed that there are about 12 hydrophobic domains in this transporter. The incomplete ORF which exists in the upstream region of the transporter gene seemed to encode an alpha-galactosidase.

Amino Acid Sequence↗

PubMed1996

Membrane topology of the melibiose permease of Escherichia coli studied by melB-phoA fusion analysis.

In order to study the secondary structure of the melibiose permease of Escherichia coli, 57 melB-phoA gene fusions were constructed and assayed for alkaline phosphatase activity. In general agreement with a previously suggested secondary structure model of melibiose permease [Botfield, M. C., Naguchi, K., Tsuchiya, T., & Wilson, T.H. (1992) J. Biol. Chem. 267, 1818], clusters of fusions exhibiting low and high phosphatase activity fusions alternate along the primary sequence. Fusions with high activity generally cluster at residues predicted to be in the periplasmic half of transmembrane domains or in periplasmic loops, while fusions with low activity cluster at residues predicted to be in the cytoplasmic half of transmembrane domains or in cytoplasmic loops. Taken together, the findings strongly support the contention that melibiose permease contains 12 transmembrane domains that traverse the membrane in zigzag fashion connected by hydrophilic loops that are exposed alternatively on the periplasmic or cytoplasmic surfaces of the membrane with the N and C termini on the cytoplasmic face of the membrane. Moreover, on the basis of the finding that the cytoplasmic half of an out-going segment is sufficient for alkaline phosphatase export to the periplasm while the periplasmic half of an in-going segment prevents it [Calamia, T., & Manoil, C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4937], the activity profile of the melibiose permease-alkaline phosphatase fusions is consistent with the predicted topology of seven of 12 transmembrane segments. However, five transmembrane domains require adjustment, and as a consequence, the size of the central cytoplasmic loop is reduced and a significant number of charged residues are shifted from a hydrophilic to a hydrophobic domain in this region of the transporter.

Alkaline Phosphatase↗

PubMed1971

Biochemical studies of melibiose metabolism in wild type and mel mutant strains of Salmonella typhimurium.

I identified two enzyme activities, alpha-galactosidase and a galactoside permease, required for melibiose metabolism by Salmonella typhimurium. These activities are very low under normal growth conditions, but their production can be induced by melibiose and gratuitously by melibiitol. Melibiose induction is severely inhibited by glucose, but the glucose effect can be countered by 3', 5' cyclic adenosine monophosphate. I isolated two phenotypic classes of mutants not able to utilize melibiose as a carbon source. One class, Car(-), is deficient in the phosphotransferase system. The other, Mel, lacks either alpha-galactosidase, galactoside permease, or both functions.

Adenine Nucleotides↗

PubMed1985

Alteration in cation specificity of the melibiose transport carrier of Escherichia coli due to replacement of proline 122 with serine.

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.

Amino Acid Sequence↗

PubMed1989

Peptide-specific antibody for the melibiose carrier of Escherichia coli localizes the carboxyl terminus to the cytoplasmic face of the membrane.

The synthetic decapeptide NH2-Cys-Val-Gly-Ala-Val-Ser-Asp-Val-Lys-Ala-COOH (designated MBct10), which corresponds to the carboxyl terminus of the melibiose carrier of Escherichia coli, was synthesized and used to raise antibodies in a rabbit. Anti-MBct10 antibodies recognizes the normal melibiose carrier but not a truncated carrier lacking 14 carboxyl-terminal amino acids. Thus the antibodies are specific for the carboxyl terminus of the carrier and not for other domains of the protein. When right-side-out and inside-out membrane vesicles were probed with anti-MBct10 serum, only the inside-out vesicles bound antibody. The carboxyl terminus of the melibiose carrier protein is therefore exposed on the cytoplasmic surface of the membrane. The co-localization of both NH2- and carboxyl termini to the cytoplasmic surface dictates that the protein cross the membrane an even number of times. These data together with hydrophobicity analysis support a topological model for the melibiose carrier with 10 or 12 transmembrane domains.

Animals↗

PubMed1984

Nucleotide sequence of the melB gene and characteristics of deduced amino acid sequence of the melibiose carrier in Escherichia coli.

The nucleotide sequence of the melB gene coding for the melibiose carrier in Escherichia coli has been determined. The melibiose carrier is predicted to consist of 469 amino acid residues, resulting in a protein with a molecular weight of 52,029. The predicted carrier protein is highly hydrophobic (70% nonpolar amino acid residues). The hydropathic profile suggests that there are 10 long hydrophobic segments in the primary structure of the carrier protein. Most of them seem to traverse the membrane. Although the hydropathic profile of the melibiose carrier is similar to that of the lactose carrier as a whole, homology in the primary structure between the two carriers is very low. Furthermore, no homology in the nucleotide sequence is found in the structural genes for the two carriers. However, the nucleotide sequences of the intergenic regions are very similar between the melibiose operon and the lactose operon. There is a typical intercistronic regulatory sequence in the 3'-flanking region of the melB as well as in that of the lacY, which suggests the presence of another gene downstream of the melB.

Amino Acid Sequence↗

PubMed2000

Physiological evidence for an interaction between helix XI and helices I, II, and V in the melibiose carrier of Escherichia coli.

In a previous study 23 residues in helix XI of the cysteine-less melibiose carrier were changed individually to cysteine. Several of these cysteine mutants (K377C, A383C, F385C, L391C, G395C) had low transport activity and they were white on melibiose MacConkey fermentation plates. After several days of incubation of these white clones on melibiose MacConkey plates a rare red mutant appeared. The plasmid DNA was then isolated and sequenced. The two second site revertants from K377C were I22S and D59A. This change of aspartic acid to a neutral residue suggests that physiologically there is an interaction between K377 and D59 (possibly a salt bridge). The revertants from A383C were in positions 20 (F20L) and 22 (I22S and I22N). Revertants of F385C were intrahelical changes (I387M and A388G) and a change in C-terminal loop (R441C). Revertants of L391C were in helix I (I22N, I22T and D19E) and helix V (A152S). Revertants of G395C were in helix I (D19E and I22N). We suggest that there is an interaction between helix XI and helices I, II, and V and proximity between these helices.

4-Chloromercuribenzenesulfonate↗

PubMed1992

Transfer of genes for utilization of starch (sta2) and melibiose (mel) to industrial strains of Saccharomyces cerevisiae by single-chromosome transfer, using a kar1 mutant as vector.

A method has been developed for the transfer of genes from other yeast strains and species to industrial yeast strains, using a haploid, kar1-1 mutant strain of Saccharomyces cerevisiae as a vector. The sta2 gene, conferring the ability to metabolize starch was transferred from an auxotrophic haploid strain of S. cerevisiae (S. diastaticus) and the melibiose-metabolism (mel) gene(s), from S. kluyveri, to the kar1-1 mutant [K5-5A; (alpha ade2 his4 can1 gal) by normal mating and protoplast fusion. From this strain, the genes were transferred to baker's yeast and brewing yeast strains, which did not utilize starch, and to baker's yeast strains, which did not utilize melibiose, by protoplast fusion, spore-cell pairing, or rare-mating. Strains that utilized starch or melibiose were obtained by all three methods. Pulsed-field gel electrophoresis preparations showed little change in the mobility of the chromosomes of the hybrids. The most probable explanation for the results obtained is that single chromosomes were transferred, first, from the donor strains to the kar1-1 haploid mutant strain, and then from the kar1-1 vector to the recipient industrial strain of S. cerevisiae. The transfer of the genes is probably accomplished through formation of disomic strains and then, in the case of the hybrids that metabolize starch, by integration of the sta2 gene into the genome of the industrial yeast strains.

Chromosomes, Fungal↗

PubMed1995

Isolation of a melibiose-binding protein from human spleen.

A melibiose-binding protein was isolated from human spleen by serial affinity chromatography on lactose-, mannose-, and melibiose-Sepharose. The purified protein agglutinated rabbit erythrocytes and re-bound to melibiose, but did not bind to murine nor human laminin. The protein was composed of approximately 58 kDa and 26 kDa polypeptides. The polypeptides were detected in buffy coat cell extracts and they were synthesized in vitro by B lymphoblastoid cells. The polypeptides did not react with anti-galaptin, anti-C-reactive protein, anti-amyloid P, anti-keratin, and anti-rat lung lectin 29 sera. The 58 kDa polypeptide reacted very weakly with anti-core-specific lectin serum and reacted with anti-IgG serum. The data suggest that the major protein isolated is an anti-Ga1 alpha 1-->6 immunoglobulin.

Animals↗

PubMed2000

The melibiose carrier of Escherichia coli: cysteine substitutions for individual residues in helix XI.

The melibiose carrier from Escherichia coli is a sugar-cation cotransport system. Previously evidence was obtained that this integral membrane protein consists of 12 transmembrane helices. Starting with the cysteine-less melibiose carrier, cysteine has been substituted individually for amino acids 374-396, which includes all of the residues in the proposed helix XI. The carriers with cysteine substitutions were studied for their transport activity and the effect of the water soluble sulfhydryl reagent p-chloromercuribenzenesulfonic acid (PCMBS). Studies were carried out on both intact cells and inside out vesicles. Cysteine substitution caused loss of transport activity in seven of the mutants (K377C, G379C, A383C, F385C, L391C, G395C and Y396C). PCMBS produced more than 50% inhibition in six of the mutants (S380C, A381C, A384C, F387C, A388C and L391C). Preincubation of the cells with melibiose protected five of these residues from the inhibitory action of PCMBS. It was concluded that the residues whose cysteine derivatives were inhibited by PCMBS probably faced the aqueous channel.

4-Chloromercuribenzenesulfonate↗

PubMed1994

Transport properties of Asp-51-->Glu and Asp-120-->Glu mutants of the melibiose carrier of Escherichia coli.

Asp-51-->Glu and Asp-120-->Glu mutants of the melibiose carrier of Escherichia coli were investigated for their cation/sugar cotransport properties. The carrier containing Glu-51 showed proton/melibiose cotransport but was extremely defective in Na+ or Li+ stimulation of sugar accumulation. On the other hand, the carrier containing Glu-120 had lost the ability to couple protons with melibiose uptake while retaining considerable Na+ or Li+ cotransport with melibiose (40-fold accumulation versus 90-fold for the wild type in the presence of Na+). It is concluded that both Asp-51 and Asp-120 are important for cation recognition.

Biological Transport↗

PubMed1996

Alteration of Na(+)-coupled transport in site-directed mutants of the melibiose carrier of Escherichia coli.

Asn-58 of the Escherichia coli melibiose carrier was replaced by Ala, Leu, Ser, and Gln. Trp-54 was replaced by Leu and a double mutant Leu-54/Ala-58 was constructed using site-directed mutagenesis. Cation/sugar cotransport and sugar-induced cation uptake were studied for each mutant. The change of Asn-58 to Ala results in a nearly complete loss of Na(+)-stimulated galactoside transport as well as sugar-stimulated Na+ uptake. Substitutions of Leu, Gln, and Ser for Asn-58 were also defective in Na(+)-stimulated sugar transport. The Trp-54 to Leu mutant shows moderate sugar accumulation with cation selectivity similar to wild-type. The double mutant Leu-54/Ala-58 shows elevated H(+)-melibiose cotransport as well as reduced Na(+)-stimulated melibiose cotransport. These results suggest that Asn-58 is important for Na+ recognition.

Amino Acid Sequence↗

PubMed1991

Melibiose permease of Escherichia coli: mutation of aspartic acid 55 in putative helix II abolishes activation of sugar binding by Na+ ions.

An aspartic residue (Asp55) located in the putative transmembrane alpha-helix II of the melibiose(mel) permease of Escherichia coli was replaced by Cys using oligonucleotide-directed, site-specific mutagenesis. Although D55C permease is expressed at 0.7 times the level of wild type permease, the mutated mel permease loses the ability to catalyse Na+ or H+ coupled melibiose transport against a concentration gradient. (3H) p-nitrophenyl-alpha-D-galactoside (NPG) binding studies demonstrated that D55C permease binds the sugar co-substrate but Na+ (or Li+) ions do no longer enhance the affinity of D55C permease for the co-transported sugar. In addition sugar binding on D55C permease but not on wild type permease is inactivated by sulfhydryl reagents and the inhibition protected by an excess of melibiose. These observations suggest 1) that the negatively-charged Asp55 residue, expected to be within the membrane embedded domain near the NH2 extremity of mel permease, is in or near the Na(+)-binding site and 2) that the cation and sugar binding sites may be overlapping.

Aspartic Acid↗

PubMed1987

Organisation of the regulatory region of the Escherichia coli melibiose operon.

The regulatory region of the Escherichia coli melibiose operon contains two divergent promoters. One promoter is responsible for the expression of the melR gene, that is essential for melibiose-dependent stimulation of the second promoter. Melibiose-induced transcription from this second promoter initiates at a start point 25 bp upstream from the start codon of the melA gene, encoding an alpha-galactosidase. The nucleotide sequence covering the divergent promoters and the melR gene is reported.

Base Sequence↗