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At least 1,207 records · Page 67Linked to original sources
PubMed1997

Evidence for phospholipid microdomain formation in liquid crystalline liposomes reconstituted with Escherichia coli lactose permease.

The well-characterized integral membrane protein lactose (lac) permease from Escherichia coli was reconstituted together with trace amounts (molar fraction X = 0.005 of the total phospholipid) of different pyrene-labeled phospholipid analogs into 1-palmitoyl-2-oleoyl-sn-glycero-3-sn-glycero-3-phospho-rac'-glycerol (POPG) liposomes. Effects of lac permease on bilayer lipid dynamics were investigated by measuring the excimer-to-monomer fluorescence intensity ratio IE/IM. Compared to control vesicles, the presence of lac permease (at a protein:phospholipid stoichiometry P/L of 1:4.000) increased the rate of excimer formation by 1-palmitoyl-2[6-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC) by approximately fivefold. Decreasing P/L from approximately 1:4.000 to 1:7.600 decreased the IE/IM for PPDPC from 0.16 to 0.05, respectively. An increase in bilayer fluidity due to permease is unlikely, thus implying that the augmented IE/IM should arise from partial lateral segregation of PPDPC in the vesicles. This notion is supported by the further 38% increase in IE/IM observed for the pyrene-labeled Cys-148 lac permease reconstituted into POPG vesicles at P/L 1:4000. The importance of the length of the lipid-protein boundary is implicated by the reduction in IE/IM resulting from the aggregation of the lac permease in vesicles by a monoclonal antibody. Interestingly, excimer formation by 1-palmitoyl-2[6-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphocholine (PPHPC) was enhanced only fourfold in the presence of lac permease. Results obtained with the corresponding pyrenyl phosphatidylglycerols and -methanols were qualitatively similar to those above, thus indicating that lipid headgroup-protein interactions are not involved. Inclusion of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamino-N-(5-fluoresce inthio- carbamoyl) (DPPF, X = 0.005) into reconstituted lactose permease vesicles containing PPDPC caused a nearly 90% decrease in excimer fluorescence, whereas in control vesicles lacking the reconstituted protein only 40% quenching was evident. The addition of 1,2-dipalmitoyl-sn-glycero-3-phospho-rac'-glycerol (DPPG) decreased IE/IM for PPDPC, revealing the driving force for the lateral segregation of this probe to become attenuated. More specifically for protein-free bilayers at XDPPG = 0.10 the rate of lateral diffusion of PPDPC in POPG is diminished, as evidenced by the 24% decrement in IE/IM, under these conditions the increase in IE/IM due to lac permease was strongly reduced, by approximately 84%. The present data are interpreted in terms of the hydrophobic mismatch theory, which predicts that integral membrane proteins will draw lipids of similar hydrophobic thickness into their vicinity. In brief, the approximate lengths of most of the predicted 12 hydrophobic, membrane-spanning alpha-helical segments of lactose permease range between 28.5 and 37.5 A and thus exceed the hydrophobic thickness of POPG of approximately 25.8 A. Therefore, to reduce the free energy of the assembly, longer lipids such as PPDPC and DPPF are accumulated in the immediate vicinity of lactose permease in fluid, liquid crystalline POPG bilayers.

Binding Sites↗
PubMed1995

A nontransportable substrate for lactose permease.

A substrate for lactose permease of Escherichia coli was synthesized that binds to the protein with a relatively high affinity, but is not transported to any detectable extent. This substrate, 6'-[(N-phenylalanylphenylalanyl)amino]hexyl 1-thio-beta-D-galactoside, is a peptide galactoside composed of a bulky aromatic dipeptide that is linked to galactose via an aminohexyl spacer. Binding of the peptide galactoside to lactose permease in cytoplasmic membranes was determined in a competition assay yielding a dissociation constant of 150 microM. Transport was measured by a counterflow assay using lipid vesicles with reconstituted lactose permease. An upper limit for the rate constant of transport was obtained as 0.02 s-1, 3 orders of magnitude smaller than the value for lactose.

Biological Transport↗
PubMed2001

Engineering conformational flexibility in the lactose permease of Escherichia coli: use of glycine-scanning mutagenesis to rescue mutant Glu325-->Asp.

Lactose/H(+) symport by lactose permease of Escherichia coli involves interactions between four irreplaceable charged residues in transmembrane helices that play essential roles in H(+) translocation and coupling [Glu269 (helix VIII) with His322 (helix X) and Arg302 (helix IX) with Glu325 (helix X)], as well as Glu126 (helix IV) and Arg144 (helix V) which are obligatory for substrate binding. The conservative mutation Glu325-->Asp causes a 10-fold reduction in the V(max) for active lactose transport and markedly decreased lactose-induced H(+) influx with no effect on exchange or counterflow, neither of which involves H(+) symport. Thus, shortening the side chain may weaken the interaction of the carboxyl group at position 325 with the guanidino group of Arg302. Therefore, Gly-scanning mutagenesis of helices IX and X and the intervening loop was employed systematically with mutant Glu325-->Asp in an effort to rescue function by introducing conformational flexibility between the two helices. Five Gly replacement mutants in the Glu325-->Asp background are identified that exhibit significantly higher transport activity. Furthermore, mutant Val316-->Gly/Glu325-->Asp catalyzes active transport, efflux, and lactose-induced H(+) influx with kinetic properties approaching those of wild-type permease. It is proposed that introduction of conformational flexibility at the interface between helices IX and X improves juxtapositioning between Arg302 and Asp325 during turnover, thereby allowing more effective deprotonation of the permease on the inner surface of the membrane [Sahin-Tóth, M., Karlin, A., and Kaback, H. R. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 10729-10732.

Amino Acid Sequence↗
PubMed1987

Size and shape of the Escherichia coli lactose permease measured in filamentous arrays.

The Escherichia coli lactose permease has been purified on cation exchanger to contain a minimal amount of phospholipids, i.e., 4-5 mol/mol of permease, in the presence of the detergent dodecyl beta-maltoside at its critical micelle concentration. This preparation is active in galactoside binding. When the detergent level is further reduced by dialysis, the lactose permease forms filaments one molecule wide and up to several micrometers long. The filaments tend to associate laterally to form sheets. Analysis of electron micrographs of negatively stained filamentous arrays indicates an average filament spacing of 51 A and a subunit period of 26-30 A along individual filaments. These values most probably correspond to the dimensions of the lactose permease molecule measured parallel to the membrane plane. In many filaments, the subunits show a stain-penetrated cleft. It suggests that the lactose permease molecule comprises two domains, which may be correlated with internal repeats between the N- and C-terminal halves of the polypeptide sequence.

Chromatography, Ion Exchange↗
PubMed2002

An isotope-edited FT-IR study of a symporter, the lactose permease.

The lactose permease of Escherichia coli transports protons and lactose across the plasma membrane and uses a transmembrane ion gradient as the energy source to drive the uphill accumulation of lactose. In this report, the effect of the electrochemical gradient on the permease has been studied. Bacteriorhodopsin was co-reconstituted with the lactose permease to provide a light-triggered electrochemical gradient. Reaction-induced Fourier transform infrared spectra were acquired, and bacteriorhodopsin contributions were subtracted. In previous work, positive bands in the 1765-1730 cm(-1) region of the reaction-induced FT-IR spectrum were attributed to the perturbation of carboxylic acid residues in the permease [Patzlaff, J. S., Brooker, R. J., and Barry, B. A. (2000) J. Biol. Chem. 275, 28695-28700]. In this study, we have globally labeled the permease with (13)C or (15)N. Isotopic labeling demonstrates that features in the reaction-induced FT-IR spectrum arise from permease carboxylic acid, amide I, and amide II vibrational modes. In addition, isotope labeling leads to a tentative assignment of spectral features to lysine, arginine, histidine, glutamine, and/or asparagine in the permease. These results indicate that the electrochemical gradient causes changes in the environment or protonation state of carboxylic acid residues in the permease and suggest an interaction between these carboxylic acid side chains and nitrogen-containing amino acid side chains. Evidence for a change in secondary structure, corresponding to an interconversion of secondary structural elements, a change in the hydrogen-bonding strength, or coupling of peptide vibrational modes, is also presented. These experiments demonstrate the usefulness of reaction-induced spectroscopy in the study of transmembrane transport.

Bacteriorhodopsins↗
PubMed1998

Sulfhydryl oxidation of mutants with cysteine in place of acidic residues in the lactose permease.

To examine further the role of charge-pair interactions in the structure and function of lactose permease, Asp237 (helix VII), Asp240 (helix VII), Glu126 (cytoplasmic loop IV/V), Glu269 (helix VIII), and Glu325 (helix X) were replaced individually with Cys in a functional mutant devoid of Cys residues. Each mutant was then oxidized with H2O2 in order to generate a sulfinic and/or sulfonic acid at these positions. Due to the isosteric relationship between aspartate and sulfinate, in particular, and the lower pKa of the sulfinic and sulfonic acid side chains, oxidized derivatives of Cys are useful probes for examining the role of carboxylates. Asp237-->Cys or Asp240-->Cys permease is inactive, as shown previously, but H2O2 oxidation restores activity to an extent similar to that observed when a negative charge is reintroduced by other means. Glu126-->Cys, Glu269-->Cys, or Glu325-->Cys permease is inactive, but oxidation does not restore active lactose transport. The data are consistent with previous observations indicating that Asp237 and Asp240 are not critical for active lactose transport, while Glu126, Glu269, and Glu325 are irreplaceable. Although Glu269-->Cys permease does not transport lactose, the oxidized mutant exhibits significant transport of beta,D-galactosylpyranosyl 1-thio-beta,D-galactopyranoside, a property observed with Glu269-->Asp permease. The observation supports the idea that an acidic residue at position 269 is important for substrate recognition. Finally, oxidized Glu325-->Cys permease catalyzes equilibrium exchange with an apparent pKa of about 6.5, more than a pH unit lower than that observed with Glu325-->Asp permease, thereby providing strong confirmatory evidence that a negative charge at position 325 determines the rate of translocation of the ternary complex between the permease, substrate, and H+.

Amino Acid Substitution↗
PubMed2000

Transgalactosylation by thermostable beta-glycosidases from Pyrococcus furiosus and Sulfolobus solfataricus. Binding interactions of nucleophiles with the galactosylated enzyme intermediate make major contributions to the formation of new beta-glycosides during lactose conversion.

The hyperthermostable beta-glycosidases from the Archaea Sulfolobus solfataricus (SsbetaGly) and Pyrococcus furiosus (CelB) hydrolyse beta-glycosides of D-glucose or D-galactose with relaxed specificities pertaining to the nature of the leaving group and the glycosidic linkage. To determine how specificity is manifested under conditions of kinetically controlled transgalactosylation, the major transfer products formed during the hydrolysis of lactose by these enzymes have been identified, and their appearance and degradation have been determined in dependence of the degree of substrate conversion. CelB and SsbetaGly show a marked preference for making new beta(1-->3) and beta(1-->6) glycosidic bonds by intermolecular as well as intramolecular transfer reactions. The intramolecular galactosyl transfer of CelB, relative to glycosidic-bond cleavage and release of glucose, is about 2.2 times that of SsbetaGly and yields beta-D-Galp-(1-->6)-D-Glc and beta-D-Galp-(1-->3)-D-Glc in a molar ratio of approximately 1 : 2. The partitioning of galactosylated SsbetaGly between reaction with sugars [kNu (M-1. s-1)] and reaction with water [kwater (s-1)] is about twice that of CelB. It gives a mixture of linear beta-D-glycosides, chiefly trisaccharides at early reaction times, in which the prevailing new glycosidic bonds are beta(1-->6) and beta(1-->3) for the reactions catalysed by SsbetaGly and CelB, respectively. The accumulation of beta-D-Galp-(1-->6)-D-Glc at the end of lactose hydrolysis reflects a 3-10-fold specificity of both enzymes for the hydrolysis of beta(1-->3) over beta(1-->6) linked glucosides. Galactosyl transfer from SsbetaGly or CelB to D-glucose occurs with partitioning ratios, kNu/kwater, which are seven and > 170 times those for the reactions of the galactosylated enzymes with 1-propanol and 2-propanol, respectively. Therefore, the binding interactions with nucleophiles contribute chiefly to formation of new beta-glycosides during lactose conversion. Likewise, noncovalent interactions with the glucose leaving group govern the catalytic efficiencies for the hydrolysis of lactose by both enzymes. They are almost fully expressed in the rate-limiting first-order rate constant for the galactosyl transfer from the substrate to the enzyme and lead to a positive deviation by approximately 2.5 log10 units from structure-reactivity correlations based on the pKa of the leaving group.

Carbohydrate Sequence↗
PubMed1979

Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: in vivo probe for transcriptional control sequences.

The lactose structural genes, without the lactose promoter, have been incorporated into the bacteriophage Mu genome to form a Mu-lac specialized transducing phage. This phage also carries a gene encoding resistance to ampicillin (Ap)[Mu(Ap, lac)]. After infection and upon establishment of lysogeny, the Mu(Ap, lac) genome can integrate into apparently random sites in the Escherichia coli chromosome. When integration occurs within a gene in the orientation of its transcription, the lactose structural genes are so situated that they become expressed solely from the promoter of that gene. Thus, expression of the lactose genes of Mu(Ap, lac) can be used as an assay for transcription of that gene and for functional and mutational studies of gene regulation.

Arabinose↗
PubMed1981

Mutations in the lacY gene of Escherichia coli define functional organization of lactose permease.

Mutations in the lacY gene of Escherichia coli have been used to analyze the functional organization of lactose permease. Deletions suggest that the NH2 terminus of lactose permease is not essential and can be replaced by residues of the cytoplasmic enzyme beta-galactosidase. Negative dominant mutations in the lacY gene can be explained by the assumption that membrane-associated lactose permease is active as a dimer or oligomer. The map positions of these mutations and other point mutations that lower or alter the sugar specificity define regions of lactose permease involved in sugar or proton binding and transport.

Amino Acid Sequence↗
PubMed1994

Some comparisons among the calming and pain-relieving effects of sucrose, glucose, fructose and lactose in infant rats.

Ultrasonic vocalizations were recorded from 10-day-old albino rats while they were isolated from their dam and siblings. Each rat received a 1.0% BW intra-oral infusion of sucrose, fructose, glucose or lactose at a concentration range of 0.22-0.66 M for 3 min and their vocalizations were determined during the infusion and for an additional 7 min. Sucrose, fructose and glucose all significantly reduced vocalizations to about 50% of baseline levels, whereas lactose, the milk sugar, was ineffective. Moreover, the dose-response function was flat for the three effective sugars. In a second experiment, the effects of these sugars on heat escape latency were measured. Sucrose, fructose and glucose each elevated the latency with which infant rats removed a paw from a 48 degrees C surface; lactose did not. These findings of lactose ineffectiveness and the flat dose-response function for the other three sugars exactly parallel those obtained for human newborns. Their implications are discussed.

Analgesics↗
PubMed2001

Toxoplasma gondii micronemal protein MIC1 is a lactose-binding lectin.

Host cell invasion by Toxoplasma gondii is a multistep process with one of the first steps being the apical release of micronemal proteins that interact with host receptors. We demonstrate here that micronemal protein 1 (MIC1) is a lactose-binding lectin. MIC1 and MIC4 were recovered in the lactose-eluted (Lac(+)) fraction on affinity chromatography on immobilized lactose of the soluble antigen fraction from tachyzoites of the virulent RH strain. MIC1 and MIC4 were both identified by N-terminal microsequencing. MIC4 was also identified by sequencing cDNA clones isolated from an expression library following screening with mouse polyclonal anti-60/70 kDa (Lac(+) proteins) serum. This antiserum localized the Lac(+) proteins on the apical region of T. gondii tachyzoites by confocal microscopy. The Lac(+) fraction induced hemagglutination (mainly type A human erythrocytes), which was inhibited by beta-galactosides (3 mM lactose and 12 mM galactose) but not by up to 100 mM melibiose (alpha-galactoside), fucose, mannose, or glucose or 0.2 mg/ml heparin. The lectin activity of the Lac(+) preparation was attributed to MIC1, because blotted MIC1, but not native MIC4, bound human erythrocyte type A and fetuin. The copurification of MIC1 and MIC4 may have been due to their association, as reported by others. These data suggest that MIC1 may act through its lectin activity during T. gondii infection.

Amino Acid Sequence↗
PubMed2004

Lactose derivatives are inhibitors of Trypanosoma cruzi trans-sialidase activity toward conventional substrates in vitro and in vivo.

Chagas' disease, caused by Trypanosoma cruzi, affects about 18 million people in Latin America, and no effective treatment is available to date. To acquire sialic acid from the host glycoconjugates, T. cruzi expresses an unusual surface sialidase with trans-sialidase activity (TcTS) that transfers the sugar to parasite mucins. Surface sialic acid was shown to have relevant functions in protection of the parasite against the lysis by complement and in mammalian host cell invasion. The recently determined 3D structure of TcTS allowed a detailed analysis of its catalytic site and showed the presence of a lactose-binding site where the beta-linked galactose accepting the sialic acid is placed. In this article, the acceptor substrate specificity of lactose derivatives was studied by high pH anion-exchange chromatography with pulse amperometric detection. The lactose open chain derivatives lactitol and lactobionic acid, as well as other derivatives, were found to be good acceptors of sialic acid. Lactitol, which was the best of the ones tested, effectively inhibited the transfer of sialic acid to N-acetyllactosamine. Furthermore, lactitol inhibited parasite mucins re-sialylation when incubated with live trypanosomes and TcTS. Lactitol also diminished the T. cruzi infection in cultured Vero cells by 20-27%. These results indicate that compounds directed to the lactose binding site might be good inhibitors of TcTS.

Animals↗
PubMed1993

Isolation, characterization and nucleotide sequence of the Streptococcus mutans lactose-specific enzyme II (lacE) gene of the PTS and the phospho-beta-galactosidase (lacG) gene.

The lacE and lacG genes from Streptococcus mutans have been isolated and characterized, and their nucleotide sequence has been determined. The lacE gene encodes the lactose-specific Enzyme II component of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). The lacG gene encodes the phospho-beta-galactosidase which cleaves the lactose phosphate that is formed by the lactose PTS. The S. mutans lacE and lacG genes are located in the same operon as the tagatose genes. S. mutans metabolizes lactose via the tagatose phosphate pathway. The deduced LacE and LacG proteins of S. mutans display high homology with the corresponding proteins from Lactococcus lactis, Staphylococcus aureus and Lactobacillus casei.

Amino Acid Sequence↗
PubMed1977

Depression of lactose hydrolysis by yeasts.

To investigate the role that the known disaccharidase depression may play in the aetiology of infant gastroenteritis caused by Candida albicans, C. albicans and the rarely pathogenic, Saccharomyces cerevisiae were studied by three different methods. Both types of yeast significantly depressed the lactose-hydrolysis activity of beta-galactosidase, and both depressed lactose hydrolysis in the ligated small intestine of infant rabbits, either in intact animals allowed to survive for 10 h, or in a physiological bath for 20 h. The depression of lactose activity was not temperature dependent; living and inactivated yeast preparations produced comparable degrees of depression of enzyme activity. It is concluded that the depression of lactose hydrolysis is not a virulence factor of C. albicans, but contributes to the often observed disaccharide intolerance associated with candida gastroenteritis in infants.

Animals↗
PubMed1994

Fusion proteins as tools for crystallization: the lactose permease from Escherichia coli.

A novel strategy is presented for the crystallization of membrane proteins or other proteins with low solubility and/or stability. The method is illustrated with the lactose permease from Escherichia coli, in which a fusion is constructed between the permease and a 'carrier' protein. The carrier is a soluble, stable protein with its C and N termini close together in space at the surface of the protein, so that the carrier can be introduced into an internal position of the target protein. The carrier is chosen with convenient spectral or enzymatic properties, making the fusion protein easier to handle than the native molecule. Data are presented for the successful construction, expression and purification of a fusion product between lactose permease and cytochrome b(562) from E. coli. The lactose transport activity of the fusion protein is similar to that of wild-type lactose permease, and the fusion product has an absorption spectrum in the visible range which is essentially identical to that of cytochrome b(562). The fusion protein has a higher proportional polar surface area than wild-type permease, and should have better possibilities of forming the strong directional intermolecular contacts required of a crystal lattice.

Journal Article↗
PubMed1998

Actinomyces serovar WVA963 coaggregation-defective mutant strain PK2407 secretes lactose-sensitive adhesin that binds to coaggregation partner Streptococcus oralis 34.

Actinomyces serovar WVA963 strain PK1259 mediates intergeneric coaggregation with several oral streptococci. These lactose-inhibitable coaggregations appear to involve a 95-kDa putative actinomyces adhesin in complex with type 2 fimbriae. A coaggregation-defective strain PK2407 lacking type 2 fimbriae synthesizes the putative adhesin but appears unable to present it properly on its surface. Antiserum was raised against surface sonicates of PK2407 and was absorbed with a different coaggregation-defective mutant PK3092 that synthesizes type 2 fimbriae but no adhesin. This absorbed antiserum specifically blocked lactose-inhibitable coaggregation of wild-type strain PK1259 and Streptococcus oralis 34 and identified a 95-kDa protein in ammonium sulfate precipitates of culture supernatant of the coaggregation-defective mutant PK2407. The 95-kDa secreted protein was bound to the streptococcal partner cells and to lactose-agarose affinity beads and was released by lactose from both the affinity beads and partner, indicating that the secreted and precipitated protein is biochemically active and may mediate coaggregation with streptococci.

Actinomyces↗
PubMed1987

The N-terminal region of Escherichia coli lactose permease mediates membrane contact of the nascent polypeptide chain.

Plasmids encoding N-terminal segments of the Escherichia coli lactose permease (also referred to as lactose carrier) have been used to analyze the biosynthesis and membrane insertion of this complex integral protein of the cytoplasmic membrane. Such truncated polypeptides were found to be stably associated with the membrane and to resemble the full-length protein with respect to their solubilization characteristics. Membrane-bound and free cytoplasmic polysomes were prepared from plasmid-bearing cells and incubated in the presence of [35S]methionine to permit completion of polypeptides initiated in vivo. Under these conditions, lactose permease was found to be radiolabeled in the fraction of membrane-bound polysomes; beta-galactosidase, used as a control, was translated almost exclusively by free polysomes. From similar experiments with N-terminal segments of lactose permease, we estimate that at most a polypeptide of 120 amino acid residues emerging from the ribosome is needed to target the nascent chain to the lipid bilayer and to mediate attachment of the ribosome to the membrane during elongation. Additional data support the idea that even shorter N-terminal sequences of 50 and 71 amino acid residues contain sufficient 'information' to provide contact with the membrane.

Amino Acid Sequence↗
PubMed1997

Establishing a model to study the regulation of the lactose operon in Lactobacillus casei.

The chromosomally encoded lactose-specific phosphoenol pyruvate-dependent phosphotransferase system (PTS) has been investigated in Lactobacillus casei ATCC 393 [pLZ15-] and it was considered an excellent system to study the regulation of the lactose operon. This chromosomal operon has been cloned and sequenced, being 99% homologous to that encoded on the plasmid pLZ64. Expression of the lactose operon in different mutants of L. casei ATCC 393 [pLZ15-] and primer extension analysis revealed that it is subject to a dual regulation: (i) glucose repression possibly mediated by CcpA and PTS elements, and (ii) induction by lactose through transcriptional antitermination.

Base Sequence↗