Browse
Submit Data
Databases
API
Help

Dataset Information

11 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Delineating electrogenic reactions during lactose/H+ symport.

ABSTRACT: Electrogenic reactions accompanying downhill lactose/H(+) symport catalyzed by the lactose permease of Escherichia coli (LacY) have been assessed using solid-supported membrane-based electrophysiology with improved time resolution. Rates of charge translocation generated by purified LacY reconstituted into proteoliposomes were analyzed over a pH range from 5.2 to 8.5, which allows characterization of two electrogenic steps in the transport mechanism: (i) a weak electrogenic reaction triggered by sugar binding and observed under conditions where H(+) translocation is abolished either by acidic pH or by a Glu325 --> Ala mutation in the H(+) binding site (this step with a rate constant of approximately 200 s(-1) for wild-type LacY leads to an intermediate proposed to represent an "occluded" state) and (ii) a major electrogenic reaction corresponding to 94% of the total charge translocated at pH 8, which is pH-dependent with a maximum rate of approximately 30 s(-1) and a pK of 7.5. This partial reaction is assigned to rate-limiting H(+) release on the cytoplasmic side of LacY during turnover. These findings together with previous electrophysiological results and biochemical-biophysical studies are included in an overall kinetic mechanism that allows delineation of the electrogenic steps in the reaction pathway.

SUBMITTER: Garcia-Celma JJ

PROVIDER: S-EPMC2907097 | biostudies-other | 2010 Jul

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

pH Regulation of Electrogenic Sugar/H+ Symport in MFS Sugar Permeases.

Project description:Bacterial sugar symporters in the Major Facilitator Superfamily (MFS) use the H+ (and in a few cases Na+) electrochemical gradients to achieve active transport of sugar into the cell. Because a number of structures of MFS sugar symporters have been solved recently, molecular insight into the transport mechanism is possible from detailed functional analysis. We present here a comparative electrophysiological study of the lactose permease (LacY), the fucose permease (FucP) and the xylose permease (XylE), which reveals common mechanistic principles and differences. In all three symporters energetically downhill electrogenic sugar/H+ symport is observed. Comparison of the pH dependence of symport at symmetrical pH exhibits broad bell-shaped pH profiles extending over 3 to 6 pH units and a decrease at extremely alkaline pH ? 9.4 and at acidic to neutral pH = 4.6-7.5. The pH dependence can be described by an acidic to neutral apparent pK (pKapp) and an alkaline pKapp. Experimental evidence suggests that the alkaline pKapp is due to H+ depletion at the protonation site, while the acidic pKapp is due to inhibition of deprotonation. Since previous studies suggest that a single carboxyl group in LacY (Glu325) may be the only side chain directly involved in H+ translocation and a carboxyl side chain with similar properties has been identified in FucP (Asp46) and XylE (Asp27), the present results imply that the pK of this residue is switched during H+/sugar symport in all three symporters.

| S-EPMC4882079 | biostudies-literature

Conservation of residues involved in sugar/H(+) symport by the sucrose permease of Escherichia coli relative to lactose permease.

Project description:Building a three-dimensional model of the sucrose permease of Escherichia coli (CscB) with the X-ray crystal structure lactose permease (LacY) as template reveals a similar overall fold for CscB. Moreover, despite only 28% sequence identity and a marked difference in substrate specificity, the structural organization of the residues involved in sugar-binding and H(+) translocation is conserved in CscB. Functional analyses of mutants in the homologous key residues provide strong evidence that they play a similar critical role in the mechanisms of CscB and LacY.

| S-EPMC2786776 | biostudies-literature

Stoicheiometry of lactose-H+ symport across the plasma membrane of Escherichia coli.

Project description:The rate of [(14)C]lactose inflow into anaerobic, non-metabolizing, Escherichia coli has been correlated with the rate of the simultaneous effective inflow of H(+). It is shown that the initial rates of the two transport processes are essentially the same, and it is concluded that the two processes are strictly coupled with a 1:1 stoicheiometry.

| S-EPMC1177623 | biostudies-other

Dependence on pH of substrate binding to a mutant lactose carrier, lacYun, in Escherichia coli. A model for H+/lactose symport.

Project description:The lacYun gene, which encodes a lactose carrier showing the uncoupled phenotype of substrate transport in Escherichia coli [Wilson, Kusch & Kashket (1970) Biochem. Biophys. Res. Commun. 40, 1409-1414], was cloned on a plasmid vector, pBR322. The binding of a substrate, p-nitrophenyl alpha-galactoside, to the lacYun carrier in membranes from the strain harbouring the lacYun clone showed a pH-dependence different from its binding to the wild-type lactose carrier. This finding indicated that the lacYun mutation confers higher affinity for H+ on the carrier, exerting its effect on the less efficient dissociation of substrate inside cells. The result coincides with the proposal [Yamato & Rosenbusch (1983) FEBS Lett. 151, 102-104] that the proton affecting the substrate binding is the coupling proton of the proton/lactose symport reaction, which allows only the ordered mechanism of binding of substrate to an H+-carrier binary complex. From the simplest model of the symport reaction, constructed on the basis of these results, the coupling site of energy in the carrier cycle of the transport reaction can be identified at the substrate-dissociation step inside cells.

| S-EPMC1138374 | biostudies-other

Use of progress curves to estimate the co-substrate-to-substrate flow ratio of a symport mechanism. Application to the isoleucine-Na+ symport of mouse ascites-tumour cells and to the lactose-proton symport.

Project description:The model envisages two components in the process, whereby Ht equivalents of co-substrate and St equivalents of substrate accumulate in the cellular compartment in time t. The first is the flow through the symport, n equivalents of co-substrate entering or leaving with each substrate equivalent. The second is the basal flow of co-substrate outside the symport. In certain specific circumstances n can be derived by plotting Ht/t against St/t. The principal requirement is that, whereas the ratio of the component flows must change in the interval t, the magnitude of the basal flow must either be zero or constant. The procedure is applied to published observations [West & Mitchell (1973) Biochem. J. 132, 587-592] on the lactose-proton symport of Escherichia coli [n = 1.075 +/- 0.064(7)] and to new observations on the isoleucine-Na+ symport of mouse ascites-tumour cells [n = 1.136 +/- 0.120(18)].

| S-EPMC1148970 | biostudies-other

The putative electrogenic nitrate-proton symport of the yeast Candida utilis. Comparison with the systems absorbing glucose or lactate.

Project description:Strain N.C.Y.C. 193 of Candida utilis was grown aerobically at 30 degrees C with nitrate as limiting nutrient in a chemostat. The washed yeast cells depleted of ATP absorbed up to 5 nmol of nitrate/mg dry wt. of yeast. At pH 4-6, extra protons and nitrate entered the yeast cells together, in a ratio of about 2:1. Charge balance was maintained by an outflow of about 1 equiv. of K+. Nitrate stimulated the uptake of about 1 proton equivalent during glycolysis or aerobic energy metabolism. Studies with 3,3'-dipropylthiadicarbocyanine indicated that the proton-linked absorption of nitrate, amino acids or glucose depolarized the yeast cells. Proton uptake along with lactate led neither to net expulsion of K+ nor to membrane depolarization.

| S-EPMC1152744 | biostudies-other

Delineating antibody recognition against Zika virus during natural infection.

Project description:Zika virus (ZIKV) is an emerging mosquito-transmitted flavivirus that shares a considerable degree of homology with dengue virus (DENV). Here, we examined longitudinal antibody response against ZIKV during natural infection in 2 convalescent individuals. By decomposing the antibody recognition into DI/DII and DIII of the E glycoprotein, we showed their development in humans followed a spatiotemporal hierarchy. Plasma binding to DI/DII appeared to peak and wane during early infection with extensive cross-reactivity with DI/DII of DENV. Binding to DIII, however, peaked early but persisted months into the infection without detectable cross-reactivity with DIII of DENV. A clear trend of increase in DIII-specific neutralizing activity was observed over the course of infection. mAbs isolated during early infection are largely DI/DII specific, weakly neutralizing, and highly cross-reactive with DENV, while those from later infection are more diverse in recognition, potently neutralizing, and ZIKV specific. The most potent neutralizing mAb targeting the DIII provided 100% protection in mice from lethal ZIKV infection and could therefore serve as a promising candidate for antibody-based therapy and prevention. The dynamic features unveiled here will assist us to better understand the pathogenesis of ZIKV infection and inform rational design of vaccines.

| S-EPMC5470883 | biostudies-literature

Protein expression dynamics during Escherichia coli glucose-lactose diauxie.

Project description:BackgroundEscherichia coli is a well-studied anaerobic bacteria which is able to regulate metabolic pathways depending on the type of sugar presented in the medium. We have studied the glucose-lactose shift in E. coli at the protein level using a recently developed mass spectrometry platform.MethodCells were grown in minimal medium containing two sugars (glucose and lactose) and analyzed using novel mass spectrometry cluster. The cluster combines the high resolving power and dynamic range of Fourier transform ion cyclotron resonance (FTICR) for accurate mass measurement and quantitation with multiple ion traps for fast and sensitive tandem mass spectrometry. The protein expression profile was followed in time across the glucose-lactose diauxic shift using label-free quantitation from the FTICR data.Results and conclusionThe entire dataset was interrogated by KEGG pathway analysis, mapping measured changes in protein abundance onto known metabolic pathways. The obtained results were consistent with previously published gene expression data, with ?-galactosidase being the most strongly induced protein during the diauxic shift.

| S-EPMC3121593 | biostudies-literature

Structure-based mechanism for Na(+)/melibiose symport by MelB.

Project description:The bacterial melibiose permease (MelB) belongs to the glycoside-pentoside-hexuronide:cation symporter family, a part of the major facilitator superfamily (MFS). Structural information regarding glycoside-pentoside-hexuronide:cation symporter family transporters and other Na(+)-coupled permeases within MFS has been lacking, although a wealth of biochemical and biophysical data are available. Here we present the three-dimensional crystal structures of Salmonella typhimurium MelBSt in two conformations, representing an outward partially occluded and an outward inactive state of MelBSt. MelB adopts a typical MFS fold and contains a previously unidentified cation-binding motif. Three conserved acidic residues form a pyramidal-shaped cation-binding site for Na(+), Li(+) or H(+), which is in close proximity to the sugar-binding site. Both cosubstrate-binding sites are mainly contributed by the residues from the amino-terminal domain. These two structures and the functional data presented here provide mechanistic insights into Na(+)/melibiose symport. We also postulate a structural foundation for the conformational cycling necessary for transport catalysed by MFS permeases in general.

| S-EPMC4026327 | biostudies-literature

Synthesis of glucooligosaccharides with prebiotic potential by glucansucrase URE 13-300 acceptor reactions with maltose, raffinose and lactose.

Project description:In the present work, we report an efficient synthesis of glucooligosaccharides (GOSs) with prebiotic potential by novel glucansucrase URE 13-300 from Leuconostoc mesenteroides URE 13 strain. The highest total yield of GOSs with degree of polymerization (DP) from 3 to 6 was obtained with maltose as an acceptor and maltose/sucrose (M/S) ratio 1-136 g/L. An efficient modulation of GOSs composition is achieved by varying the M/S ratio. At M/S = 1, 2, 4 and 7 the content of DP3 products gradually increase from 54.50 to 91.70%. When the M/S ratio was decreased the synthesis of DP>3 GOSs is predominant and reaches 75.60% (M/S = 0.25). In addition, the maltose derived GOSs with DP>3, as well as raffinose and lactose glucosylation products have a branched structure which is prerequisite for increased prebiotic potential. The synthesized GOSs were efficiently metabolized by probiotic strains of Lb. plantarum S26, Lb. brevis S27 and Lb. sakei S16, and the calculated values of specific growth rate (μ) were nearly identical to this on glucose media, when maltose derived GOSs were used as a carbohydrate source. Strain specific features were observed in the utilization of the synthesized GOSs, as well as in the production of lactic acid and acetic acid.

| S-EPMC6999593 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data