Browse
Submit Data
Databases
API
Help

Dataset Information

21 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Quantitative analysis of proton-linked transport systems. The lactose permease of Escherichia coli.

ABSTRACT: Evidence is presented that lactose uptake into whole cells of Escherichia coli occurs by symport with a single proton over the range of external pH 6.5--7.7. The proton/lactose stoicheiometry has been measured directly over this pH range by comparison of the initial rates of proton and lactose uptake into anaerobic resting cell suspensions of E. coli ML308. Further, the relationship between the protonmotive force and lactose accumulation has been studied in E. coli ML308-225 over the range of external pH 5.9--8.7. At no point was the accumulation of the beta-galactoside in thermodynamic equilibrium with the protonmotive force. It is concluded that the concentration of lactose within the cell is governed by kinetic factors rather than pH-dependent changes in the proton/substrate stoicheiometry. The relevance of these findings to the model of pH-dependent proton/substrate stoicheiometries derived from studies with E. coli membrane vesicles is discussed.

SUBMITTER: Booth IR

PROVIDER: S-EPMC1161402 | biostudies-other | 1979 Sep

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Quantitative analysis of proton-linked transport system. beta-Galactoside exit in Escherichia coli.

Project description:The exit of lactose and thiomethyl-beta-D-galactoside from Escherichia coli ML308-225 has been studied to determine the role of carrier-dependent (zero-trans efflux) and carrier-independent (leak) processes. On the basis of its sensitivity to p-chloromercuribenzene sulphonate the exit of lactose was found to be almost wholly mediated by the carrier. Consistent with this conclusion was the finding that the rate of exit of this sugar was dependent on the external pH, being considerably slower at acid pH. On the other hand exit of thiomethyl-beta-D-galactoside was found to be composed of both carrier-dependent and carrier-independent processes. Both processes exhibited first-order kinetics with the rate constants for zero-trans efflux and leak being 0.137 min-1 and 0.079 min-1, respectively. The relevance of these findings for out earlier proposal for the methods of attenuation of solute accumulation is discussed [Booth, Mitchell & Hamilton (1979) Biochem. J. 182, 687--696].

| S-EPMC1161890 | biostudies-other

Quantitative analysis of proton-linked transport systems. Glutamate transport in Staphylococcus aureus.

Project description:1. The magnitude of the protonmotive force in respiring Staphylococcus aureus was measured over the range of extracellular pH from 5.6 to 7.8. 2. The membrane potential remains constant at 150 mV, inside-negative, but the pH gradient decreases from 2.1 units, inside-alkaline, at pH 5.6 to zero at pH 7.5 and above. 3. The accumulation of glutamate in the soluble cell pool is pH-independent at a value equivalent to 100 mV. 4. The results of experiments studying co-transport of protons are consistent with a proton/glutamate stoichiometry of 2 and electrogenic transport across the pH range examined. 5. The amount of glutamate uptake is the result of a kinetic steady state between influx and efflux pathways. 6. Evidence is presented for the regulation of this kinetic steady state by the response of the initial rate of uptake to changes in the protonmotive force.

| S-EPMC1161780 | biostudies-other

Proton-coupled dynamics in lactose permease.

Project description:Lactose permease of Escherichia coli (LacY) catalyzes symport of a galactopyranoside and an H? via an alternating access mechanism. The transition from an inward- to an outward-facing conformation of LacY involves sugar-release followed by deprotonation. Because the transition depends intimately upon the dynamics of LacY in a bilayer environment, molecular dynamics (MD) simulations may be the only means of following the accompanying structural changes in atomic detail. Here, we describe MD simulations of wild-type apo LacY in phosphatidylethanolamine (POPE) lipids that features two protonation states of the critical Glu325. While the protonated system displays configurational stability, deprotonation of Glu325 causes significant structural rearrangements that bring into proximity side chains important for H? translocation and sugar binding and closes the internal cavity. Moreover, protonated LacY in phosphatidylcholine (DMPC) lipids shows that the observed dynamics are lipid-dependent. Together, the simulations describe early dynamics of the inward-to-outward transition of LacY that agree well with experimental data.

| S-EPMC3496080 | biostudies-literature

Proton-linked L-fucose transport in Escherichia coli.

Project description:1. Addition of L-fucose to energy-depleted anaerobic suspensions of Escherichia coli elicited an uncoupler-sensitive alkaline pH change diagnostic of L-fucose/H+ symport activity. 2. L-Galactose or D-arabinose were also substrates, but not inducers, for the L-fucose/H+ symporter. 3. L-Fucose transport into subcellular vesicles was dependent upon respiration, displayed a pH optimum of about 5.5, and was inhibited by protonophores and ionophores. 4. These results showed that L-fucose transport into E. coli was energized by the transmembrane electrochemical gradient of protons. 5. Neither steady state kinetic measurements nor assays of L-fucose binding to periplasmic proteins revealed the existence of a second L-fucose transport system.

| S-EPMC1148569 | biostudies-other

Energetics of ligand-induced conformational flexibility in the lactose permease of Escherichia coli.

Project description:Isothermal titration calorimetry has been applied to characterize the thermodynamics of ligand binding to wild-type lactose permease (LacY) and a mutant (C154G) that strongly favors an inward facing conformation. The affinity of wild-type or mutant LacY for ligand and the change in free energy (DeltaG) upon binding are similar. However, with the wild type, the change in free energy upon binding is due primarily to an increase in the entropic free energy component (TDeltaS), whereas in marked contrast, an increase in enthalpy (DeltaH) is responsible for DeltaG in the mutant. Thus, wild-type LacY behaves as if there are multiple ligand-bound conformational states, whereas the mutant is severely restricted. The findings also indicate that the structure of the mutant represents a conformational intermediate in the overall transport cycle.

| S-EPMC2793331 | biostudies-literature

Intermolecular thiol cross-linking via loops in the lactose permease of Escherichia coli.

Project description:Previous experiments using intermolecular thiol cross-linking to determine surface-exposed positions in the transmembrane helices of the lactose permease suggest that only positions accessible from the aqueous phase are susceptible to cross-linking. This approach is now extended to most of the remaining positions in the molecule. Of an additional 143 single-Cys mutants studied, homodimer formation is observed with both a 5-A- and a 21-A-long crosslinking agent containing bis-methane thiosulfonate reactive groups in 33 mutants and exclusively with the 21-A-long reagent in 43 mutants. Furthermore, intermolecular cross-linking has little or no effect on transport activity, thereby providing further support for the argument that lactose permease is functionally, as well as structurally, a monomer in the membrane. In addition, evidence is presented indicating that reentrance loops are unlikely in this polytopic membrane transport protein.

| S-EPMC193537 | 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

Properties of permease dimer, a fusion protein containing two lactose permease molecules from Escherichia coli.

Project description:An engineered fusion protein containing two tandem lactose permease molecules (permease dimer) exhibits high transport activity and is used to test the phenomenon of negative dominance. Introduction of the mutation Glu-325-->Cys into either the first or the second half of the dimer results in a 50% decrease in activity, whereas introduction of the mutation into both halves of the dimer abolishes transport. Lactose transport by permease dimer is completely inactivated by N-ethylmaleimide; however, 40-45% activity is retained after N-ethylmaleimide treatment when either the first or the second half of the dimer is replaced with a mutant devoid of cysteine residues. The observations demonstrate that both halves of the fusion protein are equally active and suggest that each half may function independently. To test the possibility that oligomerization between dimers might account for the findings, a permease dimer was constructed that contains two different deletion mutants that complement functionally when expressed as untethered molecules. Because this construct does not catalyze lactose transport to any extent whatsoever, it is unlikely that the two halves of the dimer interact or that there is an oligomeric interaction between dimers. The approach is consistent with the contention that the functional unit of lactose permease is a monomer.

| S-EPMC44007 | biostudies-other

Systems analysis of lactose metabolism in Trichoderma reesei identifies a lactose permease that is essential for cellulase induction.

Project description:Trichoderma reesei colonizes predecayed wood in nature and metabolizes cellulose and hemicellulose from the plant biomass. The respective enzymes are industrially produced for application in the biofuel and biorefinery industry. However, these enzymes are also induced in the presence of lactose (1,4-0-ß-d-galactopyranosyl-d-glucose), a waste product from cheese manufacture or whey processing industries. In fact, lactose is the only soluble carbon source that induces these enzymes in T. reesei on an industrial level but the reason for this unique phenomenon is not understood. To answer this question, we used systems analysis of the T. reesei transcriptome during utilization of lactose. We found that the respective CAZome encoded all glycosyl hydrolases necessary for cellulose degradation and particularly for the attack of monocotyledon xyloglucan, from which ß-galactosides could be released that may act as the inducers of T. reesei's cellulases and hemicellulases. In addition, lactose also induces a high number of putative transporters of the major facilitator superfamily. Deletion of fourteen of them identified one gene that is essential for lactose utilization and lactose uptake, and for cellulase induction by lactose (but not sophorose) in pregrown mycelia of T. reesei. These data shed new light on the mechanism by which T. reesei metabolizes lactose and offers strategies for its improvement. They also illuminate the key role of ß-D-galactosides in habitat specificity of this fungus.

| S-EPMC3648571 | biostudies-literature

Apo-intermediate in the transport cycle of lactose permease (LacY).

Project description:The lactose permease (LacY) catalyzes coupled stoichiometric symport of a galactoside and an H(+). Crystal structures reveal 12, mostly irregular, transmembrane ?-helices surrounding a cavity with sugar- and H(+)- binding sites at the apex, which is accessible from the cytoplasm and sealed on the periplasmic side (an inward-facing conformer). An outward-facing model has also been proposed based on biochemical and spectroscopic measurements, as well as the X-ray structure of a related symporter. Converging lines of evidence demonstrate that LacY functions by an alternating access mechanism. Here, we generate a model for an apo-intermediate of LacY based on crystallographic coordinates of LacY and the oligopeptide/H(+) symporter. The model exhibits a conformation with an occluded cavity inaccessible from either side of the membrane. Furthermore, kinetic considerations and double electron-electron resonance measurements suggest that another occluded conformer with bound sugar exists during turnover. An energy profile for symport is also presented.

| S-EPMC3497813 | 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