scholarly journals Energization of the transport systems for arabinose and comparison with galactose transport in Escherichia coli

1981 ◽  
Vol 200 (3) ◽  
pp. 611-627 ◽  
Author(s):  
K R Daruwalla ◽  
A T Paxton ◽  
P J Henderson
Keyword(s):  
Escherichia Coli ◽  
Transport System ◽  
Membrane Vesicles ◽  
Kinetic Studies ◽  
Transport Systems ◽  
Protonmotive Force ◽  
Alkaline Ph ◽  
Ph Change ◽  
Intact Cells ◽  
Membrane Bound

1. Strains of Escherichia coli were obtained containing either the AraE or the AraF transport system for arabinose. AraE+,AraF- strains effected energized accumulation and displayed an arabinose-evoked alkaline pH change indicative of arabinose-H+ symport. In contrast, AraE-,AraF+ strains accumulated arabinose but did not display H+ symport. 2. The ability of different sugars and their derivatives to elicit sugar-H+ symport in AraE+ strains was examined. Only L-arabinose and D-fucose were good substrates, and arabinose was the only inducer. 3. Membrane vesicles prepared from an AraE+,AraF+ strain accumulated the sugar, energized most efficiently by the respiratory substrates ascorbate + phenazine methosulphate. Addition of arabinose or fucose to an anaerobic suspension of membrane vesicles caused an alkaline pH change indicative or sugar-H+ symport on the membrane-bound transport system. 4. Kinetic studies and the effects of arsenate and uncoupling agents in intact cells and membrane vesicles gave further evidence that AraE is a low-affinity membrane-bound sugar-H+ symport system and that AraF is a binding-protein-dependent high-affinity system that does not require a transmembrane protonmotive force for energization. 5. The interpretation of these results is that arabinose transport into E. coli is energized by an electrochemical gradient of protons (AraE system) or by phosphate bond energy (AraF system). 6. In batch cultures the rates of growth and carbon cell yields on arabinose were lower in AraE-,AraF+ strains than in AraE+,AraF- or AraE+,AraF+ strains. The AraF system was more susceptible to catabolite repression than was the AraE system. 7. The properties of the two transport systems for arabinose are compared with those of the genetically and biochemically distinct transport systems for galactose, GalP and MglP. It appears that AraE is analogous to GalP, and AraF to MglP.

scholarly journals The association of proton movement with galactose transport into subcellular membrane vesicles of Escherichia coli

1983 ◽  
Vol 210 (3) ◽  
pp. 699-705 ◽  
Author(s):  
P Horne ◽  
P J F Henderson
Keyword(s):  
Escherichia Coli ◽  
Transport System ◽  
Direct Evidence ◽  
Membrane Vesicles ◽  
Protonmotive Force ◽  
Ph Values ◽  
Subcellular Membrane ◽  
System 2 ◽  
Galactose Transport ◽  
Proton Movement

1. Subcellular membrane vesicles were prepared from a strain of Escherichia coli constitutive for the GalP galactose-transport system. 2. The addition of substrates of the GalP transport system to vesicle suspensions promoted alkaline pH changes, which provided direct evidence for the coupling of sugar and proton transport. 3. Respiration-energized galactose transport was progressively inhibited at pH values above 6.0, and was abolished by agents that render the membrane permeable to protons. 4. The combined effects of valinomycin, the nigericin-like compound A217 and pH on galactose transport suggested that both delta pH and delta psi components of the protonmotive force contributed to energization of galactose transport. 5. These results substantiate the conclusion that the GalP transport system operates by a chemiosmotic mechanism.

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

1979 ◽  
Vol 182 (3) ◽  
pp. 687-696 ◽  
Author(s):  
I R Booth ◽  
W J Mitchell ◽  
W A Hamilton
Keyword(s):  
Escherichia Coli ◽  
Membrane Vesicles ◽  
Transport Systems ◽  
Protonmotive Force ◽  
Lactose Permease ◽  
Whole Cells ◽  
E Coli ◽  
Ph Dependent ◽  
Ph Range ◽  
External Ph

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.

scholarly journals Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jasmine M. Hershewe ◽  
Katherine F. Warfel ◽  
Shaelyn M. Iyer ◽  
Justin A. Peruzzi ◽  
Claretta J. Sullivan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Synthetic Biology ◽  
Membrane Protein ◽  
Membrane Vesicles ◽  
Processing Methods ◽  
Glycoprotein Synthesis ◽  
On Demand ◽  
Free Gene ◽  
Membrane Bound

AbstractCell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N-linked and O-linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities.

Proton gradient-dependent renal transport of glycine: evidence for vesicle studies

1990 ◽  
Vol 258 (2) ◽  
pp. F388-F396 ◽  
Author(s):  
H. Roigaard-Petersen ◽  
H. Jessen ◽  
S. Mollerup ◽  
K. E. Jorgensen ◽  
C. Jacobsen ◽  
...  
Keyword(s):  
Transport System ◽  
Membrane Vesicles ◽  
Proton Gradient ◽  
Transport Systems ◽  
Rabbit Kidney ◽  
Renal Transport ◽  
High Affinity ◽  
Luminal Membrane ◽  
Pars Recta ◽  
Pars Convoluta

The characteristics of renal transport of glycine by luminal membrane vesicles isolated from either proximal convoluted part (pars convoluta) or proximal straight part (pars recta) of rabbit proximal tubule were investigated. In vesicles from pars convoluta two transport systems have been characterized: a Na(+)-dependent system with intermediate affinity (half-saturation 3.64 mM) and a Na(+)-independent system that, in the presence of H+ gradient (extravesicular greater than intravesicular), can accelerate the transport of glycine into these vesicles. This is the first demonstration of H(+)-glycine cotransport across the luminal membrane of rabbit kidney proximal convoluted tubule. By contrast, in membrane vesicles from pars recta, transport of glycine was strictly dependent on Na+ and occurred via a dual transport system, namely a high-affinity (half-saturation 0.34 mM) and a low-affinity system (half-saturation 8.56 mM). The demonstration of competition between the H(+)-gradient dependent uptake of glycine, L-alanine, and L-proline, but insignificant inhibition with L-phenylalanine in vesicles from pars convoluta suggests that glycine, L-proline, and L-alanine probably share a common proton gradient-dependent transport system. In vesicles from pars recta, the Na(+)-dependent uptake of glycine was inhibited by low concentrations of L-alanine and L-phenylalanine, whereas addition of L-proline to the incubation medium did not significantly alter the uptake of glycine, suggesting that the Na(+)-dependent high-affinity system for glycine located in pars recta is shared with the high-affinity L-alanine and L-phenylalanine but not L-proline transport system.

Effect of pH on Na(+)-dependent phosphate transport in renal outer cortical and outer medullary BBMV

1990 ◽  
Vol 258 (2) ◽  
pp. F356-F363 ◽  
Author(s):  
G. A. Quamme
Keyword(s):  
Brush Border ◽  
Sodium Phosphate ◽  
Membrane Vesicles ◽  
Phosphate Transport ◽  
Transport Systems ◽  
Outer Cortex ◽  
Ph Change ◽  
High Affinity ◽  
Medullary Tissue ◽  
Maximum Uptake Rate

The influence of pH on sodium-phosphate cotransport was determined in brush-border membrane vesicles (BBMV) isolated from outer cortical and outer medullary tissue of porcine kidneys. Two transport systems are apparent in outer cortical brush-border vesicles, and one process is apparent in outer medullary vesicles at all pH values. The apparent maximum uptake rate (Vmax) of the low-affinity system in outer cortex vesicles decreased from 8.3 +/- 1.7 to 3.2 +/- 0.05 nmol.mg protein-1.min-1 with pH change of 8.0 to 6.0, and the high-affinity process changed from 1.3 +/- 0.2 to 0.1 +/- 0.01 nmol.mg protein-1.min-1. The respective affinity values (Km) also decreased 5.5 +/- 0.9 to 0.6 +/- 0.01 mM and 0.08 +/- 0.005 to 0.01 +/- 0.005 mM, respectively, with acidification. In outer medullary vesicles a decrease in pH diminished the apparent Km, 0.28 +/- 0.03 to 0.02 +/- 0.003 mM, and mean Vmax from 3.0 +/- 0.07 to 0.5 +/- 0.1 nmol.mg protein-1.min-1. The mean KNaD values were 22.1 +/- 4.2 mM in outer cortical vesicles (low-affinity system) and 58.7 +/- 7.2 mM in outer medullary vesicles (high-affinity system) and were not altered by pH, suggesting that H+ does not affect the sodium interactive site. The data suggest that the vesicles prepared from outer cortical and outer medullary tissue possess distinctive sodium-phosphate transporters that are sensitive to external H+ concentrations.

scholarly journals Proton-linked l-rhamnose transport, and its comparison with l-fucose transport in Enterobacteriaceae

1993 ◽  
Vol 290 (3) ◽  
pp. 833-842 ◽  
Author(s):  
J A R Muiry ◽  
T C Gunn ◽  
T P McDonald ◽  
S A Bradley ◽  
C G Tate ◽  
...  
Keyword(s):  
Transport System ◽  
Erwinia Carotovora ◽  
Alkaline Ph ◽  
Transport Activity ◽  
Ph Change ◽  
Kinetic Measurements ◽  
E Coli ◽  
Steady State Kinetic ◽  
Competitive Inhibitors ◽  
State Kinetic

1. An alkaline pH change occurred when L-rhamnose, L-mannose or L-lyxose was added to L-rhamnose-grown energy-depleted suspensions of strains of Escherichia coli. This is diagnostic of sugar-H+ symport activity. 2. L-Rhamnose, L-mannose and L-lyxose were inducers of the sugar-H+ symport and of L-[14C]rhamnose transport activity. L-Rhamnose also induced the biochemically and genetically distinct L-fucose-H+ symport activity in strains competent for L-rhamnose metabolism. 3. Steady-state kinetic measurements showed that L-mannose and L-lyxose were competitive inhibitors (alternative substrates) for the L-rhamnose transport system, and that L-galactose and D-arabinose were competitive inhibitors (alternative substrates) for the L-fucose transport system. Additional measurements with other sugars of related structure defined the different substrate specificities of the two transport systems. 4. The relative rates of H+ symport and of sugar metabolism, and the relative values of their kinetic parameters, suggested that the physiological role of the transport activity was primarily for utilization of L-rhamnose, not for L-mannose or L-lyxose. 5. L-Rhamnose transport into subcellular vesicles of E. coli was dependent on respiration, was optimal at pH 7, and was inhibited by protonophores and ionophores. It was insensitive to N-ethylmaleimide or cytochalasin B. 6. L-Rhamnose, L-mannose and L-lyxose each elicited an alkaline pH change when added to energy-depleted suspensions of L-rhamnose-grown Salmonella typhimurium LT2, Klebsiella pneumoniae, Klebsiella aerogenes, Erwinia carotovora carotovora and Erwinia carotovora atroseptica. The relative rates of subsequent acidification varied, depending on both the organism and the sugar. L-Fucose promoted an alkaline pH change in all the L-rhamnose-induced organisms except the Erwinia species. No L-rhamnose-H+ symport occurred in any organism grown on L-fucose. 7. All these results showed that L-rhamnose transport into the micro-organisms occurred by a system different from that for L-fucose transport. Both systems are energized by the trans-membrane electrochemical gradient of protons. 8. Neither steady-state kinetic measurements nor binding-protein assays revealed the existence of a second L-rhamnose transport system in E. coli.

scholarly journals Cation transport in Escherichia coli. IX. Regulation of K transport.

1978 ◽  
Vol 72 (3) ◽  
pp. 283-295 ◽  
Author(s):  
D B Rhoads ◽  
W Epstein
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Cation Transport ◽  
Transport Systems ◽  
Energy Requirements ◽  
Protonmotive Force ◽  
K Uptake ◽  
Kinetics Of ◽  
K Transport ◽  
External K

Kinetics of K exchange in the steady state and of net K uptake after osmotic upshock are reported for the four K transport systems of Escherichia coli: Kdp, TrkA, TrkD, and TrkF. Energy requirements for K exchange are reported for the Kdp and TrkA systems. For each system, kinetics of these two modes of K transport differ from those for net K uptake by K-depleted cells (Rhoads, D. B. F.B. Walters, and W. Epstein. 1976. J. Gen. Physiol. 67:325-341). The TrkA and TrkD systems are inhibited by high intracellular K, the TrkF system is stimulated by intracellular K, whereas the Kdp system is inhibited by external K when intracellular K is high. All four systems mediate net K uptake in response to osmotic upshock. Exchange by the Kdp and TrkA systems requires ATP but is not dependent on the protonmotive force. Energy requirements for the Kdp system are thus identical whether measured as net K uptake or K exchange, whereas the TrkA system differs in that it is dependent on the protonmotive force only for net K uptake. We suggest that in both the Kpd and TrkA systems formation of a phosphorylated intermediate is necessary for all K transport, although exchange transport may not consume energy. The protonmotive-force dependence of the TrkA system is interpreted as a regulatory influence, limiting this system to exchange except when the protonmotive force is high.

scholarly journals 2-Deoxy-d-galactose, a substrate for the galactose-transport system of Escherichia coli

1977 ◽  
Vol 168 (1) ◽  
pp. 15-22 ◽  
Author(s):  
P J F Henderson ◽  
R A Giddens
Keyword(s):  
Escherichia Coli ◽  
Transport System ◽  
Transport Systems ◽  
Mutual Inhibition ◽  
E Coli ◽  
System 2 ◽  
Galactose Transport ◽  
Proton Uptake ◽  
Inhibition Studies ◽  
Lactose Transport

The following observations showed that 2-deoxy-D-galactose is a useful tool for the isolation and elucidation of the activity of one system for galactose uptake into Escherichia coli. 1. 2-Deoxygalactose, which is not a substrate for growth of E. coli, was transported into strains of the organism induced for galactose transport. 2. By using appropriate mutants it was shown that 2-deoxygalactose is a much better substrate for the galactose-transport system than for the methyl galactoside-transport system. This was confirmed by the results of mutual inhibition studies with substrates of each transport system. 3. The glucose-, arabinose- or lactose-transport systems did not effect significant transport of 2-deoxygalactose. 4. Like other substrates of the galactose-transport system, 2-deoxygalactose promoted effective proton uptake into de-energized suspensions of appropriate E. coli strains. 5. The S183 series of E. coli mutants were found to contain a constitutive galactose-transport system, if 2-deoxygalactose transport is used as one criterion for such activity.

scholarly journals Transport of galactose, glucose and their molecular analogues by Escherichia coli K12

1977 ◽  
Vol 162 (2) ◽  
pp. 309-320 ◽  
Author(s):  
P J F Henderson ◽  
R A Giddens ◽  
M C Jones-Mortimer
Keyword(s):  
Escherichia Coli ◽  
Transport System ◽  
Proton Transport ◽  
Regulatory Mechanism ◽  
Transport Systems ◽  
British Library ◽  
Transport Activity ◽  
Phosphotransferase System ◽  
Escherichia Coli K12 ◽  
Specific Transport System

1. Strains of Escherichia coli K12 were made that are unable to assimilate glucose by the phosphotransferase system, since they lack the glucose-specific components specified by the genes ptsG and ptsM. 2. Derivative organisms lacking the methyl galactoside or galactose-specific transport system were examined for their ability to transport galactose, d-fucose, methyl beta-D-galactoside, glucose, 2-deoxy-D-glucose and methyl alpha-D-glucoside. 3. Galactose, glucose and to a lesser extent fucose are substrates for both transport systems. 4. 2-Deoxyglucose is transported on the galactose-specific but not the methyl galactoside system. 5. The ability of sugars to elicit anaerobic proton transport is associated with the galactose-specific, but not with the methyl galactoside transport activity. Hence a chemiosmotic mechanism of energization is likely to apply to the former but not to the latter. Alternatively the methyl galactoside system may be switched off under certain conditions, which would indicate a novel regulatory mechanism. 6. Details of the procedure for the derivation of strains may be obtained from the authors, and have been deposited as Supplementary Publication SUP 50074 (8 pages at the) British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1977), 161,1.

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