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E G Sedgwick

Publications and source records attributed to E G Sedgwick.

14 recordsLinked to original sources
PubMed1997

Cross-linking and N-(1-pyrenyl)maleimide labeling of cysteine mutants of proton-pumping pyridine nucleotide transhydrogenase of Escherichia coli.

The pyridine nucleotide transhydrogenase of Escherichiacoli is a proton pump composed of two subunits (alpha and beta) organized as an alpha2beta2 tetramer. The enzyme contains seven cysteine residues, five in the alpha-subunit and two in the beta-subunit. The reaction of these residues with the cross-linking agent cupric 1, 10-phenanthrolinate and with the fluorescent thiol reagent N-(1-pyrenyl)maleimide was investigated in mutants in which one or more of these cysteine residues had been mutated to serine or threonine residues. Mutation of alphaCys395 and alphaCys397 prevented disulfide bond formation to give the cross-linked alpha2 dimer. We concluded that the two alpha-subunits of the holoenzyme interface in the region of these two cysteine residues. Pyrenylmaleimide reacted with detergent-washed cytoplasmic membrane vesicles containing high levels of transhydrogenase protein to show characteristic fluorescence emission bands at 378-379, 397-398, and 419-420 nm. At higher ratios of pyrenylmaleimide:transhydrogenase (>5:1) and longer times of reaction, an eximer band at 470 nm was formed. This was attributed to interaction between noncovalently bound molecules of pyrenylmaleimide. The cysteine residues of the beta-subunit (betaCys147 and betaCys260) were covalently modified by pyrenylmaleimide. betaCys147 reacted more strongly than betaCys260 with the fluorophore, and the pyrene derivative of betaCys147 was more accessible to quenching by 5-doxylstearate, suggesting a proximity to the surface of the membrane. Covalent modification of betaCys260 resulted in inhibition of enzyme activity. The inhibition was attributed to the introduction of the bulky pyrene group into the enzyme.

Amino Acid Sequence↗
PubMed1996

The role of efflux systems and the cell envelope in fluorescence changes of the lipophilic cation 2-(4-dimethylaminostyryl)-1-ethylpyridinium in Escherichia coli.

The interaction of the fluorescent dye 2-(4-dimethylaminostyryl)-1-ethlypyridinium cation (DMP+) with cells of Escherichia coli AN120 (uncA) and AS-1 (acrA) was studied to elucidate the role of the envelope and of efflux systems in the uptake of lipophilic cations. DMP+ bound to the two strains in a different manner. With AS-1 the bound dye was displaced only to a small extent by addition of Mg2+ or other divalent cations. By contract, 50% of the DMP+ was displaced by micromolar concentrations of Mg2+ from resting cells of AN120. Energization of the cells by substrate oxidation resulted in the loss in AN120 of 50% of the bound dye and a decrease of the fluorescence in the cell suspension. With AS-1, energization caused more DMP+ to be taken up from the medium. This was associated with an increase in fluorescence in the cell suspension. The extent of the quenching by addition of Mg2+ was not increased. Right-side out vesicles from AN120, like those of AS-1, showed DMP+ fluorescence behaviour which resembled that of intact cells of AS-1. Transformation of AS-1 with plasmids encoding the E. coli Mvr and EmrAB efflux systems resulted in the DMP+ fluorescence response of this strain becoming like that of AN120. It is suggested that with strain AN120 the changes in binding of DMP+ and fluorescence intensity were associated with activation of efflux systems on cell energization. With AS-1, it is suggested that the observed fluorescence and binding changes are due to inactivation of the AcrAB efflux system by the acrA mutation. Thus, the net entry of lipophilic cations is facilitated. Energization of dye update and release is driven by an electrochemical gradient of protons. ATP is not directly involved in energizing the movement of the dye.

Adenosine Triphosphate↗
PubMed1993

Mechanism of uptake of the fluorescent dye 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation (DMP+) by phospholipid vesicles.

The fluorescent dye 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation (DMP+) is taken up by liposomes of egg phosphatidylcholine in response to the imposition of a transmembrane potential. Entry of DMP+ into the bilayer driven by the transmembrane potential is accompanied by a change in the fluorescence emission maximum of the dye. This change reflects the movement of the dye molecules from the headgroup region of the bilayer into the region of the fatty acyl chains. It is released into the external aqueous phase on discharge of the transmembrane potential. Partition of the dye into the phospholipid bilayer is favoured by the presence of negatively charged lipids, such as dioleoylphosphatidic acid and dicetyl phosphate, in the bilayer. Stearylamine opposes entry of the dye into the bilayer. Tetraphenylboron (TPB-) increases the partitioning of DMP+ into the phospholipid bilayer even in the absence of a transmembrane potential. The fluorescence emission maximum of DMP+ under these conditions is similar to that observed in the absence of TPB- following imposition of the transmembrane potential. It is suggested that TPB- facilitates the entry of DMP+ into the fatty acyl chain regions of the phospholipid bilayer.

Drug Interactions↗
PubMed1992

Differential permeability for lipophilic compounds in uncoupler-resistant cells of Escherichia coli.

The acrA strain AS-1 of Escherichia coli is more sensitive than its parent W3110 to growth inhibition by Methylene blue, sodium dodecyl sulfate and novobiocin. UR-3 is an uncoupler-resistant strain isolated from AS-1 which is resistant to growth inhibition by carbonylcyanide m-chlorophenylhydrazone (CCCP), 3,3',4',5-tetrachlorsalicylanilide (TCS) and tributyltin chloride, while remaining sensitive to the first group of compounds. A revertant of AS-1 acquired resistance to Methylene blue and sodium dodecyl sulfate but remained sensitive to uncouplers. In contrast to AS-1, proline uptake in UR-3 was resistant to uncouplers. Strain UR-3 grown in the presence of uncoupler incorporated elongation factor Tu to high levels in the outer membrane of the cell. A role for the outer membrane in the acquisition of uncoupler-resistance by UR-3 is suggested by the behaviour of the mutant to the fluorescence probe N-phenyl-1-naphthylamine. The fluorescence intensity of this probe was quenched by membrane energization in the wild-type strain W3110 but not in AS-1. UR-3 behaved like W3110, suggesting that an outer membrane barrier to neutral lipophilic compounds like N-phenyl-1-naphthylamine (NPN) and uncouplers had been restored in UR-3. By contrast, AS-1 and UR-3 both allowed energized uptake of the fluorescent lipophilic cation 2-(dimethylaminostyryl)-1-ethylpyridinium (DMP+). It is concluded that lipophilic materials must permeate the outer membrane of E. coli by at least two different routes. However, uncoupler-resistance in UR-3 appears to be more complex than the provision of an outer membrane barrier to uncouplers. Thus, uncouplers readily discharged a pH gradient established in both AS-1 and UR-3 by addition of HCl to cell suspensions.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
PubMed1992

Mechanism of energization of uptake of the fluorescent dye 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation [DMP+] into an acrA strain of Escherichia coli.

The mechanism of uptake of the fluorescent dye 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation (DMP+) into cells and vesicles of the acrA strain AS-1 of Escherichia coli was examined. Uptake was energized by substrate oxidation and discharged by uncouplers. Uptake was enhanced by the presence of tetraphenylphosphonium cation, tetraphenylboron anion and tributyltin chloride, which may inhibit the efflux system for DMP+. Uptake was inhibited by 5-methoxyindole-2-carboxylic acid (MIC). By the use of ionophores with right-side-out vesicles loaded with monovalent cations it was shown that DMP+ uptake could be driven both by the establishment of a membrane potential across the vesicle membrane and by a H+/DMP+ antiport system. Attempts to demonstrate the latter mechanism in everted membrane vesicles were unsuccessful.

Animals↗
PubMed1990

pH probes respond to redox changes in cytochrome o.

N-Phenylnaphthylamine (NPN) has been used previously to probe the fluidity or microviscosity of membrane lipids. We have shown (Sedgwick, E. G., and Bragg, P.D. (1988) FEBS Lett. 229, 127-130) that the fluorescence intensity of this probe abruptly increases upon depletion of the oxygen content of a medium by respiring cytochrome o of Escherichia coli that has been incorporated into soybean phospholipid vesicles. We now show that the pH probes pyranine and quinacrine behave similarly to NPN. The fluorescence change is not due to changes in the pH gradient across the membrane or to a change in the distribution of probe between the vesicles and the external medium. It is insensitive to uncouplers. The fluorescence change with pyranine and quinacrine occurs also with soluble cytochrome o in the absence of added phospholipid. The NPN response requires added phospholipid. Alteration of the redox state of cytochrome o with cyanide suggests that these probes respond to a change in the redox state of the cytochrome, either by alterations in binding of the probe to the cytochrome or by a change in the environment of the probe bound to the cytochrome. This behavior should be considered when pyranine or quinacrine are used to measure changes in the internal pH of membrane vesicles containing redox proteins.

Arylsulfonates↗
PubMed1990

Differential movement of ions in artificial phospholipid vesicles.

Pyranine was incorporated into sonicated unilamellar vesicles of soybean phosphatidylcholine to monitor changes in the internal pH of the vesicles. Dilution of soybean phosphatidylcholine vesicles loaded with 0.3 M KCl, KNO3 or K2SO4 into salt-free buffer resulted in rapid exchange of K+ and protons. A pseudoequilibrium distribution of ions was achieved, since addition of valinomycin, uncoupler or nigericin now caused a rapid alkalinization of the vesicle interior. Dilution into buffer containing NaCl gave a further exchange of Na+ and protons following the initial K+/proton exchange. Na+ permeation was slower than that of K+. A stable membrane potential was not generated by the ion movements. It is proposed that aqueous channels are formed through the phospholipid bilayers and that K+ and Na+ permeate through these channels as the hydrated ions.

Arylsulfonates↗
PubMed1988

The fluorescence intensity of the lipophilic probe N-phenyl-1-naphthylamine responds to the oxidation-reduction state of purified Escherichia coli cytochrome o incorporated into phospholipid vesicles.

N-Phenyl-1-naphthylamine (NPN), a reagent which has been used previously to probe the fluidity or microviscosity of the membrane lipids of intact cells of Escherichia coli, was found to respond to the redox state of purified cytochrome o incorporated into lipid vesicles formed from purified or E. coli phospholipids. NPN was bound to the proteoliposomes to produce a steady-state level of fluorescence intensity. Addition of the substrate ascorbate, in the presence of phenazine methosulfate as an electron donor, did not alter the fluorescence. However, following complete removal of oxygen from the medium by oxidation of the substrate by molecular oxygen catalyzed by cytochrome o, there was an increase in the fluorescence of NPN. This coincided with the reduction of cytochrome o. Reoxidation of the cytochrome by addition of oxygen decreased the fluorescence to steady-state levels until the oxidant had been completely reduced. The fluorescence changes were dependent on the incorporation of cytochrome o into phospholipid vesicles but were insensitive to the state of energization of the vesicle membrane.

1-Naphthylamine↗
PubMed1987

Distinct phases of the fluorescence response of the lipophilic probe N-phenyl-1-naphthylamine in intact cells and membrane vesicles of Escherichia coli.

The fluorescence of the lipophilic probe N-phenyl-1-naphthylamine (NPN) bound to intact cells of Escherichia coli is quenched by the addition of glucose, succinate, D-lactate, pyruvate, formate and glycerol. Partial recovery of fluorescence occurs on anaerobiosis. Use of mutants with defects in the ATP synthase or the respiratory chain show that quenching of fluorescence may be energized either by ATP hydrolysis or by substrate oxidation through the respiratory chain. Permeabilization of the outer membrane by treatment of intact cells with EDTA, or use of a mutant with an outer membrane permeable to lipophilic substances, results in a more rapid binding of NPN and in a decrease in quenching observed on substrate addition. NPN binds rapidly to everted membrane vesicles, but does not respond to membrane energization. It is proposed that inner membrane energization in intact cells alters the binding or environment of NPN in the outer membrane. The fluorescence recovery which occurs on anaerobiosis has two components. One component represents a reversal of the changes which occur on membrane energization. The other component of the fluorescence change is insensitive to the uncoupler CCCP and resembles the behaviour of NPN with everted membrane vesicles. It is suggested that a portion of the fluorescence events seen with NPN involves a response of the probe to changes in the inner membrane.

1-Naphthylamine↗
PubMed1987

The fluorescence intensity of the lipophilic probe N-phenyl-1-naphthylamine responds to the oxidation-reduction state of the respiratory chain in everted membrane vesicles of Escherichia coli.

N-Phenyl-1-naphthylamine (NPN), a reagent which has been used previously to probe the fluidity or microviscosity of the membrane lipids of intact cells of Escherichia coli, was found to respond to metabolic changes in everted inner membrane vesicles from this organism. NPN was bound to the vesicles to produce a steady-state level of fluorescence intensity. Addition of substrate or ATP did not alter the fluorescence. However, following complete removal of oxygen from the medium by oxidation of substrate through the respiratory chain, there was an increase in the fluorescence of NPN. Reoxidation of the components of the respiratory chain by the addition of oxygen, ferricyanide, fumarate or nitrate decreased fluorescence to the steady-state level until the oxidant had been completely reduced. The fluorescence changes were insensitive to the state of energization of the membrane. It is proposed that NPN responds to the state of reduction of components of the respiratory chain either directly by reacting with a component of the chain or indirectly through an effect transmitted to the membrane by a change in the conformation of respiratory chain components.

1-Naphthylamine↗
PubMed1986

Uncoupler-induced relocation of elongation factor Tu to the outer membrane in an uncoupler-resistant mutant of Escherichia coli.

Escherichia coli UV6, a mutant which is resistant to the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), when grown in the presence of CCCP, but not in its absence, incorporated a new protein (Mr, 42 000) into the cell envelope. This protein was found in both cytoplasmic and outer-membrane fractions. In the outer membrane it was one of three or four most abundant proteins. The protein was tightly bound to the membranes and was not solubilized by several detergents. Solubilization was achieved with sodium lauroylsarcosinate (sarkosyl). The protein was purified close to homogeneity by affinity chromatography on a column of GDP-Sepharose. It was identified as elongation factor Tu (EF-Tu) on the basis of electrophoretic mobility, profiles of peptide fragments produced by proteolysis, and by its ability to bind to GDP-Sepharose. Disruption of cells in the presence of CCCP or incubation of envelopes with EF-Tu did not result in incorporation of EF-Tu into the membranes. It is suggested that this protein is incorporated into the outer membrane as a consequence of an alteration in the normal protein biosynthetic mechanisms of the mutant induced by the presence of CCCP.

Bacterial Outer Membrane Proteins↗
PubMed1985

Electron spin resonance studies of lipid fluidity changes in membranes of an uncoupler-resistant mutant of Escherichia coli.

The fluidity of the lipids in membrane preparations from a mutant of Escherichia coli resistant to the uncoupler CCCP, grown at different temperatures with and without CCCP, was examined by electron spin resonance using the spin probe 5-doxyl stearic acid. The fluidity of the membrane lipids at the growth temperature, as estimated using electron spin resonance, was less in cells grown at lower temperatures. Precise homeoviscous adaptation was not observed. Growth in the presence of CCCP resulted in a decrease in membrane lipid fluidity, particularly in the inner (cytoplasmic) membrane. There was no change in the proportion of phosphatidylethanolamine, phosphatidylglycerol and cardiolipin in the cell envelope. However, there was an increase in the proportion of unsaturated fatty acids in membranes from cells grown with uncoupler. This was reflected in the increased fluidity of the lipids extracted from these membranes. This result is contrary to that expected from measurements of the fluidity of the lipid in these membranes. The decreased fluidity of the lipid in these membranes may be a consequence of the observed increase in the ratio of protein to phospholipid.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
PubMed1980

Energy coupling to K+ transport in a marine bacterium.

Cells of the marine bacterium Alteromonas haloplanktis 214 ATCC 19855 (previously referred to as marine pseudomonad B-16) were depleted of K+ by washing with 0.1 M MgSO4. Washing with 0.05 M MgSO4 lowered the Vmax for K+ transport compared with washing with 0.1 M with 0.05 but did not change the Km, while washing with lower concentrations of MgSO4 caused loss of ultraviolet-absorbing material from the cells. K+ uptake was a strictly aerobic process and was accompanied by proton release. When an anaerobic suspension of cells was added to incubation mixtures containing increasing amounts of O2, intracellular ATP concentrations increased as the O2 concentration increased and reached near maximum values before K+ transport began. The O2 concentration initiating K+ transport caused transport to proceed at its maximum rate. For these experiments A. haloplanktis was depleted of ATP by incubating under anaerobic conditions. Incubating with either N,N'-dicyclohexyl carbodiimide (DCCD) or arsenate failed to deplete intact cells of ATP or prevent K+ transport. The inhibitory activity of DCCD for ATPase in membrane preparations was higher at 5 mM than at other MgSO4 concentrations and increased with time. Cyanide and the uncoupling agents tetrachloro-salicylanide (TCS) and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) prevented K+ uptake while TSC and FCCP though not cyanide caused K+ to be released from K+-containing cells. It is concluded that the driving force for K+ transport in these cells is likely to be the membrane potential and that K+ transport may be gated.

Adenosine Triphosphatases↗