Edward G. Sedgwick

Effect of uncouplers on the bioenergetic properties of a carbonyl cyanide m-chlorophenylhydrazone-resistant mutant Escherichia Coli UV6

The effects of carbonyl cyanide m-chlorophenylhydrazone (CCCP) and tri-n-butyltin chloride (Bu3SnCl) on proline transport, proton uptake and the transmembrane pH gradient in intact cells have been compared in a CCCP-resistant mutant strain Escherichia coli UV6, and its parent strain, AN180. CCCP and Bu3SnCl inhibited proline uptake in AN180 but the pH gradient was affected only by CCCP. Neither uncoupler affected the pH gradient in UV6 although inhibition of proline uptake occurred at high concentrations. CCCP caused efflux of accumulated proline in both strains but Bu3SnCl was ineffective. Bu3SnCl did not prevent the efflux of proline induced by CCCP, indicating that Bu3SnCl had not inactivated the transport carrier. In contrast with the parent strain, CCCP failed to reverse the oxidation-dependent inhibition of the phosphotransferase system in UV6 even at concentrations causing inhibition of proline uptake. The phosphorylation potential of UV6 with succinate as substrate was lower than in AN180. This was associated with a 10-fold higher concentration of phosphate in succinate-grown UV6 than in AN180. These results suggest that CCCP and Bu3SnCl have different sites of action on the membrane energization system of intact cells of E. coli. A possible explanation of the differences between AN180 and UV6 is that the energization system is altered in the CCCP-resistant mutant. Both uncouplers stimulated the uptake of protons by intact cells to the same extent in UV6 as in AN180. In UV6, and in AN180 with Bu3SnCl, this was not accompanied by effects on the transmembrane pH gradient. The extent of proton uptake appeared to be related to the level of the highly anionic membrane-associated oligosaccharides in the periplasmic space. It is proposed the outer membrane acts as a partial barrier to protons and that the uncouplers can equilibrate protons between the extracellular medium and the periplasmic space in intact cells.

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.

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.

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.

Role of genetic susceptibility to latent adenoviral infection and decreased lung function.

BACKGROUND Latent adenoviral infection may amplify cigarette smoke-induced lung inflammation and therefore play an important role in the development of chronic obstructive pulmonary disease (COPD). Adenoviruses can evade the human immune response via their 19-kDa protein (19K) which delays the expression of class I human leukocyte antigen (HLA) proteins. The 19K protein shows higher affinity to HLA-B7 and A2 compared with HLA-A1 and A3. The receptor for adenovirus (CXADR) and integrin beta(5) (ITGB5) are host factors which might affect adenovirus infection. Therefore, we investigated the contribution of HLA, CXADR, and ITGB5 genetic variants to the presence of the E1A gene and to level of lung function. METHODS Study subjects were assayed for HLA-B7, A1, A2 and A3 by PCR-based assays using allele-specific primers. Polymorphisms of the CXADR and ITGB5 genes were genotyped by PCR-based restriction fragment length polymorphism assays. Detection of adenoviral E1A gene was performed by a real-time PCR TaqMan assay. RESULTS E1A positive individuals had a lower FEV(1) compared with E1A negative individuals. However, there was no significant difference in E1A positivity rate between the high (HLA-B7 and A2) and low (HLA-A1 and A3) 19K affinity groups. There was also no significant difference in FEV(1) level between each affinity group. There was no significant difference in E1A positivity rate or lung function among the CXADR and ITGB5 genotypes. CONCLUSIONS Genetic variants in HLA, CXADR and ITGB5 do not influence latent adenoviral infections and are not associated with COPD.

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.

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.

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 as 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.

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.

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.