Showing papers by "Douglas B. Kell published in 1982"

On the extent of localization of the energized membrane state in chromatophores from Rhodopseudomonas capsulata N22.

Abstract: 1. The principle of the double-inhibitor titration method for assessing competing models of electron transport phosphorylation is expounded. 2. This principle is applied to photophosphorylation by chromatophores from Rhodopseudomonas capsulata N22. 3. It is found that, in contrast to the predictions of the chemiosmotic coupling model, free energy transfer is confined to individual electron transport chain and ATP synthase complexes. 4. This conclusion is not weakened by arguments concerning, the degree of uncoupling in the native chromatophore preparation or the relative number of electron transport chain and ATP synthase complexes present. 5. Photophosphorylation is completely inhibited by the uncoupler SF 6847 at a concentration corresponding to 0.31 molecules per electron transport chain. 6. The apparent paradox is solved by the proposal, consistent with the available evidence on the mode of action of uncouplers, that uncoupler binding causes a co-operative conformation transition in the chromatophore membrane, which leads to uncoupling and which is not present in the absence of uncoupler.

Proton-coupled energy transduction by biological membranes. Principles, pathways and praxis

Abstract: A brief outline of certain features of the chemiosmotic hypothesis of the mechanism of free-energy transfer between the reactions of electron transport and adenosine triphosphate synthesis catalysed by biological membranes is given Pulses of electron transport induced by the addition of small quantities of oxygen to suspensions of the bacterium Paracoccus denitrificans lead to vectorial H+ movements into the aqueous phase external to the organisms, where they may be detected with a glass pH electrode The stoichiometry of the number of protons translocated into the bulk phase external to the organisms, per oxygen atom reduced, is essentially unchanged when the amount of oxygen reduced is varied, in a manner inconsistent with the predictions of the chemiosmotic-coupling hypothesis These and other observations lead to the view that the energy-coupling proton-transfer processes utilised in reactions such as electron-transport phosphorylation are confined to the membrane phase Mechanisms which most easily account for this are discussed

Localized energy coupling during photophosphorylation by chromatophores of Rhodopseudomonas capsulata N22

Abstract: The principle of the dual inhibitor titration method for testing models of electron-transport phosphorylation is outlined, and the method is applied to the study of photophosphorylation in bacterial chromatophores. It is concluded that energy coupling is strictly localized in nature in this system, in the sense that free energy released by a particular electron-transport chain may be used only by a particular H+-ATP synthase. Dual inhibitor titrations using the uncoupler SF 6847 and the H+-ATP synthase inhibitor oligomycin indicate that uncouplers act by shuttling rapidly between the localized energy-coupling sites.

On the Mode of Action of the Bacteriocin Butyricin 7423

Abstract: 1 The apparent transmembrane bulk-phase electrical potential (ΔΨ) of Clostridium pasteurianum was determined from the distribution ratio of the membrane-permeable cation butyltriphenylphosphonium (BuPh3P+). In glycolysing cells the highest value of ΔΨ, calculated on the assumption that there was no energy-dependent binding of BuPh3P+ to the organisms, was recorded in media containing only 2–3 mM K+ ions and, even so, was only 100–110 mV. 2 Efrapeptin, a BF1, -directed inhibitor of the membrane H+-ATPase of Cl. pasteurianum, abolished the membrane potential ΔΨ and caused complete efflux of actively-transported K+ ions. Thus protonmotive hydrolysis of ATP generated by substrate level phosphorylation is the sole means of membrane energisation in this anaerobe. 3 At low (sublethal) concentrations, butyricin 7423 stimulated K+ efflux from Cl. pasteurianum without measurably affecting its membrane potential. At lethal and supralethal concentrations of this bacteriocin, both ΔΨ and active K+ uptake were abolished. 4 Whilst the addition of valinomycin to cells of Cl. pasteurianum suspended in media of low K+ concentration generated a diffusion potential to which BuPh3P+ would respond, addition of butyricin 7423 in place of valinomycin caused no such effect. Also, unlike valinomycin, butyricin 7423 did not increase the rate of K+ efflux from non-glycolysing cells of Cl. pasteurianum. Valinomycin stimulated, but butyricin 7423 inhibited, the uptake of 86Rb+ ions by glycolysing cells of Cl. pasteurianum. 5 A mutant strain of Cl. pasteurianum (viz. strain DC 3) which possessed a H+-ATPase with diminished sensitivity both to N,N' dicyclohexylcarbodiimide and to butyricin 7423, exhibited a negligible decrease in ΔΨ and in K+ accumulation ratio in response to concentrations of butyricin 7423 that were bactericidal to the wild-type, parent organism. Even so, the bactericidal action of butyricin 7423 on Cl. pasteurianum is not adequately explained by its ability in vitro to inhibit the membrane H+-ATPase of this organism. 6 Bactericidal concentrations of butyricin 7423 neither provoked efflux of Na+ ions from Cl. pasteurianum nor exhibited any protonophorous activity. However, at artificially high concentration, butyricin 7423 catalysed the passage of Na+ ions as well as of K+ ions through multilayer lipid membranes. 7 As a non-protonophorous uncoupler, butyricin 7423 appears to act in a similar manner to that of the membrane-active colicins. Yet no evidence was obtained that butyricin 7423 at its minimum lethal concentration might form a gated ion channel in the cytoplasmic membrane of the target cell, or act as a classic ionophore.

Transmembrane respiration-driven H+ translocation is unimpaired in an eup mutant of Escherichia coli.

Abstract: Summary: Respiration-driven H+ translocation has been examined in an eup (‘energy uncoupled phenotype’) mutant of Escherichia coli and compared with that observed in its otherwise isogenic wildtype parent. Respiration-driven H+ translocation was unimpaired in the eup mutant strain. It appears that the role of the eup gene product lies in the utilization of energized protons pumped across the E. coli cytoplasmic membrane.

Respiration-driven proton translocation in Paracoccus denitrificans: role of the ‘permeant’ ion

Abstract: Scholes & Mitchell ( 1970) demonstrated that the addition of pulses of 0 2, as air-saturated KCI. to weakly-buffered anoxic suspensions of Micrococcus (now Paracoccus) denitrificans elicited the vectorial ejection of H+ into the bulk aqueous phase external to the organisms, where they could be detected with a sensitive glass electrode. Yet, in the absence of valinomycin or the SCNion, the half-time of H+ translocation was very much greater than the half-time of 0 2 reduction; the extrapolated -+H+/O ratio was also significantly less than that observed when appropriate concentrations of valinomycin or SCNwere present. It was proposed that, in the absence of such compounds, a large transmembrane potential was built up by the translocation of a small fraction of the pumped protons, and that inhibition of this bulk-to-bulk transmembrane potential by the transmembrane electrophoretic coor counter-transport of 'permeant' ions allowed measurement of the true stoichiometry of respiration-driven H + translocation. Gould & Cramer ( 1977), working with Escherichia coli, challenged this explanation of the role of valinomycin and SeNby demonstrating (in the absence of'permeant' ions) that at high cell/02 ratios, when the calculated membrane potential was energetically insignificant, the measured -+H+ /0 ratio did not remotely attain its limiting stoichiometric value. Further, the stoichiometry of H+ ejection following the addition of a second oxygen pulse immediately after the first was unchanged. We have therefore reinvestigated the role of so-called 'permeant' ions in P. denitrificans, as part of a general study of the pathway of H+ transfer in membrane energy-coupling processes (Kell, 1979; Kell & Morris, 1981a; Kell et al., 1981). P. denitrificans N.C.I.B. 8944 was grown and maintained as described previously (McCarthy eta/., 1981). Mid-exponentialphase cultures were washed three times and resuspended at approx. 3mg dry weight/ml in a 6ml reaction mixture containing 150mM-KCI/0.25 mM-glycylglycine, pH 6.5 plus 80,ug of carbonic anhydrase/mi. The potentiometric system was as described by Kell & Morris (l981b), and 0 2 pulses were delivered as air-saturated KCl in the usual way (Scholes & Mitchell, 1970). The following results were obtained: (l) The number of measurable H+ ions translocated across the bacterial membrane, dlf+, increased linearly with the size of the 0 2 pulse from 4. 7 to 4 7 ng-atom of 0. (2) More than 90% of the observed H+ had been pumped across the bacterial membrane in that they were not observed when carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, 2,uM) was present. (3) This pattern

Butyricin 7423 and the membrane H+-ATPase of Clostridium pasteurianum

Abstract: The bacteriocin butyricin 7423 inhibited the activity of the membrane H+-ATPase (BF0, F1) of vegetative cells of Clostridium pasteurianum but not that of its soluble BF1 component. In vitro studies with the H+-ATPases of mutant strains selected for diminished sensitivity (a) to butyricin 7423 and (b) to dicyclohexylcarbodi-imide, confirmed that butyricin 7423 interacts with the BF0 component of this enzyme complex. Even so, certain other mutant strains displaying decreased sensitivity to butyricin 7423 possessed H+-ATPases which in vitro showed undiminished sensitivity to inhibition by the bacteriocin. Furthermore, from the changes in intracellular ATP concentration and in the rates and net extent of efflux of intracellular 86Rb+ ions that were provoked by exposure of the parent and several of the mutant strains to butyricin 7423, it was concluded that its primary bactericidal action was not attributable to stoichiometric inhibition of the membrane H+-ATPase. High extracellular concentrations of K+ ions enabled Cl. pasteurianum to survive exposure to low concentrations of this membrane-active bacteriocin.

SHORT COMMUNICATION Transmembrane Respiration-driven H+ Translocation is Unimpaired in an eup Mutant of Escherichia coli

Abstract: Respiration-driven H+ translocation has been examined in an eup (‘energy uncoupled phenotype’) mutant of Escherichia coli and compared with that observed in its otherwise isogenic wildtype parent. Respiration-driven H+ translocation was unimpaired in the eup mutant strain. It appears that the role of the eup gene product lies in the utilization of energized protons pumped across the E. coli cytoplasmic membrane.