Search PubMed⌕ Search

Biomedical subjects

D B Kell

Publications and source records attributed to D B Kell.

At least 109 records · Page 6Linked to original sources
PubMed1982

On the mode of action of the bacteriocin butyricin 7423. Effects on membrane potential and potassium-ion accumulation in Clostridium pasteurianum.

1. The apparent transmembrane bulk-phase electrical potential (delta psi) of Clostridium pasteurianum was determined from the distribution ratio of the membrane-permeable cation butyltriphenylphosphonium (BuPh3P+). In glycolysing cells the highest value of delta psi, 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 (delta psi) 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 delta psi 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 DC3) which possessed a H+-ATPase with diminished sensitivity both to N,N'-dicyclohexylcarbodiimide and to butyricin 7423, exhibited a negligible decrease in delta psi 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.

Adenosine Triphosphatases↗
PubMed1981

Estimation with an ion-selective electrode of the membrane potential in cells of Paracoccus denitrificans from the uptake of the butyltriphenylphosphonium cation during aerobic and anaerobic respiration.

1. Aerobic respiration by cells of Paracoccus dentrificans drives the uptake of the lipophilic cation butyltriphenylphosphonium. Anaerobiosis or addition of an uncoupler of oxidative phosphorylation (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) results in efflux of the cation. Changes in the concentration of butyltriphenylphosphonium in the suspension medium were measured by using an ion-selective electrode, the construction of which is described. 2. If the uptake of butyltriphenylphosphonium is used as an indicator of membrane potential, then at pH 7.3 an estimate of about 160 mV is obtained for cells of P. dentrificans respiring aerobically in 100 mM-Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid/NaOH or 100mM-NaH2PO4/NaOH. This potential, however, is decreased by more than 20 mV in reaction media containing a high concentration of phosphate (100 mM) together with at least 1 mM-K+. 3. Anaerobic electron transport with NO3-, NO2- or N2O as terminal electron acceptor generates a membrane potential of about 150mV in described suspension media. The presence of these species under aerobic conditions, moreover, has negligible effect upon the extent of uptake of butyltriphenylphosphonium normally driven by aerobic respiration. These data indicate that none of these molecules exert a significant uncoupling effect on the protonmotive force. 4. No 204Tl+ uptake into respiring cells was detected. This adds to the evidence that 204Tl+ is not a freely permeable cation in bacterial cells and therefore not an indicator of membrane potential as has been proposed. The absence of respiration-driven 204Tl+ uptake indicates that P. denitrificans cells grown under the conditions specified in the present work do not possess K+-transport systems of either the Kdp or TrkA types that have been described in Escherichia coli.

Aerobiosis↗
PubMed1980

Formulation and some biological uses of a buffer mixture whose buffering capacity is relatively independent of pH in the range pH 4-9.

A mixture is described which has a buffering capacity which is essentially independent of pH in the range pH 4.0-9.0. It is shown how this buffer mixture may be used to determine the force-flux relationship of proton transfer between two aqueous phases separated by a phospholipid bilayer in vesicular systems and so demonstrate that this relationship is linear over a wide range of delta mu approximately H+. The buffer mixture can, furthermore, be employed to determine the volume enclosed within a vesicular preparation.

Buffers↗
PubMed1979

Comparison of permeant ion uptake and carotenoid band shift as methods for determining the membrane potential in chromatophores from Rhodopseudomonas sphaeroides Ga.

1. A comparison was made of two methods for estimating the membrane potential in chromatophores from Rhodopseudomonas sphaeroides Ga. Illuminated chromatophores generated a potential that is apparently much larger when estimated on the basis of the red-band shift of carotenoids rather than from the extent of uptake of the permeant SCN- ion. 2. In contrast, when the chromatophores were oxidizing NADH or succinate the uptake of SCN- indicated a larger membrane potential than was estimated from the carotenoid band shift. 3. The extent of SCN- uptake and the carotenoid-band shift respond differently to changes in the ionic composition of the reaction medium. 4. The effects of antimycin on the carotenoid band shift and SCN- uptake are reported. 5. It is concluded that the carotenoid band shift and the uptake of SCN- are responding to different aspects of the energized state.

Antimycin A↗
PubMed1978

The protonmotive force in bovine heart submitochondrial particles. Magnitude, sites of generation and comparison with the phosphorylation potential.

1. The magnitude of the protonmotive force in respiring bovine heart submitochondrial particles was estimated. The membrane-potential component was determined from the uptake of S14CN-ions, and the pH-gradient component from the uptake of [14C]methylamine. In each case a flow-dialysis technique was used to monitor uptake. 2. With NADH as substrate the membrane potential was approx. 145mV and the pH gradient was between 0 and 0.5 unit when the particles were suspended in a Pi/Tris reaction medium. The addition of the permeant NO3-ion decreased the membrane potential with a corresponding increase in the pH gradient. In a medium containing 200mM-sucrose, 50mM-KCl and Hepes as buffer, the total protonmotive force was 185mV, comprising a membrane potential of 90mV and a pH gradient of 1.6 units. Thus the protonmotive force was slightly larger in the high-osmolarity medium. 3. The phosphorylation potential (= deltaG0' + RT ln[ATP]/[ADP][Pi]) was approx. 43.1 kJ/mol (10.3kcal/mol) in all the reaction media tested. Comparison of this value with the protonmotive force indicates that more than 2 and up to 3 protons must be moved across the membrane for each molecule of ATP synthesized by a chemiosmotic mechanism. 4. Succinate generated both a protonmotive force and a phosphorylation potential that were of similar magnitude to those observed with NADH as substrate. 5. Although oxidation of NADH supports a rate of ATP synthesis that is approximately twice that observed with succinate, respiration with either of these substrates generated a very similar protonmotive force. Thus there seemed to be no strict relation between the size of the protonmotive force and the phosphorylation rate. 6. In the presence of antimycin and/or 2-n-heptyl-4-hydroxyquinoline N-oxide, ascorbate oxidation with either NNN'N'-tetramethyl-p-phenylenediamine or 2,3,5,6-tetramethyl-p-phenylenediamine as electron mediator generated a membrane potential of approx. 90mV, but no pH gradient was detected, even in the presence of NO3-. These data are discussed with reference to the proposal that cytochrome oxidase contains a proton pump.

Adenosine Triphosphate↗
PubMed1978

The protonmotive force in phosphorylating membrane vesicles from Paracoccus denitrificans. Magnitude, sites of generation and comparison with the phosphorylation potential.

1. The magnitude of the protonmotive force in phosphorylating membrane vesicles from Paracoccus denitrificans was estimated. The membrane potential component was determined from the uptake of S(14)CN(-), and the transmembrane pH gradient component from the uptake of [(14)C]methylamine. In each case a flow-dialysis technique was used to monitor uptake. 2. With NADH as substrate, the membrane potential was about 145mV and the pH gradient was below 0.5 pH unit. The membrane potential was decreased by approx. 15mV during ATP synthesis, and was abolished on addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. In the presence of KCl plus valinomycin the membrane potential was replaced by a pH gradient of 1.5 units. 3. Succinate oxidation generated a membrane potential of approx. 125mV and the pH gradient was below 0.5 pH unit. Oxidation of ascorbate (in the presence of antimycin) with either 2,3,5,6-tetramethyl-p-phenylenediamine or NNN'N'-tetramethyl-p-phenylenediamine as electron mediator usually generated a membrane potential of approx. 90mV. On occasion, ascorbate oxidation did not generate a membrane potential, suggesting that the presence of a third energy-coupling site in P. denitrificans vesicles is variable. 4. With NADH or succinate as substrate, the phosphorylation potential (DeltaG(p)=DeltaG(0)'+RTln[ATP]/ [ADP][P(i)]) was approx. 53.6kJ/mol (12.8kcal/mol). Comparison of this value with the protonmotive force indicates that more than 3 protons need to be translocated via the adenosine triphosphatase of P. denitrificans for each molecule of ATP synthesized by a chemiosmotic mechanism. In the presence of 10mm-KNO(3) the protonmotive force was not detectable (<60mV) but DeltaG(p) was not altered. This result may indicate either that there is no relationship between the protonmotive force and DeltaG(p), or that for an unidentified reason the equilibration of SCN(-) or methylamine with the membrane potential and the pH gradient is prevented by NO(3) (-) in this system.

Adenosine Triphosphate↗
PubMed1978

Measurement by a flow dialysis technique of the steady-state proton-motive force in chromatophores from Rhodospirillum rubrum. Comparison with phosphorylation potential.

1. In the light a transmembrane electrical potential of 100 mV has been estimated to occur in chromatophores from Rhodospirillum rubrum. The potential was determined by measuring the steady-state distribution of the permeant SCN- across the chromatophore membrane using a flow dialysis technique. The potential was not observed in the dark, nor in the presence of antimycin. It was dissipated on the addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The potential was reduced by between 15 and 20 mV when ADP and Pi were added. Hydrolysis of ATP by the chromatophores generated a membrane potential of about 80 mV. 2. Using a flow dialysis technique light-dependent uptake of methylamine was observed only in the presence of concentrations of SCN- that were 500-fold higher than were used to measure the membrane potential. It is concluded that the pH gradient across the illuminated chromatophore membrane is insignificant except in the presence of relatively high concentrations of a permeant anion like thiocyanate. Further evidence that a negligible pH gradient was generated by the chromatophores is that addition of K+ and nigericin to illuminated chromatophores did not stimulate uptake of SCN-. 3. In the light of chromatophores established and maintained a phosphorylation potential of up to 14 kcal/mol. If a phosphorylation potential of this magnitude is to be poised against a proton-motive force that comprises solely a membrane potential of approx. 100 mV, then at least five protons must be translocated for each ATP synthesised via a chemiosmotic mechanism.

Adenine Nucleotides↗