Search PubMed⌕ Search

Biomedical subjects

S J Ferguson

Publications and source records attributed to S J Ferguson.

At least 163 records · Page 9Linked to original sources
PubMed1980

The location of dissimilatory nitrite reductase and the control of dissimilatory nitrate reductase by oxygen in Paracoccus denitrificans.

1. A method is described for preparing spheroplasts from Paracoccus denitrificans that are substantially depleted of dissimilatory nitrate reductase (cytochrome cd) activity. Treatment of cells with lysozyme + EDTA together with a mild osmotic shock, followed by centrifugation, yielded a pellet of spheroplasts and a supernatant that contained d-type cytochrome. The spheroplasts were judged to have retained an intact plasma membrane on the basis that less than 1% of the activity of a cytoplasmic marker protein, malate dehydrogenase, was released from the spheroplasts. In addition to a low activity towards added nitrite, the suspension of spheroplasts accumulated the nitrite that was produced by respiratory chain-linked reduction of nitrate. It is concluded that nitrate reduction occurs at the periplasmic side of the plasma membrane irrespective of whether nitrite is generated by nitrate reduction or is added exogenously. 2. Further evidence for the integrity of the spheroplasts was that nitrate reduction was inhibited by O2, and that chlorate was reduced at a markedly lower rate than nitrate. These data are taken as evidence for an intact plasma membrane because it was shown that cells acquire the capability to reduce nitrate under aerobic conditions after addition of low amounts of Triton X-100 which, with the same titre, also overcame the permeability barrier to chlorate reduction by intact cells. The close relationship between the appearance of chlorate reduction and the loss of the inhibitory effect of O2 on nitrate reduction also suggests that the later feature of nitrate respiration is due to a control on the accessibility of nitrate to its reductase rather than on the flow of electrons to nitrate reductase.

Chlorates↗
PubMed1979

Variable proton conductance of submitochondrial particles.

The relationship between the rate of substrate oxidation and the protonmotive force (electrochemical proton gradient) generated by bovine heart submitochondrial particles has been examined. Unexpectedly, oxidation of succinate generated a higher protonmotive force than the oxidation of NADH, although the rate of proton translocation across the membrane was inferred to be considerably lower with succinate as substrate. The data suggest that the flow of electrons through site 1 of the respiratory chain may increase the conductance of the mitochondrial membrane for protons. Upon reduction of the rate of succinate oxidation by titration with malonate, the protonmotive force remained essentially constant until the extent of inhibition was greater than 75%. The general conclusion from this work is that a constant passive membrane conductance for protons cannot be assumed.

Animals↗
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

Complete assignment of aromatic 1H nuclear magnetic resonances of the tyrosine residues of hen lysozyme.

The complete assignment of the aromatic proton nuclear magnetic resonances of the three tyrosine residues in hen lysozyme is reported. These assignments were made using double resonance techniques, specific chemical modifications of one residue (Tyr-23), and by interpretation of the effects of paramagnetic lanthanide ions. Some aspects of the behaviour of the tyrosine residues are reported, including pK values, reactivity towards modifying agents and conformational mobility.

4-Chloro-7-nitrobenzofurazan↗
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↗
PubMed1977

The phosphorylation potential generated by respiring bovine heart submitochondrial particles.

A phosphorylation potential deltaGp, where deltaGp = deltaGo' + RT2.303 log ([ATP]/([ADP][Pi])), of approx. 44.3 kJ.mol-1 (10.6 kcal.mol-1) was generated by submitochondrial particles that were oxidizing either NADH or succinate. Addition of adenylyl imidodiphosphate, which should suppress adenosine triphosphatase activity of any uncoupled particles, did not raise the phosphorylation potential. Raising the Pi concentration slightly increased the magnitude of the value for [ATP]/[ADP], but this did not fully compensate for the increased Pi concentration, so that the phosphorylation potential decreased slightly as the Pi concentration was raised. The phosphorylation potential developed by submitochondrial particles is lower than that generated by phosphorylating membrane vesicles from some bacteria, and is also less than that developed externally by mitochondria, but is strikingly close to the phosphorylation potential that is generated internally by mitochondria.

Animals↗