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S J Ferguson

Publications and source records attributed to S J Ferguson.

At least 145 records · Page 8Linked to original sources
PubMed1985

Partial uncoupling, or inhibition of electron transport rate, have equivalent effects on the relationship between the rate of ATP synthesis and proton-motive force in submitochondrial particles.

The rates of electron transport and of ATP synthesis have been measured in bovine heart Mg-ATP submitochondrial particles oxidising succinate under conditions of partial attenuation of the proton-motive force by malonate or FCCP. This paper reports evidence that the relationship between the rate of ATP synthesis and the magnitude of the proton motive force is independent of the mode by which the decrease of the proton motive force is achieved.

Adenosine Triphosphate↗
PubMed1985

Identification of an essential beta chain lysine residue from bovine heart mitochondrial ATPase specifically modified with nitrobenzofurazan.

A tetrapetide containing an essential lysine residue chemically modified with the nitrobenzofurazan group has been purified from bovine heart mitochondrial ATPase. The composition of the peptide indicates that this lysine is residue 401 in the sequence of a beta chain. The modification was achieved by incubation at pH 9 of ATPase that had been previously labelled on a single essential tyrosine residue by reaction of the enzyme with 4-chloro-7-nitrobenzofurazan. The specific transfer of the nitrobenzofurazan group from the tyrosine residue to a particular lysine residue is consistent with the previously demonstrated intramolecular character of this transfer reaction.

4-Chloro-7-nitrobenzofurazan↗
PubMed1985

Nitrous oxide reduction by members of the family Rhodospirillaceae and the nitrous oxide reductase of Rhodopseudomonas capsulata.

After growth in the absence of nitrogenous oxides under anaerobic phototrophic conditions, several strains of Rhodopseudomonas capsulata were shown to possess a nitrous oxide reductase activity. The enzyme responsible for this activity had a periplasmic location and resembled a nitrous oxide reductase purified from Pseudomonas perfectomarinus. Electron flow to nitrous oxide reductase was coupled to generation of a membrane potential and inhibited by rotenone but not antimycin. It is suggested that electron flow to nitrous oxide reductase branches at the level of ubiquinone from the previously characterized electron transfer components of R. capsulata. This pathway of electron transport could include cytochrome c', a component hitherto without a recognized function. R. capsulata grew under dark anaerobic conditions in the presence of malate as carbon source and nitrous oxide as electron acceptor. This confirms that nitrous oxide respiration is linked to ATP synthesis. Phototrophically and anaerobically grown cultures of nondenitrifying strains of Rhodopseudomonas sphaeroides, Rhodopseudomonas palustris, and Rhodospirillum rubrum also possessed nitrous oxide reductase activity.

Acetylene↗
PubMed1984

Immunochemical identification of a two-subunit NADH-ubiquinone oxidoreductase from Paracoccus denitrificans.

Analysis by crossed-immunoelectrophoresis of Paracoccus denitrificans membrane vesicles has shown that only one antigen stains for NADH dehydrogenase activity. This activity could be partially purified by a combination of gel filtration and ion-exchange chromatography of membrane vesicles that had been solubilised in the non-ionic detergent Nonidet P-40. From the limited number of precipitates observed after crossed immunoelectrophoresis of this partially purified preparation of NADH dehydrogenase it was possible to excise specifically part of the precipitate that stained for NADH dehydrogenase. Excised precipitates containing NADH dehydrogenase that had been radiolabelled by growth of cells in the presence of [35S]SO2-(4) allowed the polypeptide composition of the enzyme to be determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate followed by fluorography. Two subunits were identified with estimated relative molecular masses of 48000 and 25000. Subunits of similar molecular weight are found in the flavoprotein fragment of the NADH dehydrogenase of the mammalian mitochondrial respiratory chain. The latter has general similarities with the respiratory chain in the plasma membrane of P. denitrificans.

Chromatography, Ion Exchange↗
PubMed1984

The nature of the reaction of an essential tyrosine residue of bovine heart mitochondrial ATPase with 4-chloro-7-nitrobenzofurazan and related compounds.

Bovine heart mitochondrial ATPase is inhibited after covalent modification with 4-chloro-7-nitrobenzofuroxan. The kinetics of the reaction are indistinguishable from those for the reaction of an essential tyrosine residue of the ATPase with 4-chloro-7-nitrobenzofurazan that have been described previously [Ferguson et al. (1975) Eur. J. Biochem. 54, 117-126]. 4-Fluoro-7-nitrobenzofurazan inhibits the ATPase with a pseudo-first-order rate constant that is tenfold greater than that for 4-chloro-7-nitrobenzofurazan. These data indicate that the rate-limiting step for reaction of the enzyme with these reagents is formation of a Meisenheimer complex at the C-4 position and that the modified tyrosine is probably on the surface of the protein. No evidence was found for more complex patterns of reactivity of 4-chloro-7-nitrobenzofurazan and its analogues. Both ammonium 4-chloro-7-sulphobenzofurazan and ammonium 4-fluoro-7-sulphobenzofurazan fail to react with the ATPase. The utility of these reagents as alternatives to the nitro derivatives may be limited owing to their slow reaction rates. After modification on tyrosine by 4-chloro-7-nitrobenzofurazan, the nitrobenzofurazan group can be transferred by an intramolecular process to lysine [Ferguson et al. (1975) Eur. J. Biochem. 54, 127-133]. ATPase with the lysine thus modified is shown to be reactive towards 4-chloro-7-nitrobenzofurazan in a manner indistinguishable from the native enzyme. This indicates that the intramolecular transfer occurs at sufficient distance to avoid steric hindrance to the second reaction, and that the lysine does not participate in a neighbouring group effect to enhance the reactivity of the tyrosine.

4-Chloro-7-nitrobenzofurazan↗
PubMed1983

Characterisation of membrane vesicles from Paracoccus denitrificans and measurements of the effect of partial uncoupling on their thermodynamics of oxidative phosphorylation.

1. Vesicles from Paracoccus denitrificans were prepared by applying an osmotic shock to spheroplasts derived from cells that had been grown anaerobically with succinate as carbon source and nitrate as electron acceptor. In the presence of either phenazinemethosulphate or N,N,N' N',-tetramethyl-p-phenylenediamine, the oxidation of isoascorbate supported the uptake of both S14CN- and 86Rb+ (in the presence of valinomycin), whereas NADH and succinate oxidation resulted only in S14CN- uptake. These observations show that the preparations contain both right-side-out and inside-out vesicles, and are related to the earlier proposal that the stimulation of an NADH-2,6-dichloroindophenol reductase activity by bee venom is an indicator of the proportion of right-side-out vesicles present. The implications impinge on previous conclusions [Burnell, J. N., John. P. and Whatley, F. R. (1975) Biochem. J. 150, 527-536 and FEBS Lett. 58, 215-218] about the mechanisms of sulphate and phosphate transport in P. denitrificans. 2. The relationship between the protonmotive force (delta p; transmembrane proton electrochemical gradient expressed in mV) and the phosphorylation potential (delta Gp) generated by vesicles from P. denitrificans has been studied as a function of the concentration of an uncoupler of oxidative phosphorylation. With either NADH or succinate as substrate, the uncoupler had a more pronounced effect on delta p than on delta Gp, so that the ratio delta Gp/F x delta p increased within a limited range of values of delta p close to the maximum. delta Gp/F x delta p was, however, approximately constant over the remaining range of delta p that was titrated. A fraction of 'highly coupled' vesicles, separated from the initial preparation by centrifugation through a Ficoll pad, showed similar titration behaviour. This demonstrated that heterogeneity within a vesicle preparation was not responsible for significant distortion of the true relationship between delta p and delta Gp. Values of delta p and delta Gp/F x delta p (H+/ATP) from 143-108 mV and 3.9-4.4, respectively, were determined when NADH was substrate, whereas with succinate, delta p ranged from 123-88 mV and delta Gp/F x delta p (H+/ATP) from 4.5-5.6. The variation in the value of delta Gp/F x delta p, which can be equated with a minimum value for the H+/ATP of the ATP synthase enzyme, is similar to, but less pronounced than, some of the data previously reported for mitochondria. Thus the observations with these bacterial vesicles, which represent an experimentally simpler system than mitochondria, might be taken as further evidence that measurements of the bulk phase delta p might not truly reflect the driving force for ATP synthesis sensed by the ATP synthase enzyme. However, other explanations that would make the data consistent with a chemiosmotic mechanism cannot be eliminated...

Adenosine Triphosphate↗
PubMed1983

The effects of partial uncoupling upon the kinetics of ATP synthesis by vesicles from Paracoccus denitrificans and by bovine heart submitochondrial particles. Implications for the mechanism of the proton-translocating ATP synthase.

1. Reduction in the magnitude of the respiration-dependent protonmotive force (proton electrochemical gradient in mV) of vesicles from Paracoccus denitrificans, and of submitochondrial particles, has been found to be paralleled small increases in S50% values for both ADP and Pi. For example, reduction of the protonmotive force of P. denitrificans vesicles from 145 mV to 110 mV was accompanied by an increase of S50% (ADP) from 8 microM to 18 microM, and an increase of S50% (Pi) from 0.33 mM to 1.4 mM. This result was obtained with partial uncoupling quantities of both carbonyl-cyanide p-trifluoromethoxyphenylhydrazone and of the synergistic combination of nigericin plus valinomycin in the presence of K+. In view of the similar effects of these two different methods of uncoupling it is concluded that the changes in S50% were a consequence of the diminished protonmotive force acting on the ATP synthase rather than of a secondary, direct interaction of the uncouplers with the enzyme. Changes in S50% rather than Km are described because under several sets of conditions double-reciprocal plots were nonlinear. 2. For equivalent attenuations in the rate of ATP synthesis by submitochondrial particles, 2,4-dinitrophenol caused much larger increases in S50% (ATP) than did carbonylcyanide p-trifluoromethoxyphenylhydrazone. Therefore it is concluded that the effect of 2,4-dinitrophenol was primarily a consequence of its previously recognized direct interaction with the F1 segment of the mitochondrial ATPase. The concentration range of 2,4-dinitrophenol that raised S50% (ADP) is similar to that which weakens the binding of ADP to a particular type of site on the purified F1 sector of ATP synthase. This correlation is consistent with such a site having a catalytic role during ATP synthesis. 3. A titration of the rate of ATP synthesis by vesicles of P. denitrificans with increasing quantities of carbonylcyanide p-trifluoromethoxyphenylhydrazone showed that the initial titres of the uncoupler caused large decreases in the rate of ATP synthesis for relatively small attenuations in the protonmotive force. Thus the initial 20 mV drop in the protonmotive force was accompanied by a reduction of more than 65% in the rate of ATP synthesis. Over the lowest range of values of protonmotive force that drove detectable rates of ATP synthesis however, the dependence of the rate was a less steep function of the protonmotive force. A plot of the logarithm of the rate of ATP synthesis against protonmotive force reveals a biphasic relationship. There does not appear to be a 'threshold' value of the protonmotive force below which ATP synthesis is blocked by kinetic factors. 4. The relationships of the protonmotive force with S50% values and with the rate of ATP synthesis (at near saturating concentrations of ADP and Pi) are discussed in relation to possible mechanisms for the coupling of proton translocation to ATP synthesis.

ATP Synthetase Complexes↗
PubMed1983

Electron flow to dimethylsulphoxide or trimethylamine-N-oxide generates a membrane potential in Rhodopseudomonas capsulata.

Under dark and essentially anaerobic conditions electron flow to either dimethylsulphoxide or trimethylamine-N-oxide in cells of Rhodopseudomonas capsulata has been shown to generate a membrane potential. This conclusion is based on the observation of a red shift in the carotenoid absorption band which is a well characterised indicator of membrane potential in this bacterium. The magnitude of the dimethylsulphoxide- or trimethylamine-N-oxide-dependent membrane potential was reduced either by a protonophore uncoupler of oxidative phosphorylation or synergistically by a combination of a protonophore plus rotenone, an inhibitor of electron flow from NADH dehydrogenase. These findings, together with the observation that venturicidin, an inhibitor of the proton translocating ATPase, did not reduce the membrane potential, show that electron flow to dimethylsulphoxide or trimethylamine-N-oxide is coupled to proton translocation. Thus contrary to some previous proposals dark and anaerobic growth of Rps. capsulata in the presence of dimethylsulphoxide or trimethylamine-N-oxide cannot be regarded as purely fermentative.

Anaerobiosis↗
PubMed1982

Estimation of H+-translation stoicheiometry of mitochondrial ATPase by comparison of proton-motive forces with clamped phosphorylation potentials in submitochondrial particles.

The proton-motive forces generated in submitochondrial particles by both hydrolysis of ATP and oxidation of succinate have been measured by flow dialysis and compared with the ambient phosphorylation potentials. It is concluded that three H+ are translocated for each ATP molecule hydrolysed or synthesised. By utilising rat liver mitochondria respiring with beta-hydroxybutyrate as a new system for regeneration of ATP from ADP and Pi, phosphorylation potentials were clamped at a range of values by using mixtures of particles and mitochondria in various ratios. As the rate of ATP hydrolysis by the particles was lowered, the proton-motive force decreased only slightly except at the very lowest rates, these results paralleling earlier studies on the relation between rate of respiration-driven proton translocation and proton-motive force.

Adenosine Triphosphatases↗
PubMed1982

Hydrolysis of ITP generates a membrane potential in submitochondrial particles.

ITP hydrolysis catalysed by the ATPase of submitochondrial particles from both bovine heart and rat liver is shown to be linked to the generation of a membrane potential, and therefore also to proton translocation. The magnitude of the membrane potential is similar to that observed during ATP hydrolysis at equivalent concentrations of phosphate and nucleoside tri- and diphosphates. An explanation is suggested for why in other reports ITP was found to be a poor substrate for supporting energy-linked reactions that are driven by the membrane potential.

Adenosine Triphosphatases↗
PubMed1982

Alloantibody-induced impaired neonatal expression of a red blood cell antigen associated with maternal alloimmunization.

Animals genetically capable of making certain gene products have been shown to have the production of such products completely or partially suppressed by exposure in utero or neonatally to antibodies specific for that gene product. This alloantibody-induced depression of expression of a specific antigenic determinant has yet to be shown to occur in man. The McCoya red blood cell antigen is reported to be well developed at birth. However, 2 children born to mothers with high-titer McCoya antibodies in their serum, phenotyped as McCoy (a-) at birth and, on retesting their red blood cells, 1 at 5 months and the 2nd at 11 months, both phenotyped as McCoy (a+). It is possible that this lack of expression of the McCoya antigen at birth represents a characteristic of an obligate heterozygote. However, on testing 50 random umbilical cord red blood cell samples with two potent McCoya antisera, only 1 was found to be negative. If the negative phenotype were a characteristic of an obligate heterozygote at birth, then the neonatal incidence of the McCoy (a-) phenotype should have been significantly (p less than 10(-4)) high in our random cord blood sample than observed (expected 11). The impaired expression of the McCoya antigen on the red blood cells of these 2 infants at birth, in conjunction with evidence of maternal alloimmunization, strongly suggests alloantibody-induced antigenic suppression of the McCoya antigen as a probable cause.

Adult↗
PubMed1981

Clarification of factors influencing the nature and magnitude of the protonmotive force in bovine heart submitochondrial particles.

The magnitude of the protonmotive force, and its division between pH gradient and membrane potential components has been further characterised in submitochondrial particles. In a reaction medium containing sucrose for osmotic support and 4-(2-hydroxyethyl)-1-piperazineethanesulfonate (Hepes) as buffer, with succinate as substrate, the total protonmotive force reached a maximum value of 245 mV. The presence of Cl- enhanced the pH gradient with a partial but not fully compensating decrease in the membrane potential. When submitochondrial particles were suspended in a medium of low osmolarity consisting of phosphoric acid neutralised with Tris, again with succinate as substrate, the protonmotive force was lower and did not exceed 185 mV, and the pH gradient component was equivalent to 25 mV or less. The final phosphorylation potential, delta Gp, (formula: see text); maintained by the particles was higher in the phosphate/Tris medium (46--47.7 kJ mol-1) than in the sucrose/Hepes/KCl medium (43.7 kJ mol-1). Thus, comparison of the phosphorylation potential with the protonmotive force would suggest that the mechanistic stoichiometry H+/ATP (H+ translocated per molecule of ATP synthesied) for the ATPase enzyme is 3 in the former medium and 2 in the latter, which might be taken to indicate two different types of mechanism required for ATP synthesis. However it is questioned whether a comparison of the protonmotive force with delta Gp in terms of equilibrium thermodynamics ought not to be complemented by analysis in terms of linear non-equilibrium thermodynamics. The latter treatment shows that it is possible to estimate only a value for the product of a phenomenological stoichiometry and the degree of coupling, which can be variable, but not the mechanistic stoichiometry. This treatment can also rationalise the observation of the higher delta Gp in reaction conditions where the lower values for delta p are estimated. Irrespective of possible explanations, the data show how an unprejudiced choice of reaction conditions can lead to different conclusions about the relationship between the phosphorylation potential and the protonmotive force.

Animals↗
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↗