Showing papers by "Douglas B. Kell published in 1978"

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

Abstract: 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.

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

Abstract: 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.

On the current-voltage relationships of energy-transducing membranes: phosphorylating membrane vesicles from Paracoccus denitrificans [proceedings].

Abstract: The reducing species is actually the unstable semi-reduced dRFIH· radical, which decays spontaneously to an inactive dimer (Massey & Hemmerich, 1978). Thus reduction cannot proceed beyond the stage where the second-order Pd0 reduction becomes slower than the second-order dimerization, and in the dark the solution contains no extra reducing equivalents. On cooling, the Soret band of the cytochrome shifts from 410nm (Fe2+-RH) toward 417 nm (Fe3+-RH, low spin) (Fig. 2). This reversible shift follows perfectly heating ~cool­ ing cycles, and clearly indicates the reverse electron transfer from Fe+ -RH to Pd0 as another possibility of uncoupling. The various possibilities opened by the combination of subzero temperatures and light-activation can be tested on other sites of the cytochrome P-450 cycle and other multi-component electron-transport chains.

On the Current-Voltage Relationships of Energy-Transducing Membranes: Submitochondrial Particles

Abstract: A transmembrane electrochemical proton gradient is widely held to constitute the link between electron transport and ATP synthesis in biological membranes (Boyer et al., 1977), but much debate surrounds the issue of the stoicheiometry of H+ movement that either generates (by electron transport) or dissipates (by ATP synthesis) this proton gradient (proton-motive force, l'.p) (Brand, 1977). Our estimates of both l'.p and the phosphorylation potential generated by submitochondrial particles indicate that l'.p is thermodynamically competent to serve as such a link provided that 3 H+ ions are translocated through the mitochondrial adenosine triphosphatase for each molecule of ATP synthesized (Sorgato et al., 1978). Although the thermodynamic competence of !'ip in submitochondrial particles has now been scrutinized in some detail (Azzone et al., 1978; Sorgato et al., 1978) rather less attention has been paid to two other-related matters: (1) the relationship between l'.p and the rate of proton translocation; (2) the relationship between l'.p and the rate of ATP synthesis. It was found (Sorgato et al., 1978) that the oxidation of either succinate or NADH by submitochondrial particles generated virtually the same 1'.p, despite the fact that, after correction for the slower rate of succinate oxidation, the rate of proton translocation was approximately 2-fold slower when succinate was the substrate. Decrease, by titration with malonate, of the rate of succinate oxidation to 18 % of its maximal value is now found to cause only a small decline in l'.p from approx. 140mV to approx. 135mV. [l'.p was determined in a reaction mixture of 10 mM-phosphate/Tris, 5 mM-magnesium acetate, pH 7.3, by using a flow dialysis assay for SCNuptake to estimate /11/f, the sole detectable component of l'.p under these reaction conditions (Sorgato et al., 1978).] These two sets of observations suggest either that the protic resistance of the inner mitochondrial membrane (at least in submitochondrial particles) is variable or that the membrane capacitance is strongly voltage-dependent. Thus as the rate of proton translocation is decreased there is a corresponding decrease in the rate at which protons are able to pass back across the membrane down their electrochemical gradient. This type of behaviour has also been suggested for rat liver mitochondria (Nicholls, 1974) and for chloroplast thylakoids (Schonfeld & Neumann, 1977), although it is noteworthy that with intact mitochondria a decline in l'.p was observed when the rate of succinate oxidation was decreased by only 40% (Nicholls, 1974). Titration of the rate ofNADH oxidation by submitochondrial particles with rotenone indicated that, except at the highest respiratory rates, there was an almost ohmic relationship between l'.p and the rate of proton translocation (Kell et al., 1978). This result appears to contrast markedly with that found when succinate is the substrate. The reasons for this difference remain to be elucidated, but it may be relevant to note that Nicholls ( 1977) has suggested that the second and third segments of the respiratory chain have the ability, for a given rate of electron transfer, to maintain l'.p at a higher value than that produced at the first segment between NADH and ubiquinone. A knowledge of the resistive (and capacitative) characteristics of the membrane is essential both for the relationship between oxidation and phosphorylation to be treated in terms of irreversible thermodynamics, and for the identification of l'.p as the deterdeterminant (or otherwise) of respiratory control (Boyer et al., 1977; Kupriyanov & Pobochin, 1978). The results in the present paper indicate that treatments (Hinkle et al.,