John M. Wrigglesworth

Uncoupling of intestinal mitochondrial oxidative phosphorylation and inhibition of cyclooxygenase are required for the development of NSAID-enteropathy in the rat

The pathogenesis of NSAID‐induced gastrointestinal damage is believed to involve a nonprostaglandin dependent effect as well as prostaglandin dependent effects. One suggestion is that the nonprostaglandin mechanism involves uncoupling of mitochondrial oxidative phosphorylation.

Chemiosmotic coupling in cytochrome oxidase. Possible protonmotive O loop and O cycle mechanisms.

Using the principle of specific vectorial ligand conduction, we outline directly coupled protonmotive O loop and O cycle mechanisms of cytochrome oxidase action that are analogous to protonmotive Q loop and Q cycle mechanisms of QH2 dehydrogenase action. We discuss these directly coupled mechanisms in the light of available experimental knowledge, and suggest that they may stimulate useful new research initiatives designed to elucidate the osmochemistry of protonmotive oxygen reduction in cytochrome oxidase.

A study of the effects of indometacin on liver mitochondria from rats, mice and humans.

A part of the mechanism of the gastrointestinal toxicity exhibited by non‐steroidal anti‐inflammatory drugs is believed to involve the uncoupling of mitochondrial oxidative phosphorylation. Most previous uncoupling studies have used rat liver mitochondria. There is little information on the effects of the drugs on mitochondria from other species.

Oxidised low density lipoproteins induce iron release from activated myoglobin

Recent reports have detected the presence of iron in human atherosclerotic lesions [Biochem. J. 286 (1992) 901–905]. This study provides evidence for a biochemical mechanism whereby iron is released from myoglobin by low density lipoprotein (LDL) which has become oxidised by the ferryl myoglobin species. The haem destabilisation and iron release are inhibited by monohydroxamate compounds and desferrioxamine through their ability to inhibit the propagation of LDL oxidation. Thus, iron may derive from the myoglobin released from ruptured cells in the oxidising environment of the atherosclerotic lesion.

Antimycin inhibition of the cytochrome bd complex from Azotobacter vinelandii indicates the presence of a branched electron transfer pathway for the oxidation of ubiquinol

Antimycin A and UHBDT inhibit the activity of the purified cytochrome bd complex from Azotobacter vinelandii. Inhibition of activity is non‐competitive and antimycin A binding induces a shift to the red in the spectrum of a b‐type haem. No inhibitory effects were seen with myxothiazol. Steady‐state experiments indicate that the site of inhibition for antimycin A lies on the low‐potential side of haem b 558. In the presence of antimycin A at concentrations sufficient to inhibit respiration, some direct electron transfer from ubiquinol‐1 to haem b 595 and haem d still occurs. The results are consistent with a branched electron transfer pathway from ubiquinol to the oxygen reduction site.

Activation by reduction of the resting form of cytochrome c oxidase: tests of different models and evidence for the involvement of CuB

(1) The reaction of the resting form of oxidised cytochrome c oxidase from ox heart with dithionite has been studied in the presence and absence of cyanide. In both cases, cytochrome a reduction in 0.1 M phosphate (pH 7) occurs at a rate of 8.2.10(4) M-1.s-1. In the absence of cyanide, ferrocytochrome a3 appears at a rate (kobs) of 0.016 s-1. Ferricytochrome a3 maintains its 418 nm Soret maximum until reduced. The rate of a3 reduction is independent of dithionite concentration over a range 0.9 mM-131 mM. In the presence or cyanide, visible and EPR spectral changes indicate the formation of a ferric a3/cyanide complex occurs at the same rate as a3 reduction in the absence of cyanide. A g = 3.6 signal appears at the same time as the decay of a g = 6 signal. No EPR signals which could be attributed to copper in any significant amounts could be detected after dithionite addition, either in the presence or absence of cyanide. (2) Addition of dithionite to cytochrome oxidase at various times following induction of turnover with ascorbate/TMPD, results in a biphasic reduction of cytochrome a3 with an increasing proportion of the fast phase of reduction occurring after longer turnover times. At the same time, the predominant steady state species of ferri-cytochrome a3 shifts from high to low spin and the steady-state level of reduction of cytochrome a drops indicating a shift in population of the enzyme molecules to a species with fast turnover. In the final activated form, oxygen is not required for fast internal electron transfer to cytochrome a3. In addition, oxygen does not induce further electron uptake in samples of resting cytochrome oxidase reduced under anaerobic conditions in the presence of cyanide. Both findings are contrary to predictions of certain O-loop types of mechanism for proton translocation. (3) A measurement of electron entry into the resting form of cytochrome oxidase in the presence of cyanide, using TMPD or cytochrome c under anaerobic conditions, shows that three electrons per oxidase enter below a redox potential of around +200 mV. An initial fast entry of two electrons is followed by a slow (kobs approximately 0.02 s) entry of a third electron.(ABSTRACT TRUNCATED AT 400 WORDS)

Transport of K+ and other cations across phospholipid membranes by nonesterified fatty acids

The rate of change of internal pH and transmembrane potential has been monitored in liposomes following the external addition of various cation salts. Oleic acid increases the transmembrane movement of H+ following the imposition of a K+ gradient. An initial fast change in internal pH is seen followed by a slower rate of alkalinization. High concentrations of the fatty acid enhance the rate comparable to that seen in the presence of nigericin in contrast to the effect of FCCP (carbonyl cyanide p-(tri-fluoromethoxy)phenyl hydrazone) which saturates at an intermediate value. The ability of nonesterified fatty acids to catalyze the movement of cations across the liposome membrane increases with the degree of unsaturation and decreases with increasing chain length. Li and Na salts cause a similar initial fast pH change but have less effect on the subsequent slower rate. Similarly, the main effect of divalent cation salts is on the initial fast change. The membrane potential can enhance or inhibit cation transport depending on its polarity with respect to the cation gradient. It is concluded that nonesterified fatty acids have the capability to complex with, and transport, a variety of cations across phospholipid bilayers. However, they do not act simply as proton/cation exchangers analogous to nigericin nor as protonophores analogous to FCCP. The full cycle of ionophoric action involves a combination of both functions.

The mechanism of potassium movement across the liposomal membrane

Addition of potassium to sodium-loaded asolectin liposomes induces an internal alkalinization even in the absence of ionophores. Most of the K+ entry is electrogenic, as shown by fluorescent changes in the potential-sensitive probe Oxonol V. The major part of the proton efflux observed must therefore be electrophoretic. However, in the presence of high concentrations of membrane permeable n-butyltriphenylphosphonium, potassium addition induces a residual alkalinization under conditions where no membrane potential can be observed with Oxonol V. This suggests that liposomes also catalyze direct electroneutral K+/H+ exchange, as has been theoretically predicted for cytochrome oxidase proteoliposomes (Wrigglesworth, J.M., Cooper, C.E., Sharpe, M.A. and Nicholls, P. (1990) Biochem. J. 270, 109-118). Free fatty acids present in the soybean phospholipid mixture may be responsible for such activity.

Mitochondrial function and modification of nsaid carboxyl moiety

It is widely believed that the toxic effects of non-steroidal anti-inflammatory drugs are solely due to inhibition of cyclo-oxygenase. However, this view is no longer tenable as it is possible to inhibit cyclo-oxygenase activities without mucosal injury and cyclo-oxygenase 1-deficient mice do not have gastrointestinal lesions from indomethacin. Alternatively, non-steroidal anti-inflammatory agents may initiate damage by direct mitochondrial toxicity. The current experiments were designed to relate NSAID uncoupling of mitochondrial oxidative phosphorylation to drug pK a and to determine the effect of modification of the NSAID carboxyl group.

Estimation of lipid regions in a cytochrome oxidase-lipid complex using spin labeling electron spin resonance: Distribution effects on the spin label

The distribution of lipid in the cytochrome oxidase-lipid complex from beef heart mitochondria has been studied by the spin labeling electron spin resonance technique. The spectra of a phospholipid spin label incorporated in the complex reveals an immobilized (on the ESR time scale) component in addition to the fluid component which is found in aqueous dispersions of the extracted lipids. The first component corresponds to the domain of lipid influenced by the protein, and the second component to the remaining lipid. A theory taking into account not only the sizes of the lipid regions in which the spin label molecule distributes itself, but also the different affinities of the label for the two domains, has been developed. Taking advantage of the variation in spectra obtained with increasing amounts of spin label, computer calculations have been performed to estimate the distribution of lipid in the different regions of the cytochrome oxidase-lipid complex. An extrapolation of the amount of immobilized spin-labeled phospholipid to zero concentration of label allows a calculation of the number of fatty acid residues interacting with the protein to be made. It has been found that the number of aliphatic chains influenced by the protein is higher than that calculated for a single boundary layer around the protein. The approach used in this paper can be useful for studies of protein-lipid interactions in other systems.