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I C West

Publications and source records attributed to I C West.

At least 37 records · Page 2Linked to original sources
PubMed1987

Protonmotive stoichiometry of rat liver cytochrome c oxidase: determination by a new rate/pulse method.

The stoichoimetry of vectorial H+ ejection coupled to electron flow through the cytochrome c oxidase (EC 1.9.3.1) of rat liver mitochondria was determined by a new rate/pulse method. This is a modification of the oxygen-pulse method. Electron flow through the oxidase is initiated by adding oxygen to suspensions of anaerobic mitochondria at a known and constant rate. Cytochrome c oxidase was examined directly or in combination with cytochrome c reductase (ubiquinol:ferricytochrome c oxidoreductase). In both cases the----H0+/2e- ratio was found to be constant during the time-course of oxygen reduction, and thus independent of delta pH. The stoichiometries observed were consistent with mechanistic stoichiometries of 2 and 6 for cytochrome c oxidase alone and cytochrome c oxidase together with cytochrome c reductase, respectively. The stoichiometry of cytochrome c reductase alone was also examined, by using ferricyanide in place of oxygen. The results obtained were consistent with the accepted mechanistic stoichiometry of 4 for this enzyme.

Animals↗
PubMed1986

Characterisation in vivo of the reactive thiol groups of the lactose permease from Escherichia coli and a mutant; exposure, reactivity and the effects of substrate binding.

The reactivity and accessibility of the reactive thiol groups of the native lactose permease and a mutant have been studied in a number of circumstances and with a number of reagents, in particular using the specific thiol-disulphide exchange reaction. Seven different reactive states of the thiol in the native protein have been characterised by their different second-order rate constants. Interconversion between these states is dependent on the magnitude of the protonmotive force, pH and substrate binding. In the absence of galactoside, reactivity is controlled by an ionisation with apparent pKa 9.3. This pKa is not affected by the protonmotive force, but it is lowered in the presence of external galactoside. The conformation adopted by the permease when in equilibrium with saturating galactoside appears to be different from that of the intermediate that accumulates during net turnover. In the former state, the reactivity of the thiol group is depressed, whereas in the latter state it is enhanced. The thiol group of the native protein is buried in a hydrophobic environment that has a dielectric constant considerably lower than that of water. The environment is not greatly perturbed by changes in the magnitude of the protonmotive force, but it is affected by the binding of galactoside. In a strain which carries the YUN mutation (Wilson, T.H. and Kusch, M. (1972) Biochim. Biophys. Acta 255, 786-797), two reactive thiols were characterised. The more reactive of the two is more exposed than the thiol group of the native molecule and is in an environment that has a dielectric constant close to that of water. The less reactive thiol appears to be more deeply buried than that of the native protein. Thus the mutation appears to produce a conformation change in the central portion of the polypeptide chain that results in greater exposure of the reactive thiol to the aqueous environment.

Biological Transport, Active↗
PubMed1986

Is there Ca2+(Sr2+)-3-hydroxybutyrate symport in rat-liver mitochondria? A reappraisal.

The observation in this laboratory that respiration and Sr2+ import were stimulated by the addition of 3-hydroxybutyrate to suspensions of N-ethylmaleimide-treated mitochondria respiring in state 6, after the addition of Sr2+, in a sucrose medium containing choline as substrate, led to the proposal by Moyle and Mitchell [(1977) FEBS Lett. 84, 135-140] that there is a Ca2+(Sr2+)-3-hydroxybutyrate symporter in rat liver mitochondria. However, experiments described in the present paper support a different interpretation. Under the conditions of the experiments by Moyle and Mitchell, the rate of respiration and the poise of Sr2+ accumulation are mainly limited, not by delta mu H+, but by lack of respiratory substrate. Even though N-ethylmaleimide is a potent inhibitor of 3-hydroxybutyrate dehydrogenase, we have found that, somewhat surprisingly, under the special conditions of these experiments, sufficient 3-hydroxybutyrate dehydrogenase activity remains available to account for the 3-hydroxybutyrate-dependent respiratory stimulation and Sr2+ import.

3-Hydroxybutyric Acid↗
PubMed1986

Proton translocation by cytochrome oxidase in (antimycin + myxothiazol)-treated rat liver mitochondria using ferrocyanide or hexammineruthenium as electron donor.

When O2 was injected into an anaerobic suspension of valinomycin-treated rat liver mitochondria inhibited with rotenone, antimycin, and myxothiazol, a small amount of O2 (0.23-0.33 ng-atom of O/mg of protein) was reduced extremely rapidly (within the 2 s time-resolution of the oxygen electrode). The subsequent steady-state rate of flow of electrons to oxygen was very low [less than 3 nequiv. X s-1 X (g of mitochondrial protein)-1]. In the presence of valinomycin there was a rapid ejection of protons synchronous with the rapid phase of O2 consumption corresponding to 0.38-0.61 nequiv. of H+ X (mg of mitochondrial protein)-1. When valinomycin was replaced by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) there was a rapid alkalification of the medium corresponding to 0.20-0.42 nequiv. of H+ X (mg of mitochondrial protein)-1. When 2 mM-Fe(CN)6(4-) was present to re-reduce endogenous cytochrome c, O2 consumption was still biphasic but the second phase of O2 consumption was very much more rapid [600 nequiv. X s-1 X (g of protein)-1], and resulted in the virtually complete consumption of the O2 in the pulse within 4 s. With 60 microM-Ru(NH3)6(2+) as reductant, O2 consumption was even faster [1200 nequiv. X s-1 X (g of protein)-1]. In a medium containing 150 mM-choline chloride with Ru(NH3)6(2+) as reductant, the proton per reducing equivalent stoichiometry (delta H+O/e-) was +0.95 in the presence of valinomycin and -0.94 in the presence of FCCP. In choline chloride medium containing Ru(NH3)6(2+) and valinomycin, there was an uptake of K+ ions corresponding to 1.86 K+/e-. It is concluded that nearly 1 proton is translocated outwards through cytochrome oxidase per oxidizing equivalent injected in this medium. In low ionic strength sucrose-based medium, with Ru(NH3)6(2+) as reductant, delta H+O/e- was 1.05 in the presence of valinomycin, and -0.71 in the presence of FCCP. It is concluded that the translocation of protons is accompanied by net acid production in this medium.

Animals↗
PubMed1986

Measurement of the proton-motive stoichiometry of the respiratory chain of rat liver mitochondria: the effect of N-ethylmaleimide.

The apparent proton-motive stoichiometry as measured by the oxygen-pulse technique in KCl medium is depressed by the rapid uptake of inorganic phosphate, unless endogenous phosphate is depleted or uptake is inhibited. In sucrose or choline chloride media, where the internal pH is more acid than in KCl media, uptake may be greatly diminished. In the absence of significant phosphate uptake, the observed stoichiometry of around 8, obtained with no added substrate or respiratory inhibitors, appears to be characteristic of NADH oxidation without significant participation of the proton-translocating NAD(P) transhydrogenase. A mechanistic stoichiometry of at least 8 is indicated.

Animals↗
PubMed1986

A theoretical analysis of the effect of phosphate on apparent H+/O stoichiometries in oxygen-pulse experiments with rat liver mitochondria.

It is now generally accepted that, in oxygen-pulse experiments on rat-liver mitochondria suspended in KCl-based media, the rapid import of H+ with phosphate leads to an approx. 33% lowering of apparent H+/O stoichiometry. However, in low-K+ media, N-ethylmaleimide has no effect on stoichiometry, and there appears to be no import of H+ with phosphate. In this paper the quantitative effect of extramitochondrial phosphate on apparent H+/O stoichiometry is calculated theoretically, on the basis of internal and external buffering powers. The lack of appreciable phosphate uptake in low-K+ media is quantitatively explained in terms of several factors, including the initial pH gradient and initial phosphate distribution.

Animals↗
PubMed1985

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.

Biological Transport↗
PubMed1985

Protection of respiration of a crude mitochondrial preparation in cerebral ischaemia by control of blood glucose.

There is evidence that systemic hyperglycaemia influences the evolution of cerebral ischaemia adversely, through enhancement of brain lactic acidosis. A potential protective regimen based on induction of mild hypoglycaemia with insulin was therefore studied. Respiratory control in brain mitochondria prepared from the ischaemic hemisphere of rats subjected to unilateral common carotid artery occlusion for one hour was abolished, but was partially preserved in insulin-treated rats (p = 0.01).

Animals↗
PubMed1984

The transient kinetics of uptake of galactosides into Escherichia coli.

The uptake of galactosides into Escherichia coli via the lactose permease was studied in the time range 0.01-10s by rapid mixing and quenched flow. An initial transient was observed under two conditions. Firstly, a lag in the approach to the steady state was observed at low galactoside concentrations (less than Km). Secondly, a burst of uptake was observed when anaerobic cell suspensions were mixed with aerobic substrate solutions. However, the cause of the burst of uptake appears to be a burst in the rate of respiration. The rate of galactoside uptake during this phase is 10-fold greater than during the steady state.

Antimetabolites↗
PubMed1984

When is the outer membrane of Escherichia coli rate-limiting for uptake of galactosides?

During inflow into Escherichia coli substrates must first diffuse through the porin pores in the outer membrane by simple, passive, diffusion and then be translocated across the inner membrane by a specific (active) carrier protein, or permease. A graphical procedure is outlined whereby it is possible to estimate the concentration drop across the outer membrane from simple kinetic measurements of net inflow velocity. Experiments confirm that the concentration drop across the outer membrane is proportional to rate of inflow, as expected from Fick's law. The expected rate of diffusion of 2-nitrophenylgalactoside through the outer membrane was calculated from reported values of pore radius, length and number, and the rate was found to correspond closely with the experimental results. It is pointed out that at low substrate concentrations the outer membrane is rate-limiting and that a large increase in the amount of permease in the inner membrane will cause very little change in net inflow velocity.

Cell Membrane↗
PubMed1981

The kinetics of the beta-galactoside-proton symport of Escherichia coli.

beta-Galactoside transport by Escherichia coli occurs with the concomitant uptake of a proton. The kinetics of beta-galactoside uptake at various values of external pH are interpreted in terms of a model in which both the galactoside and the proton are substrates of the transport reaction. The values of some of the kinetic constants for this two-substrate reaction were determined. The observed effects of the protonmotive force on the apparent Michaelis constant for galactoside can be explained in terms of the proton being a substrate of the transport reaction.

Biological Transport↗
PubMed1974

Proton/sodium ion antiport in Escherichia coli.

In anaerobic suspensions of Escherichia coli, after H(+) ions have been translocated outwards across the plasma membrane by a respiratory pulse, re-equilibration is catalysed by Na(+). The sudden addition of a Na(+) salt causes the effective outward translocation of H(+) by an electroneutral process. We conclude that the plasma membrane of E. coli contains a Na(+)/H(+) antiport system that normally translocates Na(+) outwards under the influence of an inwardly directed H(+)-activity gradient.

Biological Transport, Active↗