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M D Brand

Publications and source records attributed to M D Brand.

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

Thermodynamic and steady-state-kinetic investigation of the effect of NN'-dicyclohexylcarbodi-imide on H+ translocation by the mitochondrial cytochrome bc1 complex.

Steady-state kinetic measurements showed that NN'-dicyclohexylcarbodi-imide decreased the observed H+/2e ratio of H+ transport by mitochondria respiring on succinate, acting mainly at the cytochrome bc1 complex. Thermodynamic assessment of the H+/2e ratio by measuring the force ratio across the bc1 complex showed that the inhibitor did not affect H+ translocation. Possible explanations of this disagreement between methods are examined; we conclude that the inhibitor does not alter the mechanistic stoichiometry of H+ pumping by the bc1 complex.

Animals↗
PubMed1985

Electroneutral efflux of Ca2+ from liver mitochondria.

Respiring liver mitochondria were allowed to export Ca2+ on the endogenous Ca2+/nH+ antiporter in the presence of Ruthenium Red (to inhibit uptake on the Ca2+ uniporter) until a steady state was reached. Addition of sufficient of the ionophore A23187 (which catalyses Ca2+/2H+ exchange) to bring the Ca2+ and H+ gradients into equilibrium did not alter the steady state. Thermodynamic analysis showed that if a Ca2+/nH+ exchange with any value of n other than 2 was at equilibrium, addition of A23187 would have caused an easily measurable change in extramitochondrial free [Ca2+]. Therefore, the endogenous carrier of liver mitochondria catalyses electroneutral Ca2+/2H+ antiport.

Animals↗
PubMed1984

The intracellular calcium antagonist TMB-8 [8-(NN-diethylamino)octyl-3,4,5-trimethoxybenzoate] inhibits mitochondrial ATP production in rat thymocytes.

TMB-8 inhibited respiration of rat thymocytes and rat liver mitochondria, probably by inhibition of NADH dehydrogenase. TMB-8 markedly decreased both the cellular ATP concentration and the mitochondrial membrane potential in situ in thymocytes. These effects occurred at, or well below, the concentrations used in other systems to investigate the role of intracellular calcium pools in signalling events. We conclude that caution should be exercised in the interpretation of the effects of TMB-8.

Adenosine Triphosphate↗
PubMed1984

Target size analysis of rhodopsin in retinal rod disk membranes.

Radiation inactivation of rhodopsin in situ using high-energy electrons gave a value for Mr of 20,200 by spectral assay, but 47,100 by assay of rhodopsin regeneration from opsin and 11-cis-retinal (sequence Mr = 38,840). No light/dark differences were seen. We conclude: (a) radiation inactivation measures the size of the functional unit, and the single hit hypothesis does not hold in our experiments; (b) 500 nm absorbance requires only about half the rhodopsin molecule to be intact, but reconstitution of rhodopsin from opsin requires the whole molecule; (c) we find no evidence for functional interactions between rhodopsin monomers in darkness or light.

Animals↗
PubMed1984

Membrane potential of mitochondria in intact lymphocytes during early mitogenic stimulation.

The mitochondrial membrane potential (delta psi m) in intact lymphocytes was calculated by measuring the distribution of radiolabelled methyltriphenylphosphonium cation. The value obtained was 120 mV. The pH gradient across the mitochondrial membrane in situ (delta pH m) was estimated to be 73 mV (1.2 pH units). Thus the electrochemical gradient of protons was about 190 mV. Addition of the mitogen concanavalin A did not alter delta psi m, showing that, if movement of Ca2+ across the inner membrane of lymphocyte mitochondria occurs when concanavalin A is added, it is accompanied by charge-compensating ion movements.

Animals↗
PubMed1983

Mechanism of activation of pyruvate dehydrogenase by mitogens in pig lymphocytes.

The activity of pyruvate dehydrogenase in extracts of pig mesenteric lymphocytes was measured under different preincubation conditions. The mitogens concanavalin A and ionophore A23187 both increased pyruvate dehydrogenase activity. In both cases activation required extracellular Ca2+. Digitonin-permeabilized cells required 0.5 microM free Ca2+ for half-maximal activation of pyruvate dehydrogenase. The stimulation by concanavalin A in intact cells was probably not due to changes in effectors of pyruvate dehydrogenase kinase. This evidence suggests that activation of pyruvate dehydrogenase is by Ca2+ activation of pyruvate dehydrogenase phosphatase and supports the view that the cytoplasmic free [Ca2+] rises to something less than 1 microM on stimulation with mitogens.

Animals↗
PubMed1983

Chemical modification of the mitochondrial bc1 complex by N,N'-dicyclohexylcarbodiimide inhibits proton translocation.

We report here that N,N'-dicyclohexylcarbodiimide (DCCD) decreases the H/2e stoichiometry of the cytochrome bc1 complex from 3.8 +/- 0.2 (10) to 2.1 +/- 0.1 (8) but has only a minimal effect on the H/2e ratio of cytochrome oxidase under the relatively mild conditions used. The effect on the bc1 complex cannot be explained by uncoupling, by inhibition of electron transport or by selective mitochondrial damage. We conclude that DCCD is an inhibitor of proton translocation within the bc1 complex. There are three possible explanations of this effect: (a) DCCD could alter the pathway of electron flow, (b) DCCD could prevent one of the proton translocation reactions but not electron transport, (c) DCCD could prevent the conduction of the translocated proton to the external phase.

Animals↗
PubMed1983

Size changes of phosphodiesterase in bovine rod outer segments on illumination.

Light activates a 3',5'-cyclic GMP phosphodiesterase (PDE) in bovine retinal rod outer segments. The light is absorbed by rhodopsin situated in the disk membranes. PDE is a three-subunit peripheral protein on the disks and appears to be activated via a guanine nucleotide binding protein (G) in the presence of activated rhodopsin and GTP. Does the activation occur by collision coupling of G and PDE? We have studied the protein-protein interactions of PDE in situ in disk membranes by radiation inactivation. Irradiation of a protein with high-energy electrons leads to loss of activity in proportion to radiation dose and the molecular weight of the protein. We see no change in the size of PDE upon activation by light and 100 microM guanosine 5'-(beta, gamma-imidotriphosphate) (Gpp[NH]p) compared with PDE in dark with 260 microM GTP. Application of statistics to our data shows that a 27 000 change in molecular weight would be significant at the 95% level but that smaller changes would go undetected. The apparent molecular weight is 176 000 +/- 27 000 (mean +/- 95% confidence limit), in agreement with the size determined by polyacrylamide gel electrophoresis. Thus there appears to be either (i) no permanent change in PDE size on activation or (ii) a small change, undetectable by the technique, or (iii) an exchange of subunits such that no net change in molecular weight is seen.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1983

Early plasma-membrane-potential changes during stimulation of lymphocytes by concanavalin A.

1. We have monitored the plasma-membrane potential of lymphocytes by measuring the accumulation of the lipophilic cation methyltriphenylphosphonium (TPMP+) in the presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). 2. The mitogen concanavalin A causes a decrease in TPMP+ accumulation by pig lymphocytes corresponding to a 3 mV depolarization with 2 1/2 min. Concanavalin A does not alter 86Rb+ uptake in the first 30 min. 3. In contrast concanavalin A increased TPMP+ accumulation and the rate of Rb+ uptake in mouse thymocytes. This is consistent with a previous proposal that the mitogen induces a hyperpolarization of mouse thymocytes as a result of stimulation of a Ca2+-dependent K+ channel. 4. Studies with the calcium ionophore A23187 and quinine (an inhibitor of the Ca2+-dependent K+ channel) suggest that the channel is partially closed in mouse resting thymocytes but is almost fully active in pig resting cells. Thus concanavalin A hyperpolarizes mouse thymocytes by activating the Ca2+-dependent K+ channel but cannot do so in pig lymphocytes because the channel is already maximally activated. 5. The 3mV depolarization of pig cells cannot be explained by a decrease in electrogenic K+ permeability.

Animals↗
PubMed1983

Concanavalin A causes an increase in sodium permeability and intracellular sodium content of pig lymphocytes.

1. The 3mV depolarization of pig lymphocytes observed within 2 1/2 min of treatment with concanavalin A [Felber & Brand (1983) Biochem. J. 210, 885-891] is dependent on the presence of high extracellular [Na+]. 2. The concanavalin A-induced changes in membrane potential at high and low extracellular [Na+] are quantitatively explained by an increase in the electrogenic permeability coefficient for Na+ (PNa). This rises from a negligible value in resting cells to around 4% of the permeability coefficient for K+ or Cl- in stimulated cells. 3. Concanavalin A induces a 4mM increase in the Na+ content of pig lymphocytes. This increase in intracellular [Na+] is not due solely to stimulation of electrogenic Na+ influx resulting from the rise in PNa. 4. Thus concanavalin A stimulates both an electrogenic pathway for Na+ influx, resulting in a small depolarization of the plasma membrane, and a non-electrogenic Na+ influx pathway, resulting in a rise in intracellular [Na+].

Adenosine Triphosphatases↗
PubMed1982

Proton translocation by the mitochondrial cytochrome b-c1 complex is inhibited by NN'-dicyclohexylcarbodi-imide.

NN'-Dicyclohexylcarbodi-imide at low concentrations decreases the H+/2e ratio for rat liver mitochondria over the span succinate to oxygen from 5.9 +/- 0.3 (mean +/- S.E.M.) to 4.0 +/- 0.1 and for the cytochrome b-c1 complex from 3.8 +/- 0.2 to 1.9 +/- 0.1, but has little effect on the H+/2e ratio of cytochrome oxidase. The decrease in stoicheiometry is due, not to uncoupling or inhibition of electron transport, but to inhibition of proton translocation. NN'-Dicyclohexylcarbodi-imide thus 'decouples' proton translocation in the cytochrome b-c1 complex.

Animals↗
PubMed1982

Factors determining the plasma-membrane potential of lymphocytes.

1. Lymphocytes from pig mesenteric lymph node have low permeability to K+ (Rb+), Na+ and Cl-. None of these ions is in Nernst equilibrium with the plasma-membrane potential (delta psi p). 2. delta psi p can be calculated from the transmembrane distribution of the permeant cation methyltriphenylphosphonium (TPMP+) in the presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to abolish uptake into intracellular mitochondria. In normal culture medium delta psi p is 56 mV. 3. A similar potential is found in T-enriched pig cells and in mouse thymocytes. 4. The contribution of electrogenic (Na+ + K+)ATPase to delta psi p is about 7 mV. 5. The remainder of the lymphocyte delta psi p is a polyionic potential set up by K+ and Cl- with a permeability coefficient for Cl- of similar magnitude to that for K+.

Animals↗
PubMed1982

The isolation of lymphocyte mitochondria and their regulation of extramitochondrial free Ca2+ concentration.

1. A method for the isolation of functionally intact mitochondria from lymphocytes is described. It involves digitonin breakage of the plasma membrane, followed by differential centrifugation. The yield was 36 mg of mitochondrial protein/200 g of pig mesenteric lymph node (6 mg of mitochondrial protein/10(9) lymphocytes). The mitochondrial had a respiratory-control ratio of 2--3.5 with succinate as substrate. 2. Ca2+ transport by these mitochondria was investigated. They were able to regulate the extramitochondrial free [Ca2+] very precisely, by buffering any displacements from the steady-state. The exact extramitochondrial free [Ca2+] of this steady-state depended on the conditions of incubation. In a medium designed to resemble the cytoplasmic environment, with added Ca2+, lymphocyte mitochondria maintained a steady-state free [Ca2+] of 0.63 microM (pCa of 6.2). The rates of Ca2+ uptake and efflux under these conditions, with both lymphocyte and liver mitochondria, were very much lower than those in a less complex medium. 3. Lymphocyte mitochondria were shown to possess an Na+-independent Ruthenium Red-insensitive efflux pathway similar to that of liver mitochondria. Ruthenium Red totally inhibited the electrophoretic uniporter. Although Na+ had no effect on the steady-state maintained by lymphocyte mitochondria, they were shown to possess an Na+/H+ antiporter.

Animals↗
PubMed1982

Oxaloacetate- and acetoacetate-induced calcium efflux from mitochondria occurs by reversal of the uptake pathway.

1. Addition of oxaloacetate or acetoacetate to isolated rat liver mitochondria results in an efflux of Ca2+. Concomitant with this efflux is an immediate oxidation of endogenous nicotinamide nucleotides, a fall in the mitochondrial membrane potential and an increase in the rate of respiration. The primary effect in this sequence may be either (a) physiologically important stimulation of a Ca2+-efflux carrier, followed by Ca2+ re-uptake, a fall in membrane potential and increased respiration, or (b) physiologically unimportant damage to mitochondrial integrity, followed by a fall in membrane potential, increased respiration and Ca2+ efflux. 2. Ruthenium Red and EGTA will restore the increased respiratory rate to one approximating to the control rate of respiration. However, addition of lanthanide, at a concentration which inhibits the uptake but not the normal efflux of Ca2+, inhibits the rate of Ca2+ efflux induced by oxaloacetate or acetoacetate. Therefore the observed efflux is occurring by a reversal of the uptake pathway (uniporter) and thus follows the fall in membrane potential. 3. From these results we conclude that the decrease in membrane potential and increase in the rate of respiration seen during oxaloacetate- or acetoacetate-induced Ca2+ efflux cannot be accounted for by rapid Ca2+ cycling, but are due to damage to mitochondrial integrity.

Acetoacetates↗
PubMed1981

Steady-state H+/O stoichiometry of liver mitochondria.

We have measured the H+/O stoichiometry of rat liver mitochondria respiring in a steady-state, using a novel method. This involves measuring the initial rate of H+ back-flow into mitochondria after respiratory inhibition, with the assumption that this is equal to the steady-state H+-ejection rate. Division by the steady-state O2-consumption rate yields the H+/O ratio. The H+/O values obtained were: 8.3 +/- 1.0 (mean +/- S.E.M.) for 3-hydroxybutyrate: 8.2 +/- 0.7 for glutamate plus malate; 6.0 +/- 0.2 for succinate; 4.1 +/- 0.3 for ascorbate/tetramethylphenylenediamine and 3.0 +/- 0.1 for ascorbate/ferrocyanide. These values correspond to H+/O stoichiometries for electron flow to oxygen from NAD+-linked substrates, succinate and cytochrome c of 8, 6 and 2 (charge/O ratio = 4) respectively.

Animals↗
PubMed1981

The effect of insulin on plasma-membrane and mitochondrial-membrane potentials in isolated fat-cells.

1. A recently developed technique for the measurement of plasma-membrane and mitochondrial-membrane potentials in intact cells by using the distribution of 86Rb+ and [3H]methyltriphenylphosphonium+ has enabled us to characterize a novel insulin effect on fat-cell mitochondria. For control cells the plasma-membrane and mitochondrial-membrane potentials were 75 mV and 152 mV respectively. Insulin (10 mu units/ml) caused a 9 mV hyperpolarization of the plasma membrane and a 19 mV depolarization of the mitochondrial membrane. 2. The insulin-dependent mitochondrial depolarization was observed at physiological insulin concentrations (10 mu units/ml) and was apparent when the cells metabolized a wide variety of substrates. 3. Evidence from the uptake of the weak acid 5,5-dimethyloxazolidine-2,4-dione by fat-cells was interpreted as indicating that the mitochondrial pH gradient was increased by insulin. 4. Insulin alters the balance between the electrical and pH-gradient components that form the mitochondrial protonmotive force. A model is proposed.

Adipose Tissue↗
PubMed1980

The nature of the calcium ion efflux induced in rat liver mitochondria by the oxidation of endogenous nicotinamide nucleotides.

Ca2+ efflux from rat liver mitochondria can occur when endogenous nicotinamide nucleotides are oxidized. It is suggested that nicotinamide nucleotide induced by acetoacetate sensitizes the mitochondria to damaage resulting from the accumulation of Ca2+ in the presence of Pi. Thus, acetoacetate-induced Ca2+ efflux is associated with a loss of respiratory control. Both the effluxes induced by acetoacetate and by high Ca2+ accumulation are prevented by ATP plus oligomycin, although these agents do not prevent the endoagenous nicotinamide nucleotides from becoming oxidized on addition of acetoacetate. Acetoacetate addition only results in Ca2+ release if the Ca2+ and Pi concentration are above a critical value. The acetoacetate-induced Ca2+ effflux is exactly paralled by the virtually complete collapse of the membrane potential. The presence of acetoacetate decreases the concentration of total Ca2+ necessary to induced mitochondrial damage by about 130 nmol of Ca2+/mg of protein. It is concluded that acetoacetate-induced efflux occurs by reversal of the Ca2+ uniporter after the collapse of the membrane potential.

Acetoacetates↗