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M D Brand

Publications and source records attributed to M D Brand.

At least 73 records · Page 4Linked to original sources
PubMed1995

A hierarchy of ATP-consuming processes in mammalian cells.

The rates of different ATP-consuming reactions were measured in concanavalin A-stimulated thymocytes, a model system in which more than 80% of the ATP consumption can be accounted for. There was a clear hierarchy of the responses of different energy-consuming reactions to changes in energy supply: pathways of macromolecule biosynthesis (protein synthesis and RNA/DNA synthesis) were most sensitive to energy supply, followed by sodium cycling and then calcium cycling across the plasma membrane. Mitochondrial proton leak was the least sensitive to energy supply. Control analysis was used to quantify the relative control over ATP production exerted by the individual groups of ATP-consuming reactions. Control was widely shared; no block of reactions had more than one-third of the control. A fuller control analysis showed that there appeared to be a hierarchy of control over the flux through ATP: protein synthesis > RNA/DNA synthesis and substrate oxidation > Na+ cycling and Ca2+ cycling > other ATP consumers and mitochondrial proton leak. Control analysis also indicated that there was significant control over the rates of individual ATP consumers by energy supply. Each ATP consumer had strong control over its own rate but very little control over the rates of the other ATP consumers.

Adenosine Triphosphate↗
PubMed1995

Mitochondrial proton conductance and H+/O ratio are independent of electron transport rate in isolated hepatocytes.

In this paper we examine the non-linearity of the relationship between the proton electrochemical gradient across the mitochondrial inner membrane (delta p) and oxygen consumption of non-phosphorylating mitochondria in situ in hepatocytes. Models proposing to explain the non-linear relationship were tested experimentally. It was shown that the mitochondrial proton conductance and the number of protons pumped to the cytosolic side of the mitochondrial inner membrane by the electron transport complexes per oxygen atom consumed (H+/O ratio) are independent of electron transport rate in mitochondria in isolated hepatocytes. The non-linearity of the plot of delta p against the non-phosphorylating oxygen consumption is due to either a potential-dependent slippage of the proton pumps of the mitochondrial inner membrane and/or a potential-dependent leakage of protons back across the mitochondrial inner membrane.

Animals↗
PubMed1995

Top-down control analysis of systems with more than one common intermediate.

The analysis of the control of complex metabolic systems can be greatly simplified by application of the top-down approach of metabolic control analysis, in which the reactions of the system are grouped together into a small number of blocks connected by a common intermediate. The experimental application of the top-down approach has so far been limited to systems that have only a single intermediate. In this study, we demonstrate that the connectivity and summation theorems of metabolic control analysis hold with any number of intermediates between the metabolic blocks, and in doing so show that top-down analysis is valid for systems with multiple intermediates and so can be applied to most metabolic systems regardless of their complexity; an example of such an application is provided. Top-down control analysis has successfully described the control of mitochondrial respiration by dividing the system into three blocks, the respiratory chain, phosphorylation system and proton leak, all linked by a single intermediate, proton motive force. Here, we subdivide the respiratory chain into succinate consumers and cytochrome oxidase so that a second intermediate, cytochrome c redox state, is generated. Despite the fact that the redox state of cytochrome c is not measured, we solve the control over the system fluxes. In common with previous studies, we find that under conditions where there is no ATP turnover (state 4), respiration is largely controlled by proton leak, while at maximal ATP turnover (state 3) respiration is controlled by the respiratory chain and the phosphorylating system. In state 4,85% of the control by the respiratory chain resides with cytochrome oxidase. As ATP turnover increases, the respiration rate increases, and the control by the respiratory chain shifts from cytochrome oxidase to the succinate consumers, so that in state 3 83% of the control by the respiratory chain lies in the reactions between succinate and cytochrome c and only 17% resides with cytochrome oxidase.

Animals↗
PubMed1995

Proportional activation coefficients during stimulation of oxidative phosphorylation by lactate and pyruvate or by vasopressin.

A 'proportional activation' approach designed to deal with the influence of external effectors within biochemical systems is described. The proportional activation coefficient, which enables the quantitative determination of the relative stimulation of different parts of a system by a given effector, is defined. The proportional activation approach was used to calculate the relative activation of delta p-producing and delta p-consuming subsystems during stimulation of the respiration rate of cells by a variety of different effectors. Oxidative phosphorylation was stimulated by the addition of either lactate and pyruvate (10 mM and 1 mM) or vasopressin. The addition of lactate and pyruvate to suspensions of resting hepatocytes increased the respiration rate by about 50%. The delta p-consuming subsystem was stimulated about 60% as much as the delta p-producing subsystem. Quinolinic acid, commonly considered to be a specific inhibitor of gluconeogenesis, was found to block the delta p-producing oxidative subsystem as well as the delta p-consuming subsystem, indicating some nonspecific effects of this inhibitor. Addition of vasopressin to hepatocytes that were incubated in the presence of lactate and pyruvate resulted in an increase of the respiratory rate by up to 35%. The relative stimulation of the delta p-producing and delta p-consuming subsystems was essentially equal. Using the 'proportional activation approach' to analyse these and previously published data, it is shown that substrates (lactate/pyruvate and fatty acids), Ca(2+)-acting hormones (vasopressin and others) and calcium in muscles (heart muscle and skeletal muscle) activate both subsystems to a similar extent (it concerns especially Ca(2+)-dependent systems).

Animals↗
PubMed1995

Cellular oxygen consumption depends on body mass.

Hepatocytes were isolated from nine species of mammal of different body mass (and standard metabolic rate). The cells were incubated under identical conditions and oxygen consumption measured. The rate of oxygen consumption (per unit mass of cells) scaled with body mass with exponent -0.18. In general, there was a 5.5-fold decrease in oxygen consumption rate with a 12,500-fold increase in body mass. The decrease in oxygen consumption rate was not due to an increase in cell volume with increasing body mass but to a decrease in intrinsic metabolic activity of the cells. This novel finding confirms and explains the decrease in oxygen consumption rate measured in tissue slices from larger mammals by H. A. Krebs (Biochim. Biophys. Acta 4: 249-269, 1950) and recently by P. Couture and A. J. Hulbert [Am. J. Physiol. 268 (Regulatory Integrative Comp. Physiol. 37): R641-R650, 1995].

Animals↗
PubMed1995

Causes of differences in respiration rate of hepatocytes from mammals of different body mass.

Resting O2 consumption of hepatocytes isolated from mammals ranging in mass from 20-g mice to 200-kg horses decreases with increasing body mass. The substrate oxidation system increases in activity with increasing body mass and mitochondrial proton leak and phosphorylation system decrease in activity, resulting in a higher mitochondrial membrane potential in hepatocytes from larger mammals. The absolute rates of O2 consumption due to nonmitochondrial processes, substrate oxidation, mitochondrial proton leak, and the phosphorylation system decrease with increasing body mass. These decreases are due partly to a decrease in mitochondrial number per cell and partly to decrease in mitochondrial inner membrane proton leakiness and in ATP turnover by cells from larger mammals. Quantitatively, the proportion of total cell O2 consumption by nonmitochondrial processes (13%) and oxidation of substrates (87%) and the proportions used to drive mitochondrial proton leak (19%) and the phosphorylation system (68%) are the same for hepatocytes from all mammals investigated. The effect of matched decreases in the rates of proton leak and of ATP turnover is to keep the effective amount of ATP synthesized per unit of O2 consumed relatively constant with body mass, suggesting that the observed value is optimal.

Adenosine Triphosphate↗
PubMed1995

Use of top-down elasticity analysis to identify sites of thyroid hormone-induced thermogenesis.

Top-down elasticity analysis is a novel extension of top-down metabolic control analysis. It has provided researchers with a theoretical and practical platform upon which quantitative analyses of the sites of action of hormones and drugs can be based. This approach is easy to apply and involves dividing up the metabolic system in question into two or three blocks of enzyme reactions around an intermediate between the blocks of reactions. The kinetic response of each block to the intermediate is measured in the steady state in situ by determining the flux through the block at different measured intermediate concentrations. The intermediate can be manipulated by titrating the other blocks with suitable inhibitors or activators. Then, to determine which blocks of reactions are quantitatively the most important in terms of any change in the flux rate of the system, a quantitative comparison of the titration curves from the experimental preparations is made with those of the control preparations. In this minireview we will examine, as an example, the use of top-down elasticity analysis for the quantitative identification of the important sites of action of thyroid hormones on oxidative phosphorylation in hepatocytes. The experimental results show that approximately 50% of the change in resting oxygen consumption in hepatocytes from hypothyroid and hyperthyroid rats (compared with euthyroid controls) is attributable to changes in the rate of the mitochondrial proton leak; the remaining 50% is accounted for by changes in nonmitochondrial- and ATP turnover-dependent oxygen consumption in hypothyroid and hyperthyroid hepatocytes, respectively.

Adenosine Triphosphate↗
PubMed1994

Characteristics of mitochondrial proton leak and control of oxidative phosphorylation in the major oxygen-consuming tissues of the rat.

Maintenance of an electrochemical proton gradient across the mitochondrial inner membrane against the significant proton permeability of the membrane accounts for 25-30% of resting oxygen consumption in hepatocytes. It has been proposed that proton leak could be a significant contributor to resting metabolic rate in mammals if it were present in other tissues. Mitochondria were isolated from the major oxygen-consuming tissues (liver, kidney, brain and skeletal muscle) of the rat. In each tissue, the mitochondria showed significant proton leak with the same characteristic non-linear dependence on membrane potential. Liver and kidney mitochondria showed similar membrane proton permeability per mg of mitochondrial protein; brain and muscle permeabilities were greater when expressed in this way. Differences in the kinetic response of the substrate oxidation and phosphorylating systems to membrane potential were observed. The substrate oxidation system was more active in kidney, brain and skeletal muscle mitochondria than in liver mitochondria per mg of mitochondrial protein. Liver and kidney phosphorylating systems were less active than brain and skeletal muscle per mg of mitochondrial protein. The control of oxidative phosphorylation was also assessed. The distribution of control in mitochondria isolated from the four tissue types was found to be similar.

Animals↗
PubMed1994

The sum of flux control coefficients in the electron-transport chain of mitochondria.

The sum of the flux control coefficients for group-transfer reactions such as electron transport has been proposed to be two when the coefficients are calculated from experiments in which the concentrations of the electron carriers are changed (CE) but one when they are calculated from changes in the rates of the electron-transfer processes (Cv). We tested this proposal using electron transport in uncoupled beef heart, potato tuber and rat liver mitochondria. First, with ascorbate plus N,N,N',N"-tetramethyl-p-phenylenediamine as substrate, the CE flux control coefficients of ascorbate, N,N,N',N"-tetramethyl-p-phenylenediamine, mitochondria and oxygen over electron-transport rate were measured by direct titration of the concentrations of these electron carriers. CE values were close to zero, one, one and zero, respectively, giving a sum of CE flux control coefficients of approximately two. At higher concentrations of N,N,N',N'-tetramethyl-p-phenylenediamine, its CE control decreased and the sum decreased towards one as predicted. Secondly, the Cv control coefficients of groups of electron-transfer processes with succinate or ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine as substrate were measured. This was achieved by measuring the effects of KCN (or malonate or N,N,N',N'-tetramethyl-p-phenylenediamine) on system flux when intermediates were allowed to relax and on local flux when intermediates were held constant. The Cv flux control coefficients were calculated as the ratio of the effects on system flux and on local flux. The sum of the Cv flux control coefficients was approximately one. Whether a sum of one or a sum of two was obtained depended entirely on the definition of control coefficients that was used, since either sum was obtained from the same set of data depending on the method of calculation. Both definitions are valid, but they give different information. It is important to be aware of which definition is being used when analysing control coefficients in electron-transport chains and other group-transfer systems.

Animals↗
PubMed1994

Localisation of the sites of action of cadmium on oxidative phosphorylation in potato tuber mitochondria using top-down elasticity analysis.

The aim of this study was to identify the significant sites of action of cadmium on oxidative phosphorylation in potato tuber mitocondria. We simplified the system to three convenient subsystems linked via the production or consumption of a common intermediate, namely protonmotive force. The three subsystems were substrate oxidation, which produces protonmotive force, and the proton leak reactions and the phosphorylation reactions, which consume protonmotive force. By measuring the effect of cadmium on the kinetic response of each subsystem to protonmotive force (top-down elasticity analysis), we found that cadmium stimulated proton leak reactions and strongly inhibited substrate oxidation, but had no measurable effect on the phosphorylation reactions. Cadmium therefore decreases the amount of ATP produced/oxygen consumed (the effective P/O ratio) not by inhibiting the phosphorylation reactions directly, but by inhibiting the production of protonmotive force and by diverting proton flux from phosphorylation reactions to the proton leak reactions.

Adenosine Triphosphate↗
PubMed1994

Effects of cadmium on the control and internal regulation of oxidative phosphorylation in potato tuber mitochondria.

The effect of cadmium on the distribution of control over oxidative phosphorylation in potato tuber mitochondria was quantified by measuring control coefficients using top-down metabolic control analysis. Oxidative phosphorylation was divided into three subsystems, namely substrate oxidation, the phosphorylation reactions and the proton leak. The control exerted by each of these subsystems over the system fluxes, the value of the protonmotive force and the effective P/O ratio was quantified in the presence of different concentrations of free cadmium (up to 21 microM). Cadmium is known to stimulate the proton leak and inhibit the substrate oxidation reactions, but it had little effect on the distribution of control over the system variables except to shift the pattern to lower rates. Control exerted by particular subsystems appeared to change or to stay the same as cadmium was varied, depending on whether the control coefficients were presented as a function of respiration rate or protonmotive force. The regulatory strength of protonmotive force on the system variables was also calculated, as partial internal response coefficients. These coefficients changed with ATP turnover rate and with cadmium concentration, showing how the internal regulation of oxidative phosphorylation shifts under different conditions. The values of control coefficients and partial internal response coefficients show where control lies and how intermediates regulate the system variables under different conditions of ATP demand and external effector (i.e. cadmium) concentration. However, they are not useful for identifying the sites of action of external effectors, for which elasticity and regulation analysis must be used.

Cadmium↗
PubMed1994

Quantitative determination of the regulation of oxidative phosphorylation by cadmium in potato tuber mitochondria.

The effects of cadmium on respiration rate, phosphorylation rate, proton leak rate, the protonmotive force and the effective P/O ratio were determined over a range of respiratory conditions and cadmium concentrations by applying top-down regulation analysis. To quantify the effects of cadmium, we determined the overall response coefficients of these variables of oxidative phosphorylation to cadmium in different respiratory states between state 4 and state 3 and at different cadmium concentrations. The overall response coefficients to cadmium showed quantitatively how cadmium stimulated substrate oxidation rate at high cadmium concentrations near state 4 but inhibited it to different extents under all other conditions, how cadmium inhibited the rate of proton leak rate at low cadmium concentrations near state 4 but stimulated it under all other conditions, and how cadmium inhibited the rate of phosphorylation and depressed the protonmotive force and the effective P/O ratio to different extents under all conditions. Cadmium is known to stimulate the proton leak and to inhibit the substrate oxidation reactions; we calculated the elasticities of these subsystems to cadmium to quantify its effects. To describe fully how the cadmium effects on different subsystems produce the overall responses of the system to cadmium, we then calculated the partial response coefficients of the system variables to cadmium acting through each subsystem. The partial response coefficients quantify the contribution of each block to the overall effect of cadmium on each variable in any condition, and sum to the overall response coefficient in each condition. Together with the elasticity analysis and the control analysis and internal regulation analysis presented in the preceding papers [Kesseler, A. & Brand, M. D. (1994) Eur. J. Biochem. 225, pp. 897-906; Kesseler, A. & Brand, M. D. (1994) Eur. J. Biochem. 225, pp. 907-922] they completely describe how cadmium exerts its effects on oxidative phosphorylation at the system level.

Cadmium↗
PubMed1994

The causes and functions of mitochondrial proton leak.

The non-linear relationship between respiration rate and protonmotive force in isolated mitochondria is explained entirely by delta p-dependent changes in the proton conductance of the mitochondrial inner membrane and is not caused by redox slip in the proton pumps. Mitochondrial proton leak occurs in intact cells and tissues: the futile cycle of proton pumping and proton leak accounts for 26% +/- 7% of the total oxygen consumption rate or 33% +/- 7% of the mitochondrial respiration rate of isolated hepatocytes (mean +/- S.D. for 43 rats); 52% of the oxygen consumption rate of resting perfused muscle and up to 38% of the basal metabolic rate of a rat, suggesting that heat production may be an important function in the proton leak in homeotherms. Together with non-mitochondrial oxygen consumption, it lowers the effective P/O ratio in cells from maximum possible values of 2.33 (palmitate oxidation) or 2.58 (glucose oxidation) to as low as 1.1 in liver or 0.8 in muscle. The effective P/O ratio increases in response to ATP demand; the ability to allow rapid switching of flux from leak to ATP turnover may be an even more important function of the leak reaction than heat production. The mitochondrial proton conductance in isolated mitochondria and in hepatocytes is greatly modulated by thyroid hormones, by phylogeny and by body mass. Usually the reactions of ATP turnover change in parallel so that the coupling ratio is not greatly affected. Changes in proton leak in tissues are brought about in the short term by changes in mitochondrial protonmotive force and in the longer term by changes in the surface area and proton permeability of the mitochondrial inner membrane. Permeability changes are probably caused by changes in the fatty acid composition of the membrane phospholipids.

Adenosine Triphosphate↗
PubMed1994

The effects of methylprednisolone on oxidative phosphorylation in Concanavalin-A-stimulated thymocytes. Top-down elasticity analysis and control analysis.

The glucocorticoid methylprednisolone has clinically important anti-inflammatory effects at high concentrations through unknown mechanisms. Methylprednisolone at 0.2 mg/10(7) cells inhibits respiration in Concanavalin-A(ConA)-stimulated thymocytes from rats by about 20%. We have used top-down elasticity analysis to identify the blocks of reactions within oxidative phosphorylation in thymocytes whose kinetics are significantly affected by treatment with methylprednisolone. At this concentration methylprednisolone greatly inhibited the reactions of substrate oxidation and increased mitochondrial proton leak but did not significantly affect the synthesis and and turnover of ATP by the phosphorylating system. Metabolic control analysis showed that oxygen consumption by ConA-treated thymocytes was controlled largely (0.51) by the phosphorylating system but also by proton leak (0.32) and substrate oxidation (0.17); this is similar to the distribution of control in hepatocytes, suggesting that this pattern may be general in cells. Methylprednisolone lowered control by the phosphorylating system to 0.26 and raised control by substrate oxidation to 0.37. From these results we conclude that the inhibition of respiration in ConA-stimulated thymocytes by methylprednisolone at this concentration results from an inhibition of substrate oxidation and a smaller stimulation of mitochondrial proton leak, with only a minor contribution of any effects within the phosphorylating system.

Adenosine Triphosphate↗
PubMed1994

Experimental discrimination between proton leak and redox slip during mitochondrial electron transport.

By measuring the relationship between protonmotive force and the increment in oxygen consumption by mitochondria treated with submaximal amounts of uncoupler, we have experimentally tested four different models of imperfect coupling of oxidative phosphorylation. The results show that the increased rate of oxygen consumption at high protonmotive force is explained entirely by the dependence on protonmotive force of the passive proton leak conductance of the mitochondrial inner membrane. There is no measurable contribution from redox-slip reactions in the proton pumps caused by high protonmotive force. Neither is there any contribution from increased proton conductance of the membrane or increased redox slip in the respiratory chain caused by high turnover rates of the complexes.

Animals↗
PubMed1994

Liposomes from mammalian liver mitochondria are more polyunsaturated and leakier to protons than those from reptiles.

Liposomes were prepared from phospholipids extracted from liver mitochondria of the rat (Rattus norvegicus) and an agamid lizard, the bearded dragon (Amphibolurus vitticeps) and liposome proton conductance was measured at an imposed membrane potential of 160 mV as well as the fatty acid composition of the liposomes. Despite presumed changes in fatty acid composition during liposome preparation, the mammalian liposomes had a significantly lower content of the monounsaturated oleic acid and a significantly greater content of the omega-3 polyunsaturated docosahexaenoic acid. There were significant direct correlations between the liposome arachidonic and docosahexanoic acid content and bilayer proton flux and a significant inverse correlation between liposome oleic acid content and bilayer proton flux. "Apparent valinomycin-catalysed proton flux" was significantly directly correlated with liposome docosahexaenoic acid content and inversely correlated with oleic acid content. It is suggested that the high content of long-chain polyunsaturates in the mammalian mitochondrial membrane is responsible for an increased proton leak across the mitochondrial inner membrane and thus partly responsible for the high metabolic rate in endothermic mammals compared to their ectothermic reptilian predecessors.

Animals↗
PubMed1994

Hyperthyroidism stimulates mitochondrial proton leak and ATP turnover in rat hepatocytes but does not change the overall kinetics of substrate oxidation reactions.

Thyroid hormones have well-known effects on oxidative phosphorylation, but there is little quantitative information on their important sites of action. We have used top-down elasticity analysis, an extension of metabolic control analysis, to identify the sites of action of thyroid hormones on oxidative phosphorylation in rat hepatocytes. We divided the oxidative phosphorylation system into three blocks of reactions: the substrate oxidation subsystem, the phosphorylating subsystem, and the mitochondrial proton leak subsystem and have identified those blocks of reactions whose kinetics are significantly changed by hyperthyroidism. Our results show significant effects on the kinetics of the proton leak and the phosphorylating subsystems. Quantitative analyses revealed that 43% of the increase in resting respiration rate in hyperthyroid hepatocytes compared with euthyroid hepatocytes was due to differences in the proton leak and 59% was due to differences in the activity of the phosphorylating subsystem. There were no significant effects on the substrate oxidation subsystem. Changes in nonmitochondrial oxygen consumption accounted for -2% of the change in respiration rate. Top-down control analysis revealed that the distribution of control over the rates of mitochondrial oxygen consumption, ATP synthesis and consumption, and proton leak and over mitochondrial membrane potential (delta psi m) was similar in hepatocytes from hyperthyroid and littermate-paired euthyroid controls. The results of this study include the first complete top-down elasticity and control analyses of oxidative phosphorylation in hepatocytes from hyperthyroid rats.

Adenosine Triphosphate↗