A quantitative correlation between the kinetics of solutes and water translocation in Liver Mitochondria (original) (raw)
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The osmotic nature of the ion-induced swelling of rat-liver mitochondria
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1969
Analytical techniques have been developed to measure the relation between water uptake and cation uptake in rat-liver mitochondria. The water content of inner and outer mitochondfial compartments were separately measured using [mIialbumin and [~*Cisucrose. The cation contents of the same mitochondrial pellets were measured using flame photometry. These data were used to measure the water content of each compartment which was necessary, since the basic osmotic relationships are only revealed when movements into the inner and outer compartments are separately examined. The relation between the water uptake and K-uptake, either spontaneous or valinomycin-induced, was dependent on medium osmolality. The concentration of the K-solution taken up in the inner compartment was compatible with the hypothesis that transport-linked mitochondrial swelling is driven by osmotic pressure differences which have been induced by solute movement. The changes in volume of the water compartment and in ion content during phosphate-induced swelling and during incubation in the absence of substrate were analyzed using the same methods. Under these experimental conditions also, the mitochondrial inner compartment appears to be in osmotic equilibrium with the external medium.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1967
I. Water uptake coupled to ion movement has been studied in respiratoryinhibited liver mitochondria, of which the permeability to cations was increased by valinomyein, gramicidin or EDTA, and to anions by raising the pH of the medium. The movement of water was accounted for by the osmotic pressure of the penetrating solutes. 2. The rate of movement of water was inversely proportional to the concentration of solutes in the medium, and was dependent on the presence of permeating cations and anions. The above findings are interpreted within the concept of an osmotic movement of water. 3. The flow of anions through the membrane was inhibited by Ca 2+ and Mn 2+. Mitochondrial swelling was inhibited by sucrose. 4. Ion movement was independent of energy supply from metabolism. The nature of the force driving the ion movement is discussed. AND BARTLEY 13, and BARTLEY 14.
Understanding ion transport by quantification of mitochondrial swelling
Biochemical education, 2000
Suspensions of mitochondria are turbid and scatter light. An increase in the matrix volume (swelling) due to the influx of permeable solutes results in a decrease in the amount of light scattered. This property can be used to study solute fluxes across the mitochondrial inner membrane. A rapid method for isolating mitochondria is presented along with three swelling experiments using energized and non-energized mitochondria to illustrate ion transport across energy transducing membranes.
The Journal of biological chemistry, 1970
The swelling of isolated bovine heart and rat liver mitochondria has been compared in various suspending media in the absence of a source of metabolic energy. It is concluded that mitochondria will swell under these conditions in the presence of a permeant anion and a permeant cation, provided that the internal pH is not restrictive. Mitochondria show a high selectivity for cation penetration in the absence of energy, but almost no selectivity in the presence of respiration or of exogenous ATP. It is concluded that no monovalent cations penetrate the mitochondrion by simple diffusion in the absence of energy, but that an exchange diffusion system permits Na+ to be exchanged for internal H+. Lipid-soluble amines, such as NH,, can penetrate the mitochondrion and, in the presence of reagents that acidify the interior, such as free acetic acid or the protonated form of an uncoupler, can generate a cation (NH*+). The combination of NH,+ plus an uncoupler, therefore, represents a means of allowing cation accumulation in the matrix which is equivalent to the presence of the valinomycin-K+ or the gramicidin-Na+ complex. Certain anions, such as nitrate, appear to diffuse into the mitochondrion at neutral pH when cation penetration is induced by one of the above mechanisms. Bovine heart mitochondria show little, if any, tendency to admit substrate anions by the specific exchange diffusion systems found in rat liver mitochondria. All anions tested, however, are able to penetrate the bovine heart mitochondrion in the presence of a penetrating cation by a pH-dependent mechanism. This pathway for anion penetration can also be demonstrated in liver mitochondria at high pH, but it is not as striking as in the heart mitochondria in the range of pH 7.2 to 7.5. Uncoupled mitochondria swollen in the ammonium salts of nitrate, trichloracetate, fumarate, succinate, or malate can be contracted by either exogenous ATP or respiratory substrate as previously described for NH&l.
The mechanism of energy-dependent ion transport in mitochondria
The Journal of Membrane Biology, 1973
The transport of potassium, sodium and various anions in rat-liver mitochondria was studied mainly by analysis of ion content and water compartmentation of the mitochondrial pellet. A comparison of spontaneous transport with valinomycin-or gramicidin-stimulated transport is made. The rate or extent of uptake, the internal concentrations and the concentration ratio (Cin/Cout) are calculated and compared to test existing models for ion transport in mitochondria. Several models of ion transport in mitochondria are based on a cation-pump which is directed inward. This hypothesis is rejected because of the following findings: (1) Valinomycin stimulates the rate of potassium uptake but does not increase the potassium concentration ratio that can be actively maintained in a steady state (in which there is no potassium flow). (2) Valinomycin greatly stimulates the efflux of 42K from mitochondria during the process of potassium accumulation. When potassium accumulation is stimulated the flux ratio, i.e. influx/efflux, decreases; in the presence of valinomycin this ratio approaches 1. (3) In the presence of gramicidin, the concentration ratios of potassium and sodium are about the same under a variety of conditions. These findings indicate that potassium and sodium transport are passive processes of relaxation towards electro-chemical equilibrium (of the potassium and sodium). In high external potassium concentrations the extent of potassium uptake is limited by the permeation of anions; of the permeating anions multivalent acids support a higher extent than monovalent acids. It was found that succinate, acetate and oxalate which are transported together with potassium are distributed in accordance with the ApH and without any relation to the potassium concentration ratio. These findings are compatible with the hypothesis that an outward-directed proton pump creates an electrical potential gradient, which shifts the equilibrium state for the cations and drives sodium and potassium inward, and also creates proton gradient that is the driving force for anion transport.
European journal of biochemistry, 1990
Washed and purified rat-or mouse-liver mitochondria exhibiting high membrane integrity and metabolic activity were studied by flow cytometry. The electrophoretic accumulation/redistribution of cationic lipophilic probes, rhodamine 123, safranine 0 and a cyanine derivative, 3,3'-dihexyloxadicarbocyanine iodide, during the energization process was studied and was consistent with the generation of a negative internal membrane potential. An exception to this was nonylacridine orange which spontaneously bound to the mitochondrial membrane by hydrophobic interactions via its hydrocarbon chain. Energized purified mitochondria stained with potentiometric dyes exhibited both higher fluorescence and population homogeneity than the non-energized or deenergized (nigericin plus valinomycin) mitochondria. By contrast, under non-energized or deenergized conditions, the mitochondrial population exhibited fluorescence intensity heterogeneity related to the residual membrane potential; two subpopulations were evident, one of low fluorescence which may be related to the autofluorescence of the mitochondria (plus non-specific dye binding) and a second population which exhibited high fluorescence. Flow cytometry of the unpurified, simply washed, rat-liver mitochondria stained with rhodamine 123, a classically used dye, provided evidence of their heterogeneity in terms of light-scattering properties and membranepotential-related fluorescence. One third of the washed mitochondria were found to be non-functional by such assays. The fluorescence of purified rat-liver mitochondria due to the membrane potential built up by endogenous substrates indicates heterogeneity of the mitochondrial population with respect to levels of endogeneous substrates. The low-angle light scattering increases upon energization and provides some original information about the shape and modification of the inner mitochondrial conformation accompanying the energization. The heterogeneity of the rat liver mitochondrial population, from a structural, metabolic (existence of endogenous substrates) and functional (active and non-active mitochondrial population dispersion) point of view could thus be demonstrated by flow-cytometry analysis. Two animal models were examined with regard to the alteration of the mitochondrial membrane potential under the effects of drugs (rat-liver mitochondria), and the effects of ammonium toxicity (mouse-liver mitochondria). These results are promising and open new perspectives in the study of mitochondriopathies. Energy-transducing organelles, such as mitochondria, generate a high proton electrochemical gradient, as predicted by Mitchell's chemiosmotic hypothesis [l]. This proton gradient (dpH ') comprises two components : a membrane potential difference (Ay), negative interior, and a pH difference (ApH). In mitochondria, the membrane potential predominates over the dpH component.
Journal of Bioenergetics and Biomembranes, 1997
When isolated rat liver mitochondria are incubated in KCl medium, matrix volume, flux, and forces in both hypo- and hyperosmolarity are time-dependent. In hypoosmotic KCl medium, matrix volume is regulated via the K+/H+ exchanger. In hyperosmotic medium, the volume is regulated in such a manner that at steady state, which is reached within 4 min, it is maintained whatever the hyperosmolarity. This regulation is Pi- and DeltatildemuH+\Delta \tilde \mu H^ +DeltatildemuH+ -dependent, indicating Pi-K salt entry into the matrix. Under steady state, hyperosmolarity has no effect on isolated rat liver mitochondria energetic parameters such as respiratory rate, proton electrochemical potential difference, and oxidative phosphorylation yield. Hypoosmolarity decreases the NADH/NAD+ ratio, state 3 respiratory rate, and DeltatildemuH+\Delta \tilde \mu H^ +DeltatildemuH+ , while oxidative phosphorylation yield is not significantly modified. This indicates kinetic control upstream the respiratory chain. This study points out the key role of potassium on the regulation of matrix volume, flux, and forces. Indeed, while matrix volume is regulated in NaCl hyperosmotic medium, flux and force restoration in hyperosmotic medium occurs only in the presence of external potassium.