Relation between the Gradient of the ATP/ADP Ratio and the Membrane Potential across the Mitochondrial Membrane (original) (raw)
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A kinetic assay of mitochondrial ADP–ATP exchange rate in permeabilized cells
Analytical Biochemistry, 2010
We have previously described a method to measure ADP-ATP exchange rates in isolated mitochondria by recording the changes in free extramitochondrial [Mg 2+ ] reported by a Mg 2+sensitive fluorescent indicator, exploiting the differential affinity of ADP and ATP to Mg 2+ . In this manuscript we describe a modification of this method suited for following ADP-ATP exchange rates in environments with competing reactions that interconvert adenine nucleotides, such as in permeabilized cells that harbor phosphorylases and kinases, ion pumps exhibiting substantial ATPase activity and myosin ATPase activity. Here we report that addition of BeF 3 − and Na 3 VO 4 to media containing digitonin-permeabilized cells inhibit all ATP-ADP utilizing reactions, except the ANTmediated mitochondrial ATP-ADP exchange. An advantage of this assay is that mitochondria that may have been also permeabilized by digitonin do not contribute to ATP consumption by the exposed F 1 F o -ATPase, due to its sensitivity to BeF 3 − and Na 3 VO 4 . With this assay, ADP-ATP exchange rate mediated by the ANT in permeabilized cells is measured for the entire range of mitochondrial membrane potential titrated by stepwise additions of an uncoupler, and expressed as a function of citrate synthase activity per total amount of protein.
Involvement of the ADP/ATP carrier in permeabilization processes of the inner mitochondrial membrane
European Journal of Biochemistry, 1993
The effect of different agents on inner-mitochondrial-membrane permeabilization and lipoperoxidation induced by Ca2+ and the pyridine-nucleotide oxidant t-butylhydroperoxide or inorganic phosphate was investigated. Comparing the protection conferred by ADP, a substrate of the ADP/ATP carrier, dithiothreitol, a disulfide reductant and butylhydroxytoluene, a radical scavenger, it was found that ADP was always the most effective against mitochondrial damage, when present in the incubation medium from the beginning. Moreover, carboxyatractyloside, a specific inhibitor of the ADP/ATP carrier, abolished completely the protective effect of ADP on both the lipoperoxidation and mitochondrial swelling processes. Experiments where deenergized mitochondria were previously incubated with Ca2+ showed a decrease in the content of active ADP/ATP carrier, indicating a direct involvement of this protein in the formation of a non-specific Ca2+-dependent pore. Our results also eliminate the possibility of an attack of oxygen radicals on lipids or proteins of the mitochondrial membrane as the primary event triggering the permeability transition of the inner mitochondrial membrane.
ATP Synthesis and Electrical Membrane Potential in Mitochondria
European Journal of Biochemistry, 1970
The difference of the proton's electrochemical potential (A j i~) across the membranes of rat liver mitochondria was compared with the rate of phosphorylation. It was found that in high potassium concentration (100 DIM), A/i= was approx. zero and there is no phosphorylation. This finding is predicted by the chemiosmotic model. I n low potassium concentrations phosphorylation increases in parallel with the increase in Ah=. In all the experiments ApH was found to be negligibly small, thus A j i~ was effectively due to the membrane potential (A y). dy was calculated
Bioelectrochemistry and Bioenergetics, 1987
The mechanism of proton translocation across the mitochondrial Fe-F, ATP synthase was investigated by studying the electrical behaviour of the intramembrane sector, F,,, of the ATP synthase as well as by studying the role of dithiol groups of Fc in proton transport. The conductance properties of Fa were inferred by determining the current-voltage characteristics of inner mitochondrial membrane preparations in which the interaction between Ft and Fc had been selectively damaged so as to open Fa channels. Such a study indicated that open Fs channels have a gated response to the membrane potential so that proton flow through Fa is inhibited at membrane potential values below approximately 100 mV. Specific oxidation of dithiol groups to disulphides was obtained by incubating purified, urea-extracted Fa with diamide. It was found that, following such treatment, Fs could no longer be reconstituted into an energy-coupled Fe-F, ATP synthase, as apparent from the loss of sensitivity to oligomycin of ATP hydrolysis by the reconstituted enzyme. Moreover, it was found that Ft and/or the oligomycin sensitivity conferring protein protect Fa essential dithiols from diamide deleterious oxidation.
Molecular and Cellular Biochemistry, 1994
Cytosolic proteins as components of the physiological mitochondrial environment were substituted by dextrans added to media normally used for incubation of isolated mitochondria. Under these conditions the volume of the intermembrane space decreases and the contact sites between the both mitochondrial membranes increase drastically. These morphological changes are accompanied by a reduced permeability of the mitochondrial outer compartment for adenine nucleotides as it was shown by extensive kinetic studies of mitochondrial enzymes (oxidative phosphorylation, mi-creatine kinase, mi-adenylate kinase). The decreased permeability of the mitochondrial outer membrane causes increased rate dependent concentration gradients in the micromolar range for adenine nucleotides between the intermembrane space and the extramitochondrial space. Although all metabolites crossing the outer membrane exhibit the same concentration gradients, considerable compartmentations are detectable for ADP only due to its low extramitochondrial concentration. The consequences of ADP-compartmentation in the mitochondrial intermembrane space for ADP-channelling into the mitochondria are discussed. (Mol Cell Biochem 133/134. 85-104, 1994)