The thermodynamic and kinetic properties associated with intrinsic uncoupling in a six-state model of a redox proton pump have been studied by computing the flow-force relations for different degrees of coupling. Analysis of these relations shows the regulatory influence of the thermodynamic forces on the extent and relative contributions of redox slip and proton slip. Inhibition has been introduced into the model in two different ways, corresponding to possible modes of action of experimental inhibitors. Experiments relating the rate of electron transfer to delta microH at static head upon progressive inhibition of the pumps have been simulated considering (1) the limiting case that the nonzero rate of electron transfer at static head is only due to intrinsic uncoupling (no leaks) and (2) the experimentally observed case that about 30% of the nonzero rate of electron transfer at static head is due to a constant proton leakage conductance in parallel with the pumps, the rest being due to intrinsic uncoupling. The same simulations have been performed for experiments in which the rate of electron transfer is varied by varying the substrate concentration rather than by using an inhibitor. The corresponding experimental results obtained by measuring delta microH and the rate of electron transfer at different succinate concentrations in rat liver mitochondria are presented. Comparison between simulated behavior and experimental results leads to the general conclusion that the typical relationship between rate of electron transfer and delta microH found in mitochondria at static head could certainly be a manifestation of some degree of intrinsic uncoupling in the redox proton pumps.(ABSTRACT TRUNCATED AT 250 WORDS)
A system is described for the study of the synthesis of ATP by the K+ concentration gradient.The system consists of mitochondria which accumulate K+ phosphate aerobically in the presence of valinomycin and then slowly release K+ upon addition of rotenone. The release of K+ is accelerated several folds by the addition of ADP and during the phase of ADP stimulated K+ efflux a net synthesis of ATP is observed. The amount of ATP synthesised may amount to 15-20 pmoles/g protein. The reaction proceeds for about 2 min and during this period a constant ratio of 4 K+/ATP is observed. The apparent K , for ADP and Pi are 25 and 150 pM, respectively.The synthesis of ATP is strongly dependent on the Kl+/K,+ ratio ; above an external concentration of 3 mM K+ the synthesis of ATP is abolished. The rate of ATP synthesis is also markedly effected by the pH of the medium in that it is stimulated a t pH 6.5 and inhibited a t pH above 7.5.A high rate of K+ efflux is observed in the presence of ADP, after an osmotic swelling and in alkaline media. With the use of mersalyl it is shown that the K+ efflux can be largely accounted for by H+ uptake. The high rate of proton translocation and the synthesis of ATP during K + efflux are assumed to involve a reversal of the proton pump which operates the energy linked accumulation of cations.Synthesis of ATPdriven by a pH gradient has been reported by Jagendorf and Uribe [ l ] to occur in chloroplasts and has been confirmed by McCarthy and Racker [2]. These experiments have been taken to support the chemiosmotic hypothesis which predicts the synthesis of ATP by a proton motive force, constituted by both a membrane potential and a pH differential. Attempts have been made to obtain a proton driven ATP synthesis with liver mitochondria. Reid et ul. Enzymes. Glucose-6-phosphate dehydrogenase or D-glucose-6-phosphate:NADP oxidoreductase (EC 1.1.1.49); adenvlate kinase or ATP : AMP Phosphotransferase (EC 2.7.4:3) ; hexokinase or ATP : D-hexose-6phosphotransfe;ase (EC 2.7.1.1).chondria. The findings were interpreted in terms of reversal of a cation pump. The alternative explanation was, however, conceived that the K+ efflux gives rise to a membrane potential which in turn drives the ATP synthesis [8].The present investigation was devoted to the analysis of the stoicheometries of the chemiosmotic transformation and of the mechanism of H+ translocation in energized mitochondria. Evidence will be provided that the K+ efflux can induce the synthesis of large amounts of ATP with a stoicheometry of 4 K+/ATP. It will be proposed that the K+ driven ATP synthesis and the H+ translocation occur through the operation of a proton carrier [9].
METHODSRat liver was homogenized in 0.25 M sucrose, 5 mM Tris-C1 pH 7.5 and 0.5 mM EGTA. After two washings in the same medium, the mitochondria were resuspended in 0.25 M sucrose, 5 mM Tris-C1, p H 7.5.Variations of K+ concentration were measured with a Beckman cationic electrode, attached to a Beckman pH meter [lo]. The amount of K + taken up or released by the mitochon...
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