The uncoupling effect of free fatty acids on oxidative phosphorylation in mitochondria has been known for more than 35 years. The mechanism of action, however, remains controversial. In this report the physicochemical basis of uncoupling was elucidated by studying the effect of free fatty acids on the proton permeability and membrane potential of proteoliposomes containing reconstituted cytochrome c oxidase (COX). A threshold membrane potential of about 125 mV was identified for fatty acid-induced proton permeability. Only above this potential do free fatty acids translocate protons across the biological membrane. The data explain the controversial effects of long-chain fatty acids on oxidative phosphorylation as well as their role on non-shivering thermogenesis in larger mammals.
Isolated cytochrome c oxidase from Paracoccus denitrzjkans, containing either two or three subunits, was reconstituted into liposomes and the membrane potential was measured at different rates of respiration using a triphenylmethylphosponium bromide electrode. Both enzymes revealed a non-linear increase of the membrane potential with increasing respiratory rates. The ratios of the respiratory rates of the two proton pumps decreased with increasing membrane potential, suggesting slippage of proton pumping, as has been shown before with two cytochrome c oxidases from bovine heart, differing in H+/e-stoichiometries due to chemical modification The generally observed non-linear relationship between electron-transfer rates and the proton motive force (ApH', or the membrane potential in the presence of the H+/ K'antiporter, nigericin) of redox proton pumps was explained either by the dpH+ dependence of the membrane H+-leak conductance (O'Shea et al., 1984;Krishnamoorthy and Hinkle, 1984;Murphy and Brand, 1987;Hafner and Brand, 1991 ;Brown and Brand, 1991), or, at least in part, by a variable (membrane-potential dependent) H+/e-stoichiametry (slippage of proton pumps; Pietrobon et al., 1981Pietrobon et al., , 1983Zoratti et al., 1986). In a recent publication, we could support proton slippage in cytochrome c oxidase (COX) by measuring the ratio of electron-transfer rates of two different efficient proton pumps at various membrane potentials (Steverding and Kadenbach, 1991). COX from bovine heart was reduced in proton pumping efficiency by chemical modification with N-ethoxycarbonyl-2-ethoxy-1 ,Zdihydroquinoline. The observed decrease in the ratio of electron-transfer rates of the two proton pumps with increasing membrane potential was taken to indicate proton slippage in reconstituted COX from bovine heart.Mammalian COX contains three mitochondrial-encoded and ten nuclear-encoded subunits . Therefore, it was of interest to find out whether proton slippage was an intrinsic property of the catalytic core of COX, composed of the mitochondrial-encoded subunits I and 11, or was due to the nuclear-encoded subunits, which are absent in COX from bacteria. Isolated COX from Paracoccus denitrificans contains either two or three subunits, which are Correspondence to
A novel flow calorimetric technique was developed to study the energy turnover of myocardial mitochondria. Cylindrical strands of cardiac muscle (trabeculae) weighing 100-500 micrograms were isolated from guinea-pig heart and mounted in a tubular recording chamber which was continuously perfused with physiological salt solution at 37 degrees C. The temperature difference between the upstream and the downstream side of the chamber, which is proportional to the rate of heat production of the trabecula, was measured at high resolution. In this way the rate of energy expenditure of isolated cardiac muscle could be recorded continuously for several hours. When the preparations were superfused with an 'intracellular' solution containing 5 mM pyruvate and 2 mM malate as substrates, permeabilization of the sarcolemma with 25 microM digitonin induced a marked increase in the measured heat rate in the presence of 2 mM ADP. The major fraction of the ADP sensitive heat production (83%) could be blocked with 400 microM atractyloside, an inhibitor of the adeninenucleotide translocase, and by 600 microM alpha-cyano-4-hydroxycinnamate, an inhibitor of monocarboxylate/H+ co-transport. The atractyloside sensitive heat production was abolished in anoxic solution. These results suggest that the atractyloside-sensitive heat production (21.8 +/- 3.5 mW cm-3 of tissue) was attributable to oxidative phosphorylation. The mitochondria apparently remained intact after treatment with digitonin, since application of the uncoupler 2,4-dinitrophenol (DNP) produced a very large increase in heat rate. A minor fraction of the heat rate induced by ADP in permeabilized cardiac muscle preparations (17%) was not sensitive to atractyloside. This component was also seen before application of digitonin and was probably related to ectonucleotidases. In conclusion, our calorimetric technique allows investigation of the energy metabolism of myocardial mitochondria 'in situ', i.e. without destroying the microarchitecture of cardiac muscle cells.
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