Abstract
Reconstituted transhydrogenase-ATPase vesicles obtained with purified beef heart transhydrogenase and oligomycin-sensitive ATPase were investigated with respect to the mode of interaction between the two proton pumps, with special reference to the relative contributions of the membrane potential and proton gradient using valinomycin and nigericin in the presence of potassium. In the absence of ionophores and at low ATP concentrations, below 20 microM, the ATPase generated a proton motive force which was predominantly due to a membrane potential, whereas at saturating concentrations of ATP the proton gradient was the predominant component. The ATP-dependence of the rate of the ATP-driven transhydrogenase reaction showed apparent Km values in the low and high ATP concentration range of about 3 and 56 microM, respectively, with a corresponding difference in Vmax of about 3-fold. It is concluded that the reconstituted transhydrogenase can utilize both a membrane potential and a proton gradient, separately or combined, where the relative contributions of these components depend on the activity of the ATPase. In the reconstituted vesicles, the maximally active transhydrogenase is apparently driven by an electrochemical proton gradient where the membrane potential and the proton gradient contribute one-third and two-thirds, respectively. The rate-dependent relative generation of a membrane potential and pH gradient presumably reflects the proton pump characteristics of the ATPase and/or buffering/permeability characteristics of the vesicles rather than the properties of the transhydrogenase per se. These results are discussed in relation to current models for transhydrogenase-linked proton translocation.
Highlights
Reconstituted transhydrogenase-ATPase vesicles obtained with purified beef heart transhydrogenase and oligomycin-sensitive ATPase were investigated with respect to the mode of interaction between the two proton pumps, with special reference to the relative contributions of the membrane potential and proton gradient using valinomycin and nigericin in the presence of potassium
In order to further establish the correlation between concentration of ATP, ATP-driven transhydrogenase activity and inhibition by valinomycin and oligomycin, the ATPdriven transhydrogenase reaction was measured as a function of ATP concentration in the absence and in the presence of oligomycin or valinomycin (Fig. 2)
The present study has aimed at a clarification of the relative roles of the membrane potential and proton gradient in driving the ATPdriven energy-linked transhydrogenase reaction, with emphasis on qualitative rather than quantitative aspects
Summary
MM sodium phosphate containing 0.5% potassium cholate, respectively. ATPase (complex V) and a similar preparation (38-45P), both from beef heart submitochondrial particles, followed the procedure described by Stiggall et al [15]. Reconstitution of transhydrogenase by the cholate dialysis procedure (cf 6,7,15) was carried out by mixing (in this order) transhydrogenase (in 0.5% potassium cholate, 200 mM sodium phosphate, pH 7.5), varying amounts of ATPase and liposomes, 20% potassium cholate to a final concentration of l%, followed by dialysis overnight at 0 “C against 1 liter of reconstitution buffer. The final concentrations of transhydrogenase, ATPase, and phospholipids were 0.02, 1, and 10 mg/ml. NADH, and 2 mM MgCl, in 80 mM potassium phosphate, pH 7.5 These concentrations of tNADP’ and NADH were saturating under the conditions used. 100% inhibited by 10 pg of oligomycin, was taken as the difference between the activity obtained after and prior to the addition of ATP, corrected for the slight activation (less than 10%) obtained by CCCP in the absence of ATP
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