Abstract
The H+/ATP synthase from yeast mitochondria, MF0F1, was purified and reconstituted into liposomes prepared from phosphatidylcholine and phosphatidic acid. Analysis by mass spectrometry revealed the presence of all subunits of the yeast enzyme with the exception of the K-subunit. The MF0F1 liposomes were energized by acid–base transitions (ΔpH) and a K+/valinomycin diffusion potential (Δφ). ATP synthesis was completely abolished by the addition of uncouplers as well as by the inhibitor oligomycin. The rate of ATP synthesis was optimized as a function of various parameters and reached a maximum value (turnover number) of 120s−1 at a transmembrane pH difference of 3.2 units (at pHin=4.8 and pHout=8.0) and a Δφ of 133mV (Nernst potential). Functional studies showed that the monomeric MF0F1 was fully active in ATP synthesis. The turnover increased in a sigmoidal way with increasing internal and decreasing external proton concentration. The dependence of the turnover on the phosphate concentration and the dependence of KM on pHout indicated that the substrate for ATP synthesis is the monoanionic phosphate species H2PO4−.
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Schedule a callMore From: Biochimica et Biophysica Acta (BBA) - Bioenergetics
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