Proton transport coupled ATP synthesis by the purified yeast H+ -ATP synthase in proteoliposomes.
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 (DeltapH) and a K(+)/valinomycin diffusion potential (Deltaphi). 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 pH(in)=4.8 and pH(out)=8.0) and a Deltaphi 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 K(M) on pH(out) indicated that the substrate for ATP synthesis is the monoanionic phosphate species H₂PO⁻₄.
Förster
Kathrin
K
Institut für Physikalische Chemie, Universität Freiburg, Albertstrasse 23a, Freiburg, Germany.
Turina
Paola
P
Drepper
Friedel
F
Haehnel
Wolfgang
W
Fischer
Susanne
S
Gräber
Peter
P
Petersen
Jan
J
D000255:Adenosine Triphosphate / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D017136:Ion Transport; D008081:Liposomes / Q000378:metabolism; D025261:Mitochondrial Proton-Translocating ATPases / Q000302:isolation & purification / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism; D011510:Proteolipids / Q000378:metabolism*; D018892:Proton-Motive Force; D011522:Protons; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000254:growth & development; D021241:Spectrometry, Mass, Electrospray Ionization
D008081:Liposomes; D010713:Phosphatidylcholines; D011510:Proteolipids; D011522:Protons; C020485:proteoliposomes; D000255:Adenosine Triphosphate; D025261:Mitochondrial Proton-Translocating ATPases