The H+/ATP synthase from yeast mitochondria, MFOF1, was isolated, purified and reconstituted into liposomes prepared from phosphatidylcholine and phosphatidic acid. Analysis by mass spectrometry revealed the presence of all known subunits of the yeast enzyme (α3β3γδε 45ad8910HfJge) with the exception of the K-subunit. The MFOF1 liposomes were energized by acid base transitions and K+/valinomycin diffusion potentials and high rates of ATP synthesis are observed. Titration of the number of MFOF1 per liposome indicates that the monomeric enzyme is able to catalyze high rates of ATP synthesis similar to that observed under physiological conditions. ATP synthesis was abolished by addition of uncouplers, as well as by the specific inhibitor oligomycin. The rate of ATP synthesis was measured as a function of pHout, pHin and the phosphate concentration. Maximal rates (turnover number) of approx. 100 s− 1 are observed at a transmembrane pH difference of 3.2 U (at pHin = 4.8 and pHout = 8.0), in the presence of a superimposed transmembrane electric potential difference of 133 mV (Nernst potential). The apparent KM for Pi depends on the pHout.
Forster K., Turina P., Drepper F., Haehnel W., Fischer S., Graber P., et al. (2010). ATP synthesis by the isolated and reconstituted monomeric mitochondrial H+-ATP synthase from yeast [10.1016/j.bbabio.2010.04.110].
ATP synthesis by the isolated and reconstituted monomeric mitochondrial H+-ATP synthase from yeast
TURINA, MARIA PAOLA;
2010
Abstract
The H+/ATP synthase from yeast mitochondria, MFOF1, was isolated, purified and reconstituted into liposomes prepared from phosphatidylcholine and phosphatidic acid. Analysis by mass spectrometry revealed the presence of all known subunits of the yeast enzyme (α3β3γδε 45ad8910HfJge) with the exception of the K-subunit. The MFOF1 liposomes were energized by acid base transitions and K+/valinomycin diffusion potentials and high rates of ATP synthesis are observed. Titration of the number of MFOF1 per liposome indicates that the monomeric enzyme is able to catalyze high rates of ATP synthesis similar to that observed under physiological conditions. ATP synthesis was abolished by addition of uncouplers, as well as by the specific inhibitor oligomycin. The rate of ATP synthesis was measured as a function of pHout, pHin and the phosphate concentration. Maximal rates (turnover number) of approx. 100 s− 1 are observed at a transmembrane pH difference of 3.2 U (at pHin = 4.8 and pHout = 8.0), in the presence of a superimposed transmembrane electric potential difference of 133 mV (Nernst potential). The apparent KM for Pi depends on the pHout.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
