Studies on the structure and conformation of yeast mitochondrial ATPase using aurovertin and methanol as probes.
1. The isolation of the mitochondrial ATPase F1 and its beta-subunit from commercial baker's yeast (Saccharomyces cerevisiae) is described. 2. The molecular weight determined by ultracentrifugation is 340000 +/- 30000. Gel chromatography indicates a molecular weight of 300000 +/- 20000. 3. Fluorimetric titration of the isolated enzyme with aurovertin reveals two binding sites per molecule. The isolated beta-subunit binds aurovertin in a 1 : 1 stoicheiometry. It is concluded that the ATPase molecule contains two aurovertin-binding beta-subunits. 4. The stabilizing agent methanol influences both the measured Kd and the concentration of binding sites for aurovertin. These results fit a model in which both F1 and aurovertin are distributed between aqueous and methanol phases. 5. The effect of methanol on the ATPase activity can be described in terms of the model proposed by Recktenwald and Hess (Recktenwald, D. and Hess, B. (1977) FEBS Lett. 76, 25-28). It is proposed that methanol enhances the affinity of the regulatory site for ATP, but at higher concentrations prevents the interaction between the regulatory and catalytic sites. 6. Since HSO(-3), a typical effector of the assumed regulatory site of F1, has no effect on the binding of aurovertin, it is concluded that the binding site of aurovertin is not correlated with the regulatory site. 7. The inhibition of ATPase activity by aurovertin is slowly (t 1/2 = 70 s) induced during turnover conditions. 8. From the effect of methanol on the inhibition of ATPase activity by aurovertin it is concluded that under turnover conditions the conformation is such that the aurovertin-binding sites have a 6-fold higher affinity for methanol than under resting conditions.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001313:Aurovertins / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D008433:Mathematics; D000432:Methanol / Q000494:pharmacology*; D008928:Mitochondria / Q000201:enzymology*; D008970:Molecular Weight; D011487:Protein Conformation / Q000187:drug effects; D006180:Proton-Translocating ATPases; D011714:Pyrans / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D001313:Aurovertins; D011714:Pyrans; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases; D000432:Methanol