1. Functional and stoichiometric analysis of subunit e in bovine heart mitochondrial F0F1ATP synthase
- Author
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Elena Bisetto, Giovanna Lippe, Irene Mavelli, Valentina Giorgio, Paola Picotti, Vera Alverdi, Bisetto E., Picotti P., Giorgio V., Alverdi V., Mavelli I., and Lippe G.
- Subjects
Models, Molecular ,Protein Conformation ,Physiology ,Proteolysis ,ATPase ,Protein subunit ,Mitochondrion ,Mitochondria, Heart ,AQUA peptide ,Adenosine Triphosphate ,Self-association ,ATP synthase gamma subunit ,Enzyme Stability ,medicine ,Animals ,Bioorganic chemistry ,Inner membrane ,Computer Simulation ,LC-MS/MS ,Mammalian F ,Binding Sites ,medicine.diagnostic_test ,biology ,ATP synthase ,Myocardium ,Cell Biology ,Enzyme Activation ,Protein Subunits ,Proton-Translocating ATPases ,Models, Chemical ,Biochemistry ,biology.protein ,Subunit e stoichiometry ,Cattle ,Protein Binding - Abstract
The role of the integral inner membrane subunit e in self-association of F0F1ATP synthase from bovine heart mitochondria was analyzed by in situ limited proteolysis, blue native PAGE/iterative SDS-PAGE, and LC-MS/MS. Selective degradation of subunit e, without disrupting membrane integrity or ATPase capacity, altered the oligomeric distribution of F 0F1ATP synthase, by eliminating oligomers and reducing dimers in favor of monomers. The stoichiometry of subunit e was determined by a quantitative MS-based proteomics approach, using synthetic isotope-labelled reference peptides IAQL*EEVK, VYGVGSL*ALYEK, and ELAEAQEDTIL*K to quantify the b, γ and e subunits, respectively. Accuracy of the method was demonstrated by confirming the 1:1 stoichiometry of subunits γ and b. Altogether, the results indicate that the integrity of a unique copy of subunit e is essential for self-association of mammalian F0F1ATP synthase. © 2008 Springer Science+Business Media, LLC.
- Published
- 2008
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