Characterization of oligomeric forms from mammalian F0F1ATP synthase by BN-PAGE: the role of detergents.
It is now widely accepted that F0F1ATPsynthase is present in membrane, beside as monomers, in homo-dimeric and higher homo-oligomeric forms, which probably play critical roles in determining mitochondrial morphology. One-step mild detergent extraction followed by blue native electrophoresis (BN-PAGE) is a very interesting tool for studying the native membrane protein assemblies which can be associated with second/third-dimensional SDS-PAGE, immunoblotting, in-gel enzyme activity staining and mass spectrometry analyses. By combining these techniques, we resolved monomers and higher oligomeric forms of ATPsynthase from bovine heart mitochondria. However, a critical point is the choice of the detergents, which strongly influence the protein pattern of BN-PAGE. By using Triton X-100 we obtained that, in spite of the same subunit composition, monomers have a much lower specific activity than dimers and the two forms have a different pattern of tyrosine phosphorylation, suggesting that monomers and dimers are functionally distinct in membrane. In addition, enzyme self-association appeared to occur independently from the binding to ATPsynthase of the inhibitor protein IF1. Dodecylmaltoside was optimal to extract the enzyme from single biopsy samples, allowing us to demonstrate that IF1 plays a central role in regulating the enzyme activity in heart in vivo. Only low concentration of digitonin maintained significant amounts of ATPsynthase oligomers, which seemed to retain intact their native catalytic properties.
Bisetto
Elena
E
Department of Biomedical Sciences and Biotechnologies, M.A.T.I. Centre of Excellence, University of Udine, Udine, Italy.
Giorgio
Valentina
V
Di Pancrazio
Francesca
F
Mavelli
Irene
I
Lippe
Giovanna
G
D000818:Animals; D002417:Cattle; D004072:Digitonin; D019281:Dimerization; D004586:Electrophoresis / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008929:Mitochondria, Heart / Q000201:enzymology*; D017830:Octoxynol; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D017830:Octoxynol; D006180:Proton-Translocating ATPases; D004072:Digitonin