Mammalian ATPsynthase monomer versus dimer profiled by blue native PAGE and activity stain |
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Authors: | Bisetto Elena Di Pancrazio Francesca Simula Maria Paola Mavelli Irene Lippe Giovanna |
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Affiliation: | Department of Biomedical Sciences and Technologies, University of Udine, Udine, Italy. |
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Abstract: | Studies into the effects of oligomerization on F(0)F(1)ATPsynthase function are contradictory. We optimized the in-gel ATPase assay to investigate the functional differences of monomers versus dimers. In Triton X-100 extracts of heavy bovine heart mitochondria (HBHM) and mitoplasts, but not submitochondrial particles (MgATP-SMP), dimers had greater specific activity than monomers: at 30 degrees C, the dimer/monomer activity ratios were 2.3, 1.4, and 1.0, respectively. These differences in HBHM and mitoplasts extracts were enhanced at 37 degrees C but lost at 20 degrees C. In mitoplasts but not in MgATP-SMP, dimers were selectively shielded from limited chymotrypsin degradation of F(1) alpha subunit, possibly due to interactions with other proteins or ligands in the native inner membrane. Despite these differences, all three preparations had similar percentages of dimers and similar contents of the native inhibitor IF(1) in Vm (monomer) and (dimer) Vd. These results suggest that, in native membrane, monomers and dimers are functionally distinct. |
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Keywords: | Activity staining Blue native PAGE Dimerization Limited proteolysis MALDI‐TOF Mammalian F0F1ATPsynthase |
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