It is now widely accepted that F0F1 ATPsynthase 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 IF0 Dodecylmaltoside was optimal to extract the enzyme from single biopsy samples, allowing us to demonstrate that IF, 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 propertis.

Characterization of oligomeric forms from mammalian F0F 0ATP synthase by BN-page: The role of detergents / Bisetto E.; Giorgio V.; Pancrazio F.D.; Mavelli I.; Lippe G.. - In: ITALIAN JOURNAL OF BIOCHEMISTRY. - ISSN 0021-2938. - ELETTRONICO. - 56:4(2007), pp. 254-258.

Characterization of oligomeric forms from mammalian F0F 0ATP synthase by BN-page: The role of detergents

Giorgio V.;Mavelli I.;
2007

Abstract

It is now widely accepted that F0F1 ATPsynthase 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 IF0 Dodecylmaltoside was optimal to extract the enzyme from single biopsy samples, allowing us to demonstrate that IF, 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 propertis.
2007
Characterization of oligomeric forms from mammalian F0F 0ATP synthase by BN-page: The role of detergents / Bisetto E.; Giorgio V.; Pancrazio F.D.; Mavelli I.; Lippe G.. - In: ITALIAN JOURNAL OF BIOCHEMISTRY. - ISSN 0021-2938. - ELETTRONICO. - 56:4(2007), pp. 254-258.
Bisetto E.; Giorgio V.; Pancrazio F.D.; Mavelli I.; Lippe G.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/794380
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