Affordable Access

Publisher Website

A Column Centrifugation Method for the Reconstitution in Liposomes of the Mitochondrial F0F1ATP Synthase/ATPase

Authors
Journal
Protein Expression and Purification
1046-5928
Publisher
Elsevier
Publication Date
Volume
7
Issue
2
Identifiers
DOI: 10.1006/prep.1996.0022
Disciplines
  • Biology
  • Chemistry

Abstract

Abstract A method for reconstitution of membrane proteins into unilamellar liposomes is described. The model enzyme was the F 0F 1ATP synthase from mitochondria when in complex or free from its inhibitor protein. The enzymes were first solubilized with either of two detergents, i.e., n-dodecyl-β- Dmaltoside or lauryldimethylamine oxide. After solubilization, the enzymes were passed through a column of Sepharose-AH using an ADP/sodium cholate selective elution buffer. The enzymes recovered from the column were subsequently passed through a centrifuge column of Sephadex G-50 fine. The eluate contained liposomes in which the F 0F 1complex (with and without inhibitor protein) had been reconstituted. The reconstituted enzymes were capable of hydrolyzing ATP with formation of electrochemical H +gradients. They also catalyzed the ATP–P iexchange reactions. Thus the F 0F 1complex which is formed by 18 subunits can be rapidly reconstituted into liposomes in a fully functional state. Moreover the data show that the interactions between the enzyme and its inhibitor protein are not perturbed in the reconstitution procedure.

There are no comments yet on this publication. Be the first to share your thoughts.