A COLUMN CENTRIFUGATION METHOD FOR THE RECONSTITUTION IN LIPOSOMES OFTHE MITOCHONDRIAL F0F1 ATP SYNTHASE ATPASE

A method for reconstitution of membrane proteins into unilamellar lipo somes is described. The model enzyme was the F0F1 ATP synthase from mi tochondria when in complex or free from its inhibitor protein. The enz ymes were first solubilized with either of two detergents, i.e., n-dod ecyl-beta-D maltoside or lauryldimethylamine oxide. After solubilizati on, the enzymes were passed through a column of Sepharose-AH using an ADP/sodium cholate selective elution buffer. The enzymes recovered fro m the column were subsequently passed through a centrifuge column of S ephadex 6-50 fine. The eluate contained Liposomes in which the F0B1 co mplex (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-i excha nge reactions. Thus the F0F1 complex which is formed by 18 subunits ca n be rapidly reconstituted into liposomes in a fully functional state. Moreover the data show that the interactions between the enzyme and i ts inhibitor protein are not perturbed in the reconstitution procedure . (C) 1996 Academic Press, Inc.