Purified soluble bovine mitochondrial F1Fo-ATP synthase contained 2mol of ATP, 2mol of ADP and 6mol of Pi/mol. Incubation of this enzyme with 1mM [32P]Pi caused the exchange of 2mol of Pi/mol of F1Fo-ATP synthase. The labelled phosphates were not displaced by ATP. Transfer of F1Fo-ATP synthase to a buffer containing 30% (v/v) DMSO and 1mM [32P]Pi resulted in the loss of bound nucleotides with the retention of 1mol of ATP/mol of F1Fo-ATP synthase. Six molecules of [32P]Pi were incorporated by exchange with the existing bound phosphate. Removal of the DMSO by passage of the enzyme through a centrifuged column of Sephadex G-50 resulted in the exchange of one molecule of bound [32P]Pi into the bound ATP. Azide did not prevent this [32P]Pi ↔ ATP exchange reaction. The bound labelled ATP could be displaced from the enzyme by exogenous ATP. Addition of ADP to the DMSO-pretreated F1Fo-ATP synthase in the original DMSO-free buffer resulted in the formation of an additional molecule of bound ATP. It was concluded that following pretreatment with and subsequent removal of DMSO the F1Fo-ATP synthase contained one molecule of ATP at a catalytic site which was competent to carry out a phosphateŐATP exchange reaction using enzyme-bound inorganic radiolabelled phosphate. In the presence of ADP an additional molecule of labelled ATP was formed from enzyme-bound Pi at a second catalytic site. The bound phosphateŐATP exchange reaction is not readily accommodated by current mechanisms for the ATP synthase.
Research Article| January 08 2001
Phosphate exchange and ATP synthesis by DMSO-pretreated purified bovine mitochondrial ATP synthase
Philip D. BRAGG
Philip D. BRAGG 1
1Department of Biochemistry and Molecular Biology, University of British Columbia, 2146 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3
1To whom correspondence should be addressed (e-mail firstname.lastname@example.org).
Search for other works by this author on:
- Views Icon Views
- Share Icon Share
- Cite Icon Cite
Seelochan BEHARRY, Philip D. BRAGG; Phosphate exchange and ATP synthesis by DMSO-pretreated purified bovine mitochondrial ATP synthase. Biochem J 15 January 2001; 353 (2): 215–222. doi: https://doi.org/10.1042/bj3530215
Download citation file: