Phosphate exchange and ATP synthesis by DMSO-pretreated purified bovine mitochondrial ATP synthase

Purified soluble bovine mitochondrial F₁F_o-ATP synthase contained 2mol of ATP, 2mol of ADP and 6mol of P_i/mol. Incubation of this enzyme with 1mM [³²P]P_i caused the exchange of 2mol of P_i/mol of F₁F_o-ATP synthase. The labelled phosphates were not displaced by ATP. Transfer of F₁F_o-ATP synthase to a buffer containing 30% (v/v) DMSO and 1mM [³²P]P_i resulted in the loss of bound nucleotides with the retention of 1mol of ATP/mol of F₁F_o-ATP synthase. Six molecules of [³²P]P_i 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]P_i into the bound ATP. Azide did not prevent this [³²P]P_i ↔ ATP exchange reaction. The bound labelled ATP could be displaced from the enzyme by exogenous ATP. Addition of ADP to the DMSO-pretreated F₁F_o-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 F₁F_o-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 P_i at a second catalytic site. The bound phosphateŐATP exchange reaction is not readily accommodated by current mechanisms for the ATP synthase.