Phosphate exchange and ATP synthesis by DMSO-pretreated purified bovine mitochondrial ATP synthase.
Purified soluble bovine mitochondrial F(1)F(o)-ATP synthase contained 2 mol of ATP, 2 mol of ADP and 6 mol of P(i)/mol. Incubation of this enzyme with 1 mM [(32)P]P(i) caused the exchange of 2 mol of P(i)/mol of F(1)F(o)-ATP synthase. The labelled phosphates were not displaced by ATP. Transfer of F(1)F(o)-ATP synthase to a buffer containing 30% (v/v) DMSO and 1 mM [(32)P]P(i) resulted in the loss of bound nucleotides with the retention of 1 mol of ATP/mol of F(1)F(o)-ATP synthase. Six molecules of [(32)P]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 [(32)P]P(i) into the bound ATP. Azide did not prevent this [(32)P]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 F(1)F(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 F(1)F(o)-ATP synthase contained one molecule of ATP at a catalytic site which was competent to carry out a phosphate-ATP 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 phosphate-ATP exchange reaction is not readily accommodated by current mechanisms for the ATP synthase.
Beharry
S
S
Department of Biochemistry and Molecular Biology, University of British Columbia, 2146 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3.
Bragg
P D
PD
D000227:Adenine Nucleotides / Q000737:chemistry; D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000096:biosynthesis* / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D003470:Culture Media; D004121:Dimethyl Sulfoxide / Q000032:analysis / Q000494:pharmacology; D008928:Mitochondria / Q000201:enzymology*; D010710:Phosphates / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000302:isolation & purification*
D000227:Adenine Nucleotides; D003470:Culture Media; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D004121:Dimethyl Sulfoxide