Pi binding by the F1-ATPase of beef heart mitochondria and of the Escherichia coli plasma membrane

Pi binding by the F-1-ATPase of beef heart mitochondria and of the Escherichia coli plasma membrane (E. coli F-1) was examined by two methods: the centrifuge column procedure [Penefsky, H.S. (1977) J. Biol. Chem. 252, 2891-2899] and the Paulus pressure dialysis cell [Paulus, H. (1969) Anal. Biochem. 32, 91-100]. The latter is an equilibrium dialysis-type procedure. Pi binding by beef heart F, could be determined by either procedure. However, direct binding of Pi to E. coli F, could be determined adequately only in the Paulus cell which indicated more than two binding sites per mol of enzyme with a K-d in the range of 0.1 mM. It is concluded that previous failure to observe Pi binding to E. coli F, with the centrifuge column procedure is due to a rapid rate of dissociation of Pi from the E. coli enzyme which results in loss of Pi during transit of the enzyme-Pi complex through the column. (c) 2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.