Monoclonal antibody modification of the ATPase activity of Escherichia coli F1 ATPase

Abstract

Monoclonal antibodies (mAbs) have been made against each of the five subunits of ECF1 (.alpha., .beta., .gamma., .delta., and .epsilon.), and these have been used in topology studies and for examination of the role of individual subunits in the functioning of the enzyme. All of the mAbs obtained reacted with ECF1, while several failed to react with ECF1F0, including three mAbs against the .gamma. subunit (.gamma.II, .gamma.III, and .gamma.IV), one mAb against .delta., and two mAbs against .epsilon. (.epsilon.I and .epsilon.II). These topology data are consistent with .gamma., .delta., and .epsilon. subunits being located at the interface between the F1 and F0 parts of the complex. Two forms of ECF1 were used to study the effects of mAbs on the ATPase activity of the enzyme: ECF1 with the .epsilon. subunit tightly bound and acting to inhibit activity and ECF1* in which the .delta. and .epsilon. subunits had been removed by organic solvent treatment. ECF1* had an ATPase activity under standard conditions of 93 .mu.mol of ATP hydrolyzed min-1 mg-1, cf. an activity of 7.5 units mg-1 for our standard ECF1 preparation and 64 units mg-1 for enzyme in which the .epsilon. subunit had been removed by trypsin treatment. The protease digestion of ECF1* reduced activity to 64 units mg-1 in a complicated process involving an inhibition of activity by cleavage of the .alpha. subunit, activation by cleavage of .gamma., and inhibition with cleavage of the .beta. subunit. mAbs to the .gamma. subunit, .gamma.II and .gamma.III, activated ECF1 by 4.4- and 2.4-fold, respectively, by changing the affinity of the enzyme for the .epsilon. subunit, as evidenced by density gradient centrifugation experiments. The .gamma.-subunit mAbs did not alter the ATPase activity of ECF1*- or trypsin-treated enzyme. The .alpha.-subunit mAb (.alpha.I) activated ECF1 by a factor of 2.5-fold and ECF1F0 by 1.3-fold, but inhibited the ATPase activity of ECF1* by 30%.