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 the 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 mumol 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%.

Defining the Domain of Binding of F1 Subunit ε with the Polar Loop of F0 Subunit c in theEscherichia coli ATP Synthase

“…The mouse monoclonal antibody to subunit ⑀ (13-A7, ⑀II; Ref. 45) was a gift from Dr. R. Capaldi (University of Oregon, Eugene, OR).…”

The Stalk Region of the Escherichia coli ATP Synthase

“…Proteins were transferred from the gel to polyvinylidene difluoride (Millipore Corp.) as described previously (12). The mouse monoclonal antibody to ␥, anti-␥ III , was characterized previously (26). The rabbit antiserum to subunit c was described by Girvin et al (27).…”

Characterization of the Interface between γ and ε Subunits of Escherichia coli F1-ATPase

“…Mutants ␥S8C, ␥T106C, and ␥V286C are described by . ECF 1 isolated from these mutants was labeled with CM 1 or NEM as described by Tang et al (1994) before being treated with trypsin in MOPS buffer (50 mM MOPS, pH 7.0, 0.5 mM EDTA, and 10% glycerol), plus additional 5 mM ATP at room temperature for 1 h. The monoclonal antibodies used in this study were prepared and characterized as described by Aggeler et al (1990).…”