The inhibitory subunit (ε) of the F1 adenosine triphosphatase (ATPase) was purified to homogeneity from the ML 308-225 and K12(λ) strains of Escherichia coli. No tryptophan or cysteine was detected in the subunit from either strain. The highly active e from both strains was found to be a globular protein with a Stokes' radius of 18-19 Å. Circular dichroism spectra suggested an a-helix content of ~40%. The molecular weight of ε was ~ 15000-16000 by sedimentation equilibrium centrifugation in the presence and absence of guanidinium hydrochloride, molecular sieve chromatography, and gel electrophoresis in the presence of sodium dodecyl sulfate and 8 M urea. The s20, w of ε was ~ 1.6 s-1 Inhibition of the purified F1 ATPase by e displayed noncompetitive kinetics with a Ki of ~ 10 nM. The inhibition of the ATPase was rapidly reversed by diluting the enzyme-e mixture. [125I]ε which was incorporated into ECF1 was readily displaced by unlabeled ε. ε had no significant effect on the ATPase activity of “native” or reconstituted everted membrane vesicles under a variety of assay conditions. Combining the ε-inhibited F1 ATPase with its hydrophobic portion in everted membrane vesicles reconstituted the reversible proton-translocating ATPase and restored nearly full ATPase activity. These results suggest that e inhibits the enzyme only when the F1 ATPase becomes detached from its hydrophobic subunits.
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