Purification of Membrane Attachment and Inhibitory Subunits of the Proton Translocating Adenosine Triphosphatase from Escherichia coli

The portion of Escherichia coli adenosine triphosphatase (ATPase) which is peripheral to the membrane (ECF₁) is composed of five separate polypeptides referred to as α,β,γ,δ and ϵ. Treating purified ECF₁ with pyridine precipitated the three larger polypeptides (α,β,γ), but the two smaller ones (δ and ϵ), which represent only about 10% of ECF₁, remained in solution. After removing the pyridine, both δ and ϵ were active and both were obtained in essentially pure form after chromatography on a single molecular-seive column. ϵ strongly inhibited the ATPase activity of ECF₁, indicating that e has a regulatory role in the enzyme. ϵ inhibited ECF₁ missing δ, indicating that δ is not required for inhibition by ϵ. However, enzyme containing just the α and β subunits, which was prepared by treating ECF₁ with a protease, was fully active hydrolytically but not at all sensitive to inhibition by α. This result suggests that the γ polypeptide is required for the inhibition of the ATPase by ϵ. δ restored the capacity of ECF₁ missing δ to recombine with ECF₁-depleted membrane vesicles. The ECF₁,which became attached to the vesicles by the added δ, was functional in energy transduction, as evidenced by the coupling of ATP hydrolysis to the transhydrogenase reaction in the vesicles. The rebinding of ECF₁ missing δ was directly proportional to the amount of δ added until all the ECF₁ receptors in the membranes were occupied. δ may be a stalk which connects the F₁ headpiece to the membrane, since the attachment of ECF₁ to the membrane exhibited an absolute dependence on δ. Although 5 is known to have an apparent molecular weight of about 20 000 by gel electrophoresis in the presence of sodium dodecyl sulfate, the active δ eluted from a molecular-seive column with an apparent molecular weight of about 35 000, suggesting that in the active form δ is a dimer or rather elongated in shape. The active ϵ subunit eluted from the same column with an apparent molecular weight of about 16 000.