Some effects of trypsin on the subunits of the membrane ATPase from Escherichia coli

Five-subunit (α, β, γ, δ, ε) F₁-ATPase from E. coli (ECF₁) was reconstituted by combining 2 mixtures of inactive subunits (an αβ fraction and a γε-rich one) and the purified δ subunit. The combination of αβ and γε-rich fractions was sufficient for reconstituting ATPase activity, which was achieved by dialyzing the subunits at 23° C in the presence of Mg-ATP. Addition of the purified δ subunmit to the reconstituted ATPase restored coupling factor activity to the enzyme. The reconstituted enzyme was as active as the native enzyme in restoring ATP-driven transhydrogenase activity to F₁-depleted vesicles. Incubation of the γε-rich fraction with trypsin decreased markedly the reconstitution of ATPase activity, whereas treatment of the αβ fraction with trypsin under the same conditions had no significant effect. ECF₁ containing only the α and β subunits, which was prepared by trypsin digestion, was highly active hydrolytically but remained inactive as a coupling factor even after the addition of the γε-rich fraction and δ. The inhibition of ECF₁ by the purified ε subunit was reversed by trypsin. Thus, while trypsin inactivates the γ and ε subunits of ECF₁, the α and/or β subunits are altered in a way which only inactivates the coupling factor activity of the enzyme without affecting its ATPase activity or the reconstitution of ATPase activity after cold inactivation. Since exposure of the α and β subunits to trypsin does not appear to alter their interaction with added γ and ε, it may be that the loss of coupling factor activity is due to a modification in α or β which disrupts their interaction with the membrane attachment subunit (δ).