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).
|Original language||English (US)|
|Title of host publication||Progress in Clinical and Biological Research|
|Number of pages||8|
|State||Published - 1978|
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