Purification and properties of reconstitutively active and inactive adenosinetriphosphatase from Escherichia coli

The Mg²⁺ and Ca²⁺ stimulated adenosine triphosphatase (ATPase) (EC 3.6.1.3; ATP phosphohydrolase) (bacterial coupling factor) was purified from two strains of E. coli by two different procedures: the method of Neslon, Kanner, and Gutnick, and by a modified procedure described in this paper. The ATPase purified from E. coli K12 (λ) by the first procedure had 4 subunits (α, β, γ, and ε). It did not bind to a deficient membrane, nor did it reconstitute ATP driven transhydrogenase activity. The modified procedure yielded 5 subunits (α, β, γ, δ, and ε). This ATPase could bind to a deficient membrane and reconstitute ATP driven transhydrogenase. This finding suggests that the δ subunit is required for the reaction with the membrane. The molecular weight of the 4 subunit ATPase was significantly lower than that of the 5 subunit ATPase, as judged by equilibrium centrifugation. The specific ATPase activities of both preparations were about the same. These two procedures were also applied to E. coli ML308 to 225.