Interaction between the soluble F₁ ATPase and its naturally occurring inhibitor protein: Studies using hydrophilic high‐performance liquid chromatography and immunoelectron microscopy

Binding of the isolated ATPase (F₁) to its naturally occurring inhibitor protein was studied by two novel, independent techniques. High‐pressure gel permeation chromatography revealed one tight binding site (K_d= 0.46 μM) for the inhibitor on F₁, and a number of weak, non‐specific sites. Use of an antibody directed against a non‐binding region of the inhibitor protein demonstrated the formation of inhibitor/F₁/immunoglobulin G complexes of 1:1:1 and 2:2:1 stoichiometry, but not of the putatively more stable cyclic 4:2:2 complexes. It was concluded that, despite the presence of three β‐subunits, only one site per F₁ molecule is available for binding its inhibitor protein.