Percent cholesterol absorption in normal women and men quantified with dual stable isotopic tracers and negative ion mass spectrometry

Amino acid substitutions at many positions in the a subunit of F1F0 ATP synthase result in impaired proton translocation and altered catalytic activity. In this work, we demonstrate that amino acid substitutions in the a subunit affect the epsilon subunit. In mutant F1F0 ATP synthases, the epsilon subunit was studied by determining its sensitivity to proteolysis and by chemical crosslinking under conditions of active turnover and in quiescent enzyme. Like native F1F0 ATP synthase, the epsilon subunit in enzymes carrying either the aarg-210-->ile or agly-218-->asp substitutions proved resistant to trypsin digestion during ATP hydrolysis. In each case, the epsilon subunit was rapidly digested in the presence of a nonhydrolyzable ligand, but this did not result in the activation of hydrolytic activity typically seen in wild-type enzyme. In enzyme carrying the aala-217-->arg substitution, the trypsin digestion of the epsilon subunit occurred regardless of ligand and was accompanied by a limited hydrolytic activation. Relative to the native F1F0 ATP synthase, the aala-217-->arg substitution resulted in reduced efficiency of crosslinking between the epsilon and beta subunits using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. These observations indicate that the structural changes resulting from amino acid substitutions in the a subunit are propagated to the epsilon subunit and are specific to the individual substitutions.