Bile acids stimulate ATP hydrolysis in the purified cholesterol transporter ABCG5/G8.

ABCG5 and ABCG8 are half-size ABC transporters that function as heterodimers (ABCG5/G8) to reduce sterol absorption in the intestines and increase sterol excretion from the liver. Previous studies demonstrated that bile acids increased ABCG5/G8 specific cholesterol efflux in cell models. In this study we tested the effects of bile acids on ATP hydrolysis in Pichia pastoris purified ABCG5/G8 and found that they stimulated hydrolysis ∼20-fold in wild-type ABCG5/G8 but not in a hydrolysis-deficient mutant. Nonconjugated cholate supported the highest ATPase activity in ABCG5/G8 (256 ± 9 nmol min−1 mg−1). ATP hydrolysis was also stimulated by other conjugated bile acids and a mixture of bile acids resembling human bile with activities ranging from 129 ± 4 to 147 ± 14 nmol min−1 mg−1. The kinetic parameters, inhibitor profiles, and lipid requirements of bile acid stimulated ATP hydrolysis were characterized. Cholate-stimulated ATP hydrolysis was maximal at concentrations of ≥10 mM MgATP and had a relatively hig...