Bile Acids Stimulate ATP Hydrolysis in the Purified Cholesterol Transporter ABCG5/G8

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• 作者：Brandy J. Harvey Johnson ; Jyh-Yeuan Lee ; Amanda Pickert ; Ina L. Urbatsch
• 刊名：Biochemistry
• 出版年：2010
• 出版时间：April 27, 2010
• 年：2010
• 卷：49
• 期：16
• 页码：3403-3411
• 全文大小：840K
• 年卷期：v.49,no.16(April 27, 2010)
• ISSN：1520-4995

文摘

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 high K_M (MgATP) of 1 mM. Orthovanadate, BeFx, and AlFx effectively inhibited ABCG5/G8 at concentrations of 1 mM. Various lipid mixtures supported bile acid-stimulated ATP hydrolysis, which increased when cholesterol was present. The data demonstrate that bile acids together with lipids and cholesterol increase ATP hydrolysis in purified ABCG5/G8. Bile acids may promote an active conformation of purified ABCG5/G8 either by global stabilization of the transporter or by binding to a specific site on ABCG5/G8.