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 approximately 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 >or=10 mM MgATP and had a relatively high K(M) (MgATP) of approximately 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.
Johnson
Brandy J Harvey
BJ
Department of Cell Biology and Biochemistry and Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA.
Lee
Jyh-Yeuan
JY
Pickert
Amanda
A
Urbatsch
Ina L
IL
D000071018:ATP Binding Cassette Transporter, Subfamily G, Member 5; D000071019:ATP Binding Cassette Transporter, Subfamily G, Member 8; D018528:ATP-Binding Cassette Transporters / Q000302:isolation & purification / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001647:Bile Acids and Salts / Q000494:pharmacology*; D002417:Cattle; D017124:Conserved Sequence; D017931:DNA Primers; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008074:Lipoproteins / Q000302:isolation & purification / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
C057215:8.2K CTP protein, Bos taurus; C416945:ABCG5 protein, human; C416946:ABCG8 protein, human; D000071018:ATP Binding Cassette Transporter, Subfamily G, Member 5; D000071019:ATP Binding Cassette Transporter, Subfamily G, Member 8; D018528:ATP-Binding Cassette Transporters; D001647:Bile Acids and Salts; D017931:DNA Primers; D008074:Lipoproteins; D026901:Membrane Transport Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases