文摘
Mutations in the ATP-binding cassette (ABC) transporters ABCG5 and ABCG8 lead tositosterolemia, a disorder characterized by sterol accumulation and premature atherosclerosis. ABCG5and ABCG8 are both half-size transporters that have been proposed to function as heterodimers in vivo.We have expressed the recombinant human ABCG5 and ABCG8 genes in the yeast Pichia pastoris andpurified the proteins to near homogeneity. Purified ABCG5 and ABCG8 had very low ATPase activities(<5 nmol min-1 mg-1), suggesting that expression of ABCG5 or ABCG8 alone yielded nonfunctionaltransporters. Coexpression of the two genes in P. pastoris greatly increased the yield of pure proteins,indicating that the two transporters stabilize each other during expression and purification. CopurifiedABCG5/G8 displayed low but significant ATPase activity with a Vmax of ~15 nmol min-1 mg-1. TheATPase activity was not stimulated by sterols. The catalytic activity of copurified ABCG5/G8 wascharacterized in detail, demonstrating low affinity for MgATP, a preference for Mg as a metal cofactorand ATP as a hydrolyzed substrate, and a pH optimum near 8.0. AlFx and BeFx inhibited MgATPhydrolysis by specific trapping of nucleotides in the ABCG5/G8 proteins. Furthermore, ABCG5/G8 elutedas a dimer on gel filtration columns. The data suggest that the hetero-dimer is the catalytically activespecies, and likely the active species in vivo.
