RECONSTITUTION AND FURTHER CHARACTERIZATION OF THE CHOLESTEROL TRANSPORT ACTIVITY OF THE SMALL-INTESTINAL BRUSH-BORDER MEMBRANE

The sterol (free and esterified cholesterol) transport activity of the small-intestinal brush border membrane was solubilized with the short -chain detergent diheptanoylphosphatidylcholine and reconstituted to a n artificial membrane system (proteoliposomes), The resulting proteoli posomes were identified as unilamellar membrane vesicles ranging in si ze between 50 and 200 nm with a broad maximum at 70-110 nm. That the s terol transport protein was indeed incorporated into the lipid bilayer was shown by density gradient centrifugation on a Ficoll gradient: th e proteoliposomes yielded a single band with an apparent density of 1. 035 g/mL, By subjecting solubilized brush border membrane vesicles (BB MV) to gel filtration on Sephadex G-200 prior to reconstitution a 7-fo ld enrichment of the sterol transport activity was achieved relative t o the original BBMV. The experimental evidence presented lends strong support to the notion that the sterol transport protein is an integral protein of the brush border membrane which is anchored in the lipid b ilayer by at least one hydrophobic domain. The active center(s) is (ar e) exposed to the external side of the membrane. Anchoring of this pro tein to the lipid bilayer by a glycosylphosphatidylinositol moiety is unlikely. The reconstituted proteoliposomes behaved very similarly to the original BBMV in terms of facilitated sterol uptake. Using these p roteoliposomes, a hitherto unknown activity of the brush border membra ne was discovered. Long-chain triacylglycerols can be tal;en up by thi s membrane as such and need not be hydrolyzed prior to absorption.