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 an artificial membrane system (proteoliposomes). The resulting proteoliposomes were identified as unilamellar membrane vesicles ranging in size between 50 and 200 nm with a broad maximum at 70-110 nm. That the sterol transport protein was indeed incorporated into the lipid bilayer was shown by density gradient centrifugation on a Ficoll gradient: the proteoliposomes yielded a single band with an apparent density of 1.035 g/mL. By subjecting solubilized brush border membrane vesicles (BBMV) to gel filtration on Sephadex G-200 prior to reconstitution, a 7-fold enrichment of the sterol transport activity was achieved relative to 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 bilayer by at least one hydrophobic domain. The active center(s) is (are) exposed to the external side of the membrane. Anchoring of this protein 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 proteoliposomes, a hitherto unknown activity of the brush border membrane was discovered. Long-chain triacylglycerols can be taken up by this membrane as such and need not be hydrolyzed prior to absorption.
Boffelli
D
D
Laboratorium für Biochemie, Eidgenössische Technische Hochschule Zürich, Switzerland.
Weber
F E
FE
Compassi
S
S
Werder
M
M
Schulthess
G
G
Hauser
H
H
D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000302:isolation & purification; D002784:Cholesterol / Q000378:metabolism*; D007421:Intestine, Small / Q000378:metabolism* / Q000648:ultrastructure*; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008567:Membranes, Artificial; D008871:Microvilli / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D011510:Proteolipids / Q000378:metabolism; D011817:Rabbits; D014304:Triolein / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism
D002352:Carrier Proteins; D008081:Liposomes; D008565:Membrane Proteins; D008567:Membranes, Artificial; D010716:Phosphatidylinositols; D011510:Proteolipids; D014304:Triolein; D002784:Cholesterol; D010738:Type C Phospholipases