Abstract
Subjecting rabbit small intestinal brush border membrane vesicles (BBMV) to freeze-thaw cycles releases water-soluble lipid exchange (transfer) proteins into the supernatant. They differ widely in apparent molecular weight and catalyze cholesterol, phosphatidylcholine, and phosphatidylinositol exchange between two populations of small unilamellar lipid vesicles. In order to determine their interrelations, the smallest water-soluble lipid exchange protein was purified to homogeneity by gel filtration on Sephadex G-75 and cation exchange chromatography on Mono S. It is a basic protein of apparent molecular mass of 13 +/- 0.5 kDa. The purified protein was used to raise polyclonal antibodies. Polyclonal antibodies were also produced against a lipid exchange protein of apparent molecular mass of 100-120 kDa. By comparing lipid exchange, lipid binding, and immunological properties of the water-soluble lipid exchange proteins it can be shown that the 13-kDa (peak 3) protein is related to the 100-120 kDa (peak 1) protein; the properties of these two proteins are different from those of the peak 2 lipid exchange protein of apparent molecular mass of 22 kDa. Based on the immunological cross-reactivity observed between the 13 and 100-120 kDa and the lipid binding properties of these two proteins, a working hypothesis is proposed: both proteins are probably part of an integral membrane protein of the brush border membrane that facilitates cholesterol and phosphatidylcholine absorption in this membrane. Evidence derived from immunogold labeling of BBMV supports the notion that this protein is located on the external (luminal) side of the brush border membrane. The analogous behavior of rabbit and human small intestinal brush border membrane in terms of lipid absorption and the release of water-soluble lipid exchange proteins is discussed.
Highlights
From the Laboratorium fiir Biochemie, Eidgenossische Technische Hochschule, ETH-Zentrum, CH-8092 Zurich, Switzerland and the Wepartement fiir Innere Medizin, Medizinische Poliklinik, Universitatsspital, CH-8091 Zilrich, Switzerland
Evidence derived from immunogold labeling of brush border membrane vesicles (BBMV) supports the notion that this protein is located on the external side of the brush border membrane
We reported that the absorption of cholesterol by brush border membrane vesicles (BBMV)1 made from rabbit small intes
Summary
From the Laboratorium fiir Biochemie, Eidgenossische Technische Hochschule, ETH-Zentrum, CH-8092 Zurich, Switzerland and the Wepartement fiir Innere Medizin, Medizinische Poliklinik, Universitatsspital, CH-8091 Zilrich, Switzerland. Subjecting rabbit small intestinal brush border membrane vesicles (BBMV) to freeze-thaw cycles releases water-soluble lipid exchange (transfer) proteins into the supernatant. Mter subjecting BBMV to proteolytic treatment, cholesterol absorption was shown to be a passive process characterized by half-times of the order of hours even with bile salt micelles as donor particles [2]. It involves cholesterol desorption from the donor, diffusion of monomeric cholesterol through the aqueous phase, and incorporation of cholesterol into the bilayer of BBM [1]. The passive process is a true first-order reaction indicating that cholesterol desorption is the rate-limiting step [1]
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