Characterization of lipid exchange proteins isolated from small intestinal brush border membrane.

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.

Membrane proteins exposed on the external side of the intestinal brush-border membrane have fusogenic properties.

The intestinal brush-border membrane contains one or several membrane proteins that mediate fusion and/or aggregation of small unilamellar egg phosphatidylcholine vesicles. The fusion is accompanied by a partial loss of vesicle contents. Proteolytic treatment of the brush-border membrane with proteinase K abolishes the fusogenic property. This finding suggests that the fusogenic activity is associated with a membrane protein exposed on the external or luminal side of the brush-border membrane. Activation of intrinsic proteinases of the brush-border membrane liberates water-soluble proteins (supernate proteins). These proteins behave in an analogous way to intact brush-border membrane vesicles; they induce fusion of egg phosphatidylcholine vesicles and render the egg phosphatidylcholine bilayer permeable to ions and small molecules (Mr less than or equal to 5000). Furthermore, supernate proteins mediate phosphatidylcholine and cholesterol exchange between two populations of small, unilamellar phospholipid vesicles. Supernate proteins are fractionated on Sephadex G-75 SF yielding three protein peaks of apparent Mr greater than or equal to 70,000, Mr = 22,000 and Mr = 11,500. All three protein fractions show similar phosphatidylcholine-exchange activity, but they differ in their effects on the stability of egg phosphatidylcholine vesicles. The protein fraction with an apparent Mr greater than or equal to 70,000 has the highest fusogenic activity while the protein fraction of apparent Mr = 11,500 appears to be most effective in rendering the egg phosphatidylcholine bilayer permeable.

Cholesterol-transfer protein located in the intestinal brush-border membrane. Partial purification and characterization.

Cholesterol absorption by small intestinal brush border membrane vesicles from taurocholate mixed micelles is a second-order reaction. From a comparison of reaction rates and order before and after proteinase K treatment of brush-border membrane vesicles, it is concluded that cholesterol absorption is protein-mediated. It is shown that the desorption of cholesterol from taurocholate mixed micelles is by a factor of about 10(4) faster than that from egg phosphatidylcholine bilayers. When brush border membrane vesicles are stored at room temperature, intrinsic proteinases are activated and proteins are liberated from the brush border membrane. These proteins collected in the supernatant catalyze cholesterol and phosphatidylcholine exchange between two populations of small unilamellar phospholipid vesicles. One of the active proteins present in the supernatant is purified by a two-step procedure involving gel filtration on Sephadex G-75 SF and affinity chromatography on a Nucleosil-phosphatidylcholine column. The protein thus obtained is pure by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. It has an apparent molecular weight of slightly less than 14,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and a value of 11,500 determined by gel filtration on Sephadex G-75 SF.

The uptake of phosphatidylcholine by small intestinal brush border membrane is protein-mediated.

Brush border membrane vesicles prepared from rabbit small intestine are essentially free of basolateral membranes and nuclear, mitochondrial, microsomal and cytosolic contaminants. The resulting brush border membrane is unstable due to intrinsic lipases and proteinases. The PC transfer between small unilamellar lipid vesicles or mixed lipid micelles as the donor and the brush border membrane vesicles as the acceptor is protein-mediated. After proteolytic treatment of brush border membrane with papain or proteinase K the PC transfer activity is lost and the kinetics of PC uptake are similar to those measured with erythrocytes under comparable conditions. Evidence is presented to show that the PC transfer activity resides in the apical membrane of the enterocyte and not in the basolateral part of the plasma membrane. Furthermore, the activity is localized on the external surface of the brush border membrane exposed to the aqueous medium with its active centre probably not in direct contact with the lipid bilayer of the membrane. Proteins released from brush border membrane by proteolytic treatment catalyze PC exchange between different populations of small unilamellar vesicles. Furthermore, these protein(s) bind(s) PC forming a PC-protein complex.

Uptake of cholesterol by small intestinal brush border membrane is protein-mediated.

Absorption of cholesterol by small intestinal brush border membrane from either mixed micelles or small unilamellar vesicles is protein-mediated. It is a second-order reaction. The kinetic data are consistent with a mechanism involving collision-induced transfer of cholesterol. With micelles as the donor particle, there is net transfer of cholesterol while with small unilamellar vesicles as the donor, cholesterol is evenly distributed between the two lipid pools at equilibrium. The cholesterol absorption by brush border membrane from both mixed micelles and small unilamellar vesicles reveals saturation kinetics. Proteolytic treatment of brush border membrane with papain releases about 25% of the total membrane protein. As a result, the cholesterol uptake by brush border membrane changes from a second-order reaction to a first-order one. The reaction mechanism changes from collision-induced cholesterol uptake to a mechanism involving diffusion of monomeric cholesterol through the aqueous phase. The protein(s) released into the supernatant by papain treatment of brush border membrane exhibit(s) cholesterol exchange activity between two populations of small unilamellar vesicles. The supernate-protein(s) bind(s) the spin-labeled cholesterol analogue 3-doxyl-5 alpha-cholestane.

A new electron spin resonance assay for membrane asymmetry and entrapped volume of unilamellar lipid vesicles based on photoreduced flavin adenine dinucleotide.

A new ESR assay has been developed for the characterization of unilamellar lipid vesicles. It is based on the reduction by photogenerated FADH2 of amphiphilic spin-labels having the spin in the polar group. FADH2 is generated in situ under anaerobic conditions from its oxidized form (FAD) by photoreduction in the presence of excess EDTA as the reducing agent. Photoreduction is induced by exposing the FAD/EDTA mixture to white light of a commercial slide projector. FADH2 as an impermeable agent reduces spin-label molecules located on the outer layer of the bilayer that are readily accessible in a first fast reaction; spin-label located on the inner layer of the bilayer is reduced in a second slow reaction. The ESR assay is suitable for the routine characterization of unilamellar membrane vesicles: it allows the determination of the vesicle size, the entrapped volume, the bilayer asymmetry, the bilayer integrity, and the vesicle stability. The ESR assay developed is of general applicability: it can be used with charged and uncharged bilayers which may be labeled with either neutral or charged spin-labels. An assessment of the new ESR assay is given in comparison to the existing ascorbate method which uses sodium ascorbate as the reducing agent. Various other potential reducing agents for spin-labels have been tested and found unsuitable for the ESR assays discussed here.

Cholesterol oxidase as a structural probe of biological membranes: its application to brush-border membrane.

Cholesterol present in intact brush-border membrane vesicles made from rabbit small intestine is a poor substrate for cholesterol oxidase (EC 1.1.3.6, from Nocardia sp. and Nocardia erythropolis). It becomes susceptible to oxidation by the enzyme only after the addition of detergent, e.g., Triton X-100, in quantities sufficient to disrupt the membrane. This is also true for cholesterol present in bilayers of small unilamellar phosphatidylcholine or phosphatidylserine vesicles made by ultrasonication. The data presented here on intestinal brush-border membrane are in good agreement with results reported on other biological membranes, e.g., from erythrocytes and vesicular stomatitis virus, but are somewhat different from those on rat intestinal brush-border membrane. Our results on phospholipid bilayers agree well with published work on model membranes. From the work presented we conclude that, with our present understanding, cholesterol oxidase can hardly be used to probe the distribution of cholesterol in biological membranes. A prerequisite for using the enzyme successfully as such a probe would be the understanding of the factors controlling the interaction of the enzyme with its substrate cholesterol. The question under which conditions cholesterol oxidase could be useful for probing the distribution and preferred location of cholesterol in biological membranes is discussed.

Structure-activity relationship in vinculin: an IR/attenuated total reflection spectroscopic and film balance study.

Surfacing and membrane-penetrating ability of vinculin and bovine serum albumin have been studied on a macroscopic level by means of a Langmuir film balance and on a molecular level by means of infrared attenuated total reflection spectroscopy. It is suggested that the driving force of the nonspontaneous process of membrane penetration by native vinculin is the spontaneous formation of rigid vinculin monolayers in the membrane. Lateral adhesion of vinculin molecules results from the formation of intermolecular pleated-sheet structures. Vinculin surface activity was found to result from an alpha-helical segment oriented approximately perpendicular to plane of the membrane. There is a conformational equilibrium between this helix and random structure. High ionic strength (110 mM) favors helix formation that leads to the greater than 100-fold enhancement of surfacing velocity relative to the velocity observed at a lower ionic strength (10 mM). Vinculin has a second helical segment oriented parallel to the plane of the membrane that is in a conformational equilibrium with the pleated-sheet structure.