Permeability enhancement of lipid vesicles to nucleotides by use of sodium cholate: Basic studies and application to an enzyme-catalyzed reaction occurring inside the vesicles

摘要

The effect of the bile salt sodium cholate on the permeability properties of lipid vesicles (liposomes) to mononucleotides at pH 8.0 (0.1 M Tris-HCl) in the presence of 5 MM MgCl2 was investigated. The vesicles mainly used were unilamellar with diameters of about 100 nm, prepared from POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). Depending on the total amount of cholate and POPC, the vesicles could be loaded with ADP (adenosine 5'-diphosphate), UMP (uridine 5'-monophosphate), UDP (uridine 5'-diphosphate), or UTP (uridine 5'-triphosphate), under conditions where bilayer solubilization did not occur, R-e much less than R-e(sat), with R-e being the molar ratio of cholate in the vesicle bilayer to POPC under conditions of the uptake experiments (R-e) or under conditions of bilayer saturation with cholate (R-e(sat)), as determined by light scattering (turbidity) measurements. The uptake experiments were preceded by a detailed study of the interaction between cholate and the POPC vesicles. Most importantly, freeze-fracture electron microscopy showed that vesicle solubilization begun at R-e values lower than R-e(sat) estimated turbidimetrically. This finding indicates that care has to be taken if vesicle solubilization studies are based only on the measurements of changes in turbidity. Although a number of investigations have been devoted in the past to the cholate-induced release of water-soluble molecules from vesicles, the present work is probably the first detailed study on the reverse process. Nucleotide uptake by the vesicles at subsolubilizing cholate concentration was confirmed in the case of ADP by using vesicles containing entrapped Micrococcus luteus polynucleotide phosphorylase, which catalyzed the endovesicular polymerization of exovesicularly added ADP to poly-A. These latter experiments show that enzyme-containing vesicles can be used as nanoreactor systems in which the permeability of the bilayers is selectively altered by a surfactant, allowing the uptake of substrate molecules but not the release of the entrapped enzyme or reaction product. [References: 79]