Reconstitution of functional water channels in liposomes containing purified red cell CHIP28 protein. The effect of the unstirred layer on human red cell water permeability. Membrane water and solute permeability determined quantitatively by self-quenching of an entrapped fluorophore. Membrane thickness, lipid phase and sterol type are determining factors in the permeability of membranes to small solutes. Water permeability of asymmetric planar lipid bilayers. Reconstituting the barrier properties of a water-tight epithelial membrane by design of leaflet-specific liposomes. Functional reconstitution and characterization of AqpZ, the E. J., Kozono, D., Calamita, G., Maloney, P. Relation of growth of Streptococcus lactis and Streptococcus cremoris to amino acid transport. The role of futile cycles in the energetics of bacterial growth. Weak acid permeation in synthetic lipid vesicles and across the yeast plasma membrane. Osmotic properties and water permeability of phospholipid liquid crystals. Drug-like Properties: Concepts, Structure Optimization, Design and Methods from ADME to Toxicity (Academic, 2008).īangham, A. Intrinsic membrane permeability to small molecules. Coupling efficiency of secondary active transporters. Structures and general transport mechanisms by the major facilitator superfamily (MFS). Collectively, these procedures provide a comprehensive toolbox for determining membrane permeability coefficients in a variety of experimental systems, and typically take 2–3 d. Data on membrane permeation are obtained using either conventional or stopped-flow kinetic fluorescence measurements on instruments available in most research institutes and are analyzed with a suite of user-friendly MATLAB scripts ( ). We describe the preparation of synthetic vesicles of varying lipid composition and determination of vesicle size distribution by dynamic light scattering. We also present analogous procedures to probe weak acid and base permeability in vesicles and cells by using the read-out of encapsulated or expressed pH-sensitive probes. Encapsulation of the fluorescent dye calcein into lipid vesicles allows monitoring of volume changes upon osmotic shifts of the medium via (de)quenching of the fluorophore, which we interpret using a well-defined physical model that takes the dynamics of the vesicles into account to calculate the permeability coefficients of solutes. This protocol describes stopped-flow fluorimetry measurements performed on lipid vesicles and living yeast cells to estimate the osmotic permeability of water and solutes across (bio)membranes. The quantification of passive solute permeation is possible with radio-isotope distribution experiments, spectroscopic measurements and molecular dynamics simulations. The passive permeability of cell membranes is of key importance in biology, biomedical research and biotechnology as it determines the extent to which various molecules such as drugs, products of metabolism, and toxins can enter or leave the cell unaided by dedicated transport proteins.
0 kommentar(er)
