| Summary: | A universal characteristic of cells is the membrane boundary that separates the interior cytoplasm from the external environment. This thesis examines the net transport of molecules such as certain metal ions, modified amino acids and peptides across the membranes of model (liposome) systems in response to transmembrane pH gradients. This net transport arises from the membrane permeable nature of the neutral form of the molecule or ion complex. For comparison, permeability studies were conducted for several types of amino acids which cannot readily adopt a neutral form.
The first section demonstrates that iron (primarily in the form of Fe+2) and barium can be accumulated into EPC and DSPC-cholesterol (55:45 mole %) large unilamellar vesicles (LUV's) in response to a transmembrane pH gradient (interior acidic) and in the presence of the ionophore A23187. It is shown that the maximally loaded Fe- and Ba-containing LUVs exhibit increased densities in that a significant fraction of the maximally loaded LUV's can be pelleted by low speed centrifugation and the Ba+2-loaded systems can be directly visualized by cryo-electron microscopy. The second area of investigation concerned the uptake of derivatives of lysine and a pentapeptide (Ala-Met-Leu-Trp-Ala), in which the C-terminal carboxyl functions have been converted to methyl esters or amides, in response to transmembrane pH gradients in LUV systems. It is shown that the presence of a pH gradient(interior acidic) results in the rapid and efficient accumulation of these basic amino acid and peptide derivatives into LUVs in a manner consistent with permeation of the neutral (deprotonated) form. The permeability coefficient of the neutral form of lysine methyl ester was 2.1 X 10"2 cm.s"1. It is suggested that this property may have general implications for mechanisms of transbilayer translocation of peptides, such as signal sequences, which exhibit weak base characteristics. The third part of the thesis concerned the permeability properties of neutral, hydrophobic, polar and charged amino acids, which did not have weak base properties. The rates of efflux of glycine, lysine, phenylalanine, serine and tryptophan were determined after they were passively entrapped in EPC and DMPC LUVs. The permeability coefficients for the neutral, polar and charged amino acids were approximately-1 10-12 cm s for EPC vesicles, while those for DMPC vesicles were approximately 10-11 cm s"1. The LUVs were10-100 times more permeable to the hydrophobic amino acids. Variations in pH had only minor effects on the permeability coefficients. Permeation rates for the amino acids studied were 108slower than those of the modified amino acids indicating that different mechanisms of efflux, including permeation of the neutral or charged forms of the amino acids or transient defects, may be responsible. The final area of study involved examining the influence of hydrophobicity and charge distribution on the uptake of modified peptides, which were weak bases. Here, the ability of small (2-3 amino acid) peptides, composed exclusively of basic (lysine) and hydrophobic (tryptophan) amino acids, to accumulate into LUV systems was investigated. In the case of the dipeptides Trp-Lys-Amide and Lys-Trp-Amide, remarkable differences in the rate constants associated withnet transport were observed. In EPC: cholesterol LUV systems exhibiting a ApH of 3 units (pH1=4.0; pH0=7.0), for example, the rate constant for the uptake of the Lys-Trp-Amide was some 5 X 103 faster than for the Trp-Lys-Amide. This difference could not be attributed to changes in the membrane-water partition coefficients. Related effects were observed for the tripeptides composed of one lysine and two tryptophan residues. It is concluded that different charge
distributions in short peptides of identical amino acid composition can profoundly influence the ability of these groups to associate with and permeate across lipid bilayers. === Medicine, Faculty of === Biochemistry and Molecular Biology, Department of === Graduate
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