Biophysical studies of the interactions of several bioactive lipids in model membranes.

摘要

The use of model systems affords the researcher an opportunity to study the complex topic of membrane dynamics and structure under controlled conditions. The investigation into an array of the properties of a small group of bioactive lipids was undertaken, and is summarized in Chapter 1. Isothermal titration calorimetry, compression of lipid monolayers, detergent solubility, and cyclodextrin-mediated efflux assays were used to investigate some of the properties of cholesterol analogs and oxidized sterols, lysophosphatidic acid (LPA), and lipopolysaccharide (LPS).; In Chapter 2, the properties of a photoactivatable derivative and fluorescent analogs of cholesterol were examined to assess their ability to faithfully mimic the parent molecule in model membrane systems. The photoactivatable probe, 6-photocholesterol, behaved in a manner similar to cholesterol. Two structurally related novel fluorescent cholesterol analogs were also examined: one, in which a BODIPY fluorophore is attached via an ester linkage, differed from cholesterol with respect to lipid-lipid interactions and may localize into non-raft domains in fluorescence studies; the other, coupled to the fluorophore without polar atoms, possessed properties that resembled those of cholesterol and appears to have utility as a membrane mimetic probe. The enantiomer of cholesterol interacted with phospholipids in monolayers and vesicles in a similar manner as cholesterol, as discussed in Chapter 3.; Results reported in Chapter 4 reveal significant variations between cholesterol and several of its oxidized products with regard to condensation of 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine monolayers and support of the formation of detergent-resistant lipid rafts.; Chapter 5 presents an isothermal titration calorimetric study of the equilibrium binding of LPA, a structurally simple lipid molecule with wide ranging biological activities, to the lipid-binding domain of the actin-severing protein gelsolin, denoted as P2. A strong dependence for LPA-gelsolin interactions on salt concentration and LPA structure was found, suggesting a high degree of specificity of the peptide for this lipid. P2 has a higher affinity for LPS from P. aeruginosa than for 1-oleoyl-LPA.; Calorimetric measurements of the critical micelle concentrations (CMC) of LPA and sphingosylphosphorylcholine (SPC) are presented in Chapter 6. A dependence on salt concentration and structure was revealed for LPA, but no such salt sensitivity was observed for SPC. However, reduction of the double bond in SPC lowered the CMC by a factor of three.