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Egg phosphatidylcholine was oxidized by atmospheric oxygen under UV light for 16 hours, and the oxidized products formed were fractionated with high-pressure liquid chromatography in reverse phase. Three fractions that appeared at retention times of 19 minutes, 21 minutes and 24 minutes respectively (fraction 19, fraction 21 and fraction 24) were isolated and stabilized by reduction with triphenylphosphine. Zinc diffusion across 1-palmitoyl-2 oleoyl-sn-glycero-3-phosphocholine (POPC) liposome bilayers mixed with the isolated oxidized fractions was measured. The rate constant for zinc diffusion through the POPC liposome was highest in fraction 19 followed by fraction 21 and fraction 24.NMR data suggests that all oxidized fractions were derived from the major egg polyunsaturated PC, 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine. The primary oxidized product, fraction 24 contains a mixture of isomers in which the linoleoyl group has formed the 9-hydroxy-10,12-trans-cis diene and trans-trans diene or the 13-hydroxy12,10-trans-cis diene and trans-trans diene. The primary oxidized products on further oxidation, result in secondary oxidized products, contained in fraction 21 and fraction 19.Experimental data indicates that the major components of fraction 21 are the 9-hydroxy12,13-epoxy-l0-trans-monoene (and 13-hydroxy-9,10-epoxy-11-trans-monoene) and the major components of fraction 19 are the 9,12,13-trihydroxy-l0-trans-monoene (and 9,10,13-trihydroxy-1 l-trans-monoene). / Department of Chemistry

Includes bibliographical references.

Thesis (Ph.D.)--University of Delaware, 2006. / Principal faculty advisors: Dionisios G. Vlachos and Jeremy S. Edwards, Dept. of Chemical Engineering. Includes bibliographical references.

Two methods were used to produce exogenous lipid/bR complexes. A detergent method (Huang et al., 1980) reconstituted bR into DMPC or DMPG bilayers, free of all endogenous purple membrane lipids as shown by high resolution ³¹ P-NMR. A novel biological detergent-free method employed bovine liver non-specific lipid transfer protein (nsTP) to mediate addition of DMPC to purple membrane, while retaining 76 - 86% of the endogenous purple membrane phospholipids. The variations with temperature of 2H-NMR quadrupole splittings for the DMPC choline α- and β-methylene CD2 segments were similar to those for protein-free lipid (Gaily et al., 1975) implying that temperature dependent changes in segmental amplitudes of motion within the choline group are preserved in the presence of bR. Incorporation of small quantities of bR increased the amplitudes of segmental motion within the choline headgroup relative to that of pure lipid, but increasing the bR content induced an ordering effect. The choline α- and β-methylene segment quadrupole splittings showed a linear variation with protein content at constant temperature. This is consistent with freeze fracture electron microscopy data, which shows the bR particles to be dispersed at all lipid/protein ratios, when quenched from temperatures above the phase transition. Applying a fast two site exchange model to the ²H-NMR data, values between 12 and 15 were calculated for the number of boundary lipids for bR (26,000 M_r) in DMPC bilayers free of purple membrane lipids. From ESR data, for delipidated bR in DMPC and DMPG bilayers at temperatures above the phase transition, the number of boundary lipids calculated were 18 - 21, which is consistent with the bR being monomeric, as also observed in DMPC bilayers with all the purple membrane lipids retained (Cherry et al., 1978). The purple membrane lipids thus appear to mediate crystallization of the bR particles into a hexagonal lattice at temperatures slightly below the exogenous lipid phase transition.

Thesis (M. Sc.)--Carleton University, 2000. / Includes bibliographical references. Also available in electronic format on the Internet.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22472号 / 工博第4733号 / 新制||工||1739(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 梅田 眞郷, 教授 浜地 格, 教授 跡見 晴幸 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

The influence of the membrane environment on the α1-adrenoceptor has been investigated by examining the effect of phospholipase digestion on the binding of [3H]prazosin to aortic and hepatic membranes. Membrane digestion by phospholipase A2 and phospholipase C was found to markedly reduce prazosin binding to the α1-adrenoceptor whereas phospholipase D had comparatively little effect. In addition, there were differences between membrane preparations since the aortic α1-adrenoceptor was less sensitive to phospholipase A2 and phospholipase C than the hepatic receptor. The results support a major role for hydrophobic groups and the negatively charged, hydrophilic phosphate moiety of phospholipids in the interaction between prazosin and the α1-adrenoceptor.

Membrane proteins play a vital role in cellular processes. In this thesis, we use KcsA, a prokaryotic potassium channel, as a model to investigate the gating mechanism of ion channels and the effect of anionic lipids on the channel activity using solid-state NMR spectroscopy. KcsA activity is known to be highly dependent on the presence of negatively charged lipids. Multiple crystal structures combined with biochemistry assays suggest that KcsA is co-purified with anionic lipids with phosphatidylglycerol headgroup. Here, we identified this specifically bound, isotopically labeled lipid in the protein 13C-13C correlation spectra. Our results reveal that the lipid cross peaks show stronger intensity when the channel is in the inactivated state compared to the activated state, which indicates a stronger protein-lipid interaction when KcsA is inactivated. In addition, our data shows that including anionic lipids into proteoliposomes leads to a weaker potassium ion affinity at the selectivity filter. Considering ion loss as a model of inactivation, our results suggest anionic lipids promote channel inactivation. However, the surface charge is not the only physical parameter that regulates channel gating or conformational preference. We found that the channel adapted to different conformations when reconstituted into liposome either made of DOPC or DOPE, two zwitterionic lipids. Also, we were able to stabilize the open-conformation of KcsA in 3:1 DOPE/DOPG liposome at pH 4.0 and acquired several multi-dimensional solid-state NMR experiments for site-specific resonance assignments. This is the first time that we obtain wild-type full-length KcsA structural information on the transient state. The structure is not only important for understanding channel gating, but can also serve as a homology model for investigating drug binding with more complicated potassium channels such as human voltage gated channel (hERG).