The purpose of this study is the characterization of the membrane-water interfaces of both sarcoplasmic reticulum membrane (SR) and charged lipid bilayers under varied properties of the surrounding aqueous solution. In this work we studied the electrokinetic properties of liposomes and SR vesicles as well as the interaction of lipophilic ions with these membranes. The study of electrokinetic properties is based on the measurements of electrophoretic mobility of SR membrane vesicles and PC/PG liposomes. Electrophoretic mobility of SR vesicles was measured as a function of ionic strength for six pH values (pH 4.0, 4.7, 5.0, 6.0, 7.5, and 9.0). Electrophoretic mobility of single-layered and multi-layered PC/PG liposomes was measured at neutral pH as a function of ionic strength. For interpretation of electrophoretic mobility studies, SR vesicles (at pH 4, 7, and 9) and multi-layered and single-layered liposome sizes were determined using photoelectron microscopy. The study of the interaction of lipophilic ions with these membranes is based on (1) measurements of their partition coefficients described in terms of an ion partition model based on the Langmuir adsorption model and (2) electrophoretic mobility measurements of SR vesicles and PC liposomes in suspension with varied concentration of lipophilic ions. SR-water and PC-water partition coefficients were measured as a function of concentration for two anions tetraphenylborate (TePB-) and pentabromophenol (PBP-) and two cations (Imipramine+, and Clomipramine+). The anions belong to a class of pesticides and the cations are drugs once prescribed as anti-depressants. Partition into the SR membrane was shown to be significantly greater for all lipophilic ions except TePB-, which only showed this effect at the higher lipophilic ion range of the data. The PC-water partition coefficient was also measured for TePP+. Since the lipid bilayer of SR is not significantly different than that of PC liposomes, we believe the differences in partition are due to excess lipophilic ions being absorbed to the proteins of SR. The electrokinetics of charged PCPG liposomes, and PC liposomes with absorbed lipophilic ions could be understood in terms of the charge being located below their surface and screened by counter-ions inside the polar head-group region. We call this model the "permeable surface model". The assumptions of this model are that (1) the charge exists on a plane at a depth, d, below the surface of the liposome within the lipid head-group region and (2) small ions (Na+, K+, Cl-) are able to penetrate the lipid head-group region with a molar membrane-water partition coefficient of 0.4. Using this model we were able to obtain the depth of sorption of lipophilic ions in PC liposomes. We found values of 0.13 nm for TePB-, 0.5 nm for PBP-, 0.12 nm for Imipramine+, 0.17 nm for Clomipramine and 0.25 nm for TePP+. The depth of lipophilic ions in PC is a valuable quantity for the study of the effect of lipophilic ions on membrane function. For PCPG mobility we found the charged plane due to PG lipids was 0.2 nm for single-layered liposomes and 0.1 nm for multi-layered liposomes. This is consistent with the relative size of PC and PG head groups The dependence of SR mobility on pH was found to be directly correlated with the total charge of the A, P, and N domains of the Ca2+-ATPase as determined by the amino acid residues and their corresponding pKa values in water. We found that detached charged plane model, a new model developed in our group, could be fit to the mobility of SR as a function of ionic strength while other soft particle models failed. The assumptions of this model are that (1) the friction caused by protruding proteins on the surface of SR can be represented by a homogeneous retardation layer of thickness D and softness parameter λRL, and (2) the charge of the APN domain can be represented as a plane of charge embedded in the retardation layer at a distance s from the membrane surface. The best-fit values for λRL, and s were not consistent for different pH value studies. The detached charged plane model was unable to predict the mobility of SR vesicles in the presence of lipophilic ions if we assumed that the lipophilic ions were sorbing to the detached charged plane that represents the native charge of the APN domains of SR. At high lipophilic ion concentration the experimental mobilities consistently were greater in magnitude than the values predicted by the model. We concluded that there is significant absorption of lipophilic ions to the proteins in SR membrane, and that the lipophilic ion sorption sites are not the same as the detached plane of charge that represents the native charge of the APN domain.
| Identifer | oai:union.ndltd.org:pdx.edu/oai:pdxscholar.library.pdx.edu:open_access_etds-1077 |
| Date | 01 January 2012 |
| Creators | Satterfield, Laura Elizabeth |
| Publisher | PDXScholar |
| Source Sets | Portland State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Dissertations and Theses |
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