In this study, electrophoretic mobilities of native and two types of trypsin digested sarcoplasmic reticulum vesicles have been determined by microelectrophoresis using a Doppler Electrophoretic Light Scattering Analyzer to investigate the influence of hydrodynamic drag, caused by the Ca2+, Mg2+ -ATPase protruding from the surface of native sarcoplasmic reticulum vesicles. After the prolonged digestion (protein:trypsin ratio of 20 for 3 hours at 25°C), the ATPase was cleaved and removed from the sarcoplasmic reticulum membrane as shown with SDS gel electrophoresis and an ATPase activity assay. Ionic strength and pH dependence of mobility showed a nearly pH independent increase in initial surface charge density after prolonged digestion. Adsorption isotherms for native, short (protein:trypsin ratio of 200 for 2 minutes at 25°C), and prolonged digested sarcoplasmic reticulum vesicles were recorded for TPhP+ (tetraphenylphosphonium), PCP- (pentachlorophenol), and Ca2+, and fitted to the Langmuir adsorption model. The most important result from the adsorption isotherms is that adsorption of the three ions did not increase significantly after prolonged digestion. From this it can be concluded that hydrodynamic drag does not have a measurable influence on electrophoretic mobility of sarcoplasmic reticulum vesicles and therefore cannot account for the big differences in mobility between sarcoplasmic reticulum vesicles and a comparable artificial membrane system (phosphatidylcholine/phosphatidylserine liposomes), which were observed in this lab earlier.
A thermodynamic analysis of adsorption was done for PCP- adsorption to phosphatidylcholine liposomes, TPhP+ adsorption to phosphatidylcholine liposomes, and TPhP+ adsorption to sarcoplasmic reticulum vesicles, by recording adsorption isotherms at 10°C, 25°C, 40°C, and 55°C. The adsorption of PCP- to phosphatidylcholine liposomes was clearly driven by enthalpy. In contrast, the adsorption of TPhP+ to phosphatidylcholine liposomes and sarcoplasmic reticulum vesicles was characterized by a positive enthalpy and a still larger negative entropy term. The thermodynamic analysis of ion adsorption shows that the driving forces of adsorption are very similar for sarcoplasmic reticulum vesicles and the chosen artificial membrane system (phosphatidylcholine liposomes) in spite of the significant lower adsorption of biological membranes compared to artificial membrane systems.
| Identifer | oai:union.ndltd.org:pdx.edu/oai:pdxscholar.library.pdx.edu:open_access_etds-2795 |
| Date | 01 July 1994 |
| Creators | Schilling, Andreas |
| Publisher | PDXScholar |
| Source Sets | Portland State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Dissertations and Theses |
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