Browsing by Subject "Membran"

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Oszillation und Musterbildung in reinen und Polypeptid-dotierten Bilayern
(2017) Gaugele, Sebastian; Hanke, Wolfgang
The question of how lipids and proteins interact in bilayers and membranes and why cells and organelles have different lipid compositions in their membranes has been in focus of research for decades. Which role these different lipid compositions play for interaction between integrated and associated proteins has not yet been clarified until today. This thesis with the title "Oszillation und Musterbildung in reinen und Polypeptid-dotierten Bilayern" focuses on forces that influence the interaction of lipids and proteins. In both, living cells as well as in bilayer experiments, the protein-lipid interaction is a complex process involving a huge number of molecules. Different test series with temperature and amperage have been carried out using electrophysiological experiments with bilayers and fluorescence in vesicles. The results were used to determine similarities and differences that indicate certain characteristics of the setup and the interaction among the molecules. Since the question of the interaction between proteins and lipids is a very complex process, the already well-known pore former alamethicin has been used for this work. Alamethicin was used in the both types of experiments, bilayer and vesicles, with different lipid compositions consisting of DOPC, DOPE, DOPS and DMPC. The lipids were solved in ethanol first and then added with certain concentrations of alamethicin in two test chambers which were connected to each other by a small hole. In this hole the bilayer was formed by the addition of aqueous KCl solution. After that the current-clamp-method with different currents was used and subjected to the bilayer. The resulting voltage profiles were observed for oscillation patterns. Subsequently, the obtained data were subjected to the method of Fourier-Transformation to separate the results from noise and interference as well as overlays which resulted from the multi-channel system of alamethicin. The Fourier-Transformation has shown a large number of heterogeneous oscillation patterns. Due to the great heterogeneity of the results that did not allow direct comparisons of individual data with each other the average time-period of oscillation was calculated. This data were used to form data clouds where regression lines were generated. In addition the slopes of the lines were calculated for comparison. The results show that the orientation and slope angle of the slopes directly dependent on the concentrations of alamethicin and KCl. It was found that the addition of a small amount of negatively charged lipid like DOPS to a mixture of DOPC and DOPE has a big impact on the results and can change the orientation of the slopes of the average time-period of oscillation. Similar results were found while using asolectin bilayer containing inositol that is negatively charged, too. As a result, it was concluded that contrary to previous assumptions of some earlier work groups, bilayers are not pure hydrophobic barrier layers between two aqueous phases but highly complex structures that affect the gating of its integrated pores in different ways. The insert and operation of alamethicin strongly depends on the used lipids. Moreover a major dependency from the used salt concentration of the alamethicin pores and bilayer stability was found. The experiments with DMPC and temperature showed that the chain length of the lipids play an important role for integrated proteins on the one hand. On the other hand it could be confirmed that the phase transition of lipids can comprise a wide range of temperature of several degree Celsius depending on the experimental conditions. It could also be confirmed that alamethicin remains active in the liquid-crystal domains during phase transition of a bilayer. In addition it was found that temperature dependent changes appear in voltage oscillation patterns that affect both the average time-period and its resulting slopes in angle and orientation.
Posttranslationale Modifikationen der IL-6-Typ-Zytokin-Rezeptoren gp130 und LIFR und ihr Einfluss auf die Assoziation mit Detergenz-resistenten Membranmikrodomänen (DRM)
(2008) Ziegler, Inna; Graeve, Lutz
Post-translational modification of proteins is an important event in the regulation of cellular functions. Glycosylation or palmitoylation, but also ligand binding can affect the localization of proteins in membrane microdomains and thus affect signal transduction. The aim of this study was to analyze how posttranslational modifications of LIFR and the common signal transducer gp130 impact the translocation to detergent resistant membranes (DRMs, lipid rafts). Palmitoylation of cysteine residues within the transmembrane domain of a protein is considered to be one process that assists in the localization of proteins to DRMs. Gp130 has two cysteine residues C711 and C725 in its transmembrane domain. My studies indicate that these cysteine residues have no significant influence on lipid raft association of gp130. Contrary to our expectations, after isolation of DRMs with Brij 58 and Triton X-100 an increase of raft association of the C->A-mutants was detected. Partial DRM association of LIFR was confirmed by using Brij 58 and Triton X-100 protocols. Furthermore, two different N-glycosylation types of that receptor could be detected. The mannose-rich (precursor) species is preferentially found in non-DRMs and is degraded by Endo-Glycosidase Hf. The hybrid-type (mature) tends towards an association with DRMs. My results indicate that only the mature-type of LIFR was phosphorylated after LIF binding to the receptor complex in 3T3-L1 and HepG2 cells. Combined with other data from our workgroup these findings suggest that only the mature-type of LIFR is expressed at the plasma membrane surface and involved in signal transduction. After stimulation with LIF an increase of LIFR tyrosine phosphorylation was observed in DRMs in HepG2 cells. However, phosphorylation of gp130 was detected only in non-DRMs fractions after stimulation with LIF. The inconsistency of these results can be explained with methodical problems. Furthermore, the translocation of phosphorylated receptors described above could not confirmed in 3T3-L1 cells. In this cell line, the activation of gp130 and LIFR occurs in detergent-resistant membranes. These findings indicate differences between cell lines with respect to receptor activation and translocation within the plasma membrane on the one hand and demonstrate a differential sensitivity of raft subdomains to extraction by different detergents on the other hand.