4. Conclusions Our studies show that there are several factors affecting the result of the proton pumping experiment, starting from vesicle preparation to the detergent
removal. The most important of these factors concerns the permeability and stability of the LUVs which strongly affects the proton pumping activity and hence pH gradient of the resulting BR-vesicles. Leaking vesicles display lower pH gradient due to the proton leakage from the membrane. Further, it is important to use lipids with high purity and to ensure complete removal of the detergent. Finally, one should examine the vesicles by DLS to verify Inhibitors,research,lifescience,medical their homogeneity and size. The degree of orientation of the BR incorporated into the LUVs also affects the proton pumping efficiency. It has been shown that 95% inside-out orientation will be achieved using the detergent-mediated reconstitution method. However, this percentage Inhibitors,research,lifescience,medical strongly depends on the experimental conditions, for example, detergent to lipid ratio and the time point, where BR will be added to the LUVs . Overall, our observations are in agreement with Inhibitors,research,lifescience,medical the earlier preliminary results with labeled penetratin by Björklund et al. . Use of an ionophore nigericin is another alternative to create acidic pH inside the vesicles . It works by exchanging K+ for H+ across the vesicle membrane and creating
a transmembrane pH gradient. However, the effect
of Inhibitors,research,lifescience,medical nigericin is dependent on the presence of high concentrations of a K+ salt inside the vesicles. To create a transmembrane salt gradient, metal ions have to be removed from outside the vesicles by passing through the columns equilibrated by high concentrations of, for example, sucrose. High-concentrated sugar and metal ions may destabilize the vesicles resulting in leakage of the protons and hence decreasing Inhibitors,research,lifescience,medical the pH gradient. The light-induced BR proton pumping experiment has the advantages that (1) it does not require any special buffer which alters the vesicle stability, (2) one is able to control pumping activity by the illumination time period, and (3) several experiments Histone demethylase can be carried out with the same sample repeating dark-illumination cycles. The present studies also suggest a general mechanism by which positively charged molecules, other than peptides, may enter into cells by endocytotic uptake followed by escape from the ALK signaling pathway acidified endosome. Acknowledgments This study was supported by the Swedish Research Council (to A. Gräslund) and the Swedish Foundation for Strategic Research (Project no. MDB09-0015). The authors want to thank Professor Esteve Padrós from Universitat Autònoma de Barcelona for the generous gift of the strain S9 of H. salinarum. The authors also want to acknowledge the funding from the European Union (Marie Curie Action PIOF-GA-2009-237120 to A. Perálvarez-Marín).