The long term objective of this research is to understand the factors that influence the movement of amphipathic and lipophilic molecules across and between membranes. We have discovered that adding cytochrome b5, an amphipathic integral membrane protein, to externally amidinated vesicles of PC/PE causes a net flux of PE from the inside to the outside. This transfer was determined by TNBS labelling. To further elucidate the mechanism of this protein-induced bilayer destabilization, we plan to carry out the following experiments: (1) Determine how all the other lipids in these vesicles redistribute, using mainly nonspecific phospholipid exchange protein (PLEP); (2) Study the effect of salt, temperature, vesicle size and composition on cytochrome b5 induced by flip flop; (3) Determine the selectivity of flip flop with very polar lipids, such as glycolipids. Chemical and isotopic labelling will be used for these studies; (4) Determine if modified forms of cytochrome b5 such as derivatives with shortened amphipathic tails induce flip flop. We also intend to find out in specific terms what factors cause or prevent cytochrome b5 from properly inserting into the """"""""tight"""""""" configuration. To see if the reconstitution pathway is important, cytochrome b5 will be introduced into vesicles in four ways: a) by adding octamers of cyt b5 in high salt; b) by adding octamers in low salt; c) by adding monomers; d) by intervesicle transfer. The effect of detergent and other integral membrane proteins will be tested, to see how these molecules effect the tight insertion of cytochrome b5. If tight insertion is obtained, we will correlate this effect with the ability of these other molecules to promote flip flop of other amphiphiles, such as N-fatty acyl peptides. In this way we can determine whether tight insertion results from a general destabilization of the bilayer or requires a specific interaction of cyt b5 with the other integral membrane proteins or detergent. These studies should also enable us to test the hypothesis that the barrier to tight inserting is the difficulty in transferring the polar residues of the tail across the bilayer.
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