Reconstitution of rhodopsin and the cGMP cascade in polymerized bilayer membranes.

The successful reconstitution of rhodopsin, the rod outer segment (ROS) G protein, and the ROS phosphodiesterase (PDE) into partially polymerized bilayer membranes is described. Purified bovine rhodopsin (Rh) was inserted into performed partially polymerized lipid vesicles. Sonicated vesicles composed of approximately equal moles of dioleoylphosphatidylcholine (DOPC) (or 1-palmitoyl-2-oleoyl-phosphatidylcholine) and 1,2-bis(octadeca-2,4-dienoyl)phosphatidylcholine (DENPC) were photolyzed with 254-nm light to polymerize the DENPC and form domains of DOPC and polyDENPC in the vesicle wall.

Preparation and characterization of polymerized liposomes.

Each of these classes of polymerizable lipids offers useful approaches to the preparation of polymerized liposomes. The lipid diacetylenes can be very photosensitive and yield highly colored rigid photopolymers. The reaction is sensitive to lipid structure, membrane packing, and temperature; however, the reaction is difficult to drive to completion.

Rod outer segment phosphodiesterase binding and activation in reconstituted membranes.

Light exposure of rhodopsin in rod outer segment (ROS) membranes activates several cyclic GMP phosphodiesterase (PDE) molecules via a GTP-binding protein (G protein). Both PDE and G protein are surface-associated (peripheral) enzymes, which may be extracted from ROS by hypotonic media, individually purified, and recombined in isotonic media with purified rhodopsin-phospholipid vesicles to yield membranes of low dark and high light phosphodiesterase activity. In isotonic media, the PDE strongly associates with phospholipid membranes as well as with ROS and rhodopsin-phospholipid membranes.

Photoinduced calcium release from rhodopsin-phospholipid membrane vesicles.

Brief blue-green light exposure of rhodopsin-phospholipid membrane vesicles that contained divalent cations released the cations from the vesicles. The photoinduced release is due to an increase in permeability of the membrane. The quantity of ions released depends on the initial ionic concentration inside the vesicles.

Light-enhanced cross-linking of rhodopsin in rod outer segment membranes as detected by chemical probes.

Bovine rod outer segment membranes were treated with cross-linking reagents before and after light exposure. Bleached membranes showed enhanced cross-linking with difluorodinitrobenzene or methyl acetimidate compared to dark-adapted membranes. The light-induced enhancement of cross-linking may be due to increased association of rhodopsin monomers in the light and/or due to increased reactivity of amino and sulfhydryl groups of bleached rhodopsin.