High-resolution freeze-etching replica images of the disk and the plasma membrane surfaces in purified bovine rod outer segments

Molecular organization of the photoreceptor disk membrane was revealed by the freeze-deep etching replica method using purified and successively rinsed bovine rod outer segment (ROS). Various membrane particles with different shape and sizes were found on cytoplasmic surface (PS face) as well as on both P and E fracture faces, which are presumed to be peripheral membrane proteins such as transducin, phosphodiesterase, guanylate cyclase and so on. Membrane particles seen on PS face were catalogued in size. The histogram on their number and size showed that they were classified at least into two major groups, the group of particles about 50 nm(2) in size and the group of particles about 115 nm(2) in size. The distribution density of the 115 nm(2) particle was 1200 mu m(-2) in native state, but it decreased to 125 mu m(-2) after washing with hypotonic buffer solution. Namely, the group of the 115 nm(2)-particle seems to be mainly composed of peripheral membrane proteins. Rinsing with the sucrose free buffer at the final step of the purification procedure enabled us to observe three types of filaments localized in ROS (filaments connecting disk to disk at the margin, filaments connecting disk to the plasma membrane, filaments associated with PS face of disk membrane); and also to find characteristic domains with crystal arrangement of particles on the external surface (ES face) of ROS plasma membrane.