9] Preparation and characterization of sealed bovine rod cell outer segments

Abstract

Publisher Summary This chapter discusses the preparation and characterization of sealed bovine rod cell outer segments. Mammalian rod (photoreceptor) cell outer segments consist of closely packed membrane disks surrounded by the plasma membrane. They can easily be purified because they are large, have a very low buoyant density (resulting from a high lipid content), and detach easily from the parent photoreceptor cell. Earlier procedures for isolating rod outer segments (ROS) yielded preparations with a low or variable content of these proteins, probably because most of the plasma membranes were extensively disrupted or because the discontinuous gradients that are usually employed did not separate sealed from disrupted ROS. The sealed ROS can be separated from disrupted ROS and disk membranes by use of continuous isopycnic density gradients, a method that takes advantage of the differences in contents and osmotic properties of the two kinds of particles. ROS are identified by their high rhodopsin content. The characteristic density of sealed ROS, their clean separation from disrupted ROS, and the characteristic ratios of protein to rhodopsin in the two fractions are usually sufficient for the identification of the sealed fraction; however, the following other methods may be useful.