10] Preparation of osmotically intact rod outer segment disks by Ficoll flotation

Abstract

This chapter discusses the preparation of osmotically intact rod outer segment disks by ficoll flotation. Rhodopsin, in retinal rods, is located primarily in the membranes of flattened disks found stacked within the rod outer segments (ROS). This structural organization serves both to provide a long absorbing pathlength of rhodopsin molecules for incident photons and to orient rhodopsin molecules for maximal light absorption efficiency. Disks have been isolated from ROS that are obtained by flotation on either sucrose or metrizamide. The ROS are burst in an aqueous hypotonic solution of 5% Ficoll, under which conditions the disks swell but do not burst. The density of the swollen disks is determined by the weighted contributions of the membranes and intradiskal trapped volume. The fact that the disks float in 5% Ficoll implies that they are osmotically active. This is confirmed by thin section electron microscopy and by the contrast in refraction between the disks and the Ficoll solution as seen with a phase contrast microscope. Disks prepared by Ficoll flotation have been shown to be intact with respect to permeation by concanavalin A, 2.6 and at 0°, by lipid labeling reagents.