11] Purification of transducin

Abstract

This chapter discusses purification of transducin. Transducin is the heterotrimeric guanosine 5'-triphosphate (GTP)-binding protein (G protein) which, in vertebrate retinal rods, couples the membrane photoreceptor rhodopsin to the intracellular effector enzyme cGMP phosphodiesterase. Transducin is specifically localized in the disk membranes of the retinal rod outer segments (ROS), where it represents about 10% of the total protein content. Although it remains preferentially membrane bound in situ and when the ROS preparations are kept in isotonic buffer (e.g., with 150 mM KCI or NaCl and 1 to 5 mM MgCl2), inactive GDP-bound transducin, TαGDP-Tβγ, can be fully solubilized from dark-adapted ROS membranes on lowering the buffer ionic strength, in the absence of detergent. On illumination of the ROS membrane, if no guanine nucleotide is added to the medium, transducin undergoes tight binding to photoexcited rhodopsin (R*). Addition of GTP or a nonhydrolyzable analog can then induce the dissociation of active GTP-bound transducin from R* and quantitative release of the TαGDP subunit from the membrane, on which Tβγ still remains bound if the buffer is of isotonic ionic strength. The dependence of transducin solubility on ROS membrane illumination and in the presence of GTP, is the basis of very simple and efficient techniques of isolation and purification of transducin subunits. The chapter describes the preparation of cattle ROS membranes, the most common material for large-scale extraction of transducin.