A gel filtration approach to the study of ribosome-membrane interactions

Abstract

1. 1. Gel filtration on agarose can be used to investigate ribosome-membrane interactions without exposing the materials to the high, and possibly perturbing, hydrostatic pressures experienced during centrifugation procedures to separate free ribosomes from membrane vesicles. 2. 2. After treatment of microsomes with degranulating agents, degranulated membranes are isolated from Sepharose 2B columns at the void volume, while displaced ribosomes elute at the total column volume. This provides a convenient method for monitoring degranulation in vitro. 3. 3. Centrifugation of rough microsomes or ribosomes into dense pellets or layers, followed by resuspension, leads to preparations which will not pass rapidly or quantitatively through Sepharose 2B columns. 4. 4. Methods are described for the isolation of degranulated microsomes and ribosomes which are eluted rapidly from Sepharose 2B at the void volume and total column volume, respectively. These materials are suitable for the investigation of ribosome-membrane binding in vitro, using a gel filtration separation to monitor binding. 5. 5. Incubation of 3H-labelled ribosomes with degranulated microsomes in vitro, leads to specific binding, demonstrated by the elution of the bound ribosomes at the void volume.