The isolation of polysomes from rat liver: Contamination by membrane proteins with high affinity to ribosomes

Abstract

The contamination of polysomes isolated from rat liver by methods involving solubilization of the endoplasmic reticulum by sodium deoxycholate or by Triton X-100 has been studied. When Triton X-100 was used, high concentrations of salt were necessary to prevent the adsorption of extraneous material to the polysomes. With sodium deoxycholate the salt concentration was not critical. When polysomes were mixed with endoplasmic reticulum solubilized by Triton X-100 at low salt concentration, a considerable amount of extraneous material became adsorbed to the polysomes. No similar adsorption was observed when the polysomes were mixed under the same conditions with the soluble fraction of the cytoplasm. [ 3H]Leucine was rapidly incorporated into the adsorbing material. Polyacrylamide gel electrophoresis of the proteins from the adsorbed material showed that a number of different proteins were present. The proteins from the endoplasmic reticulum showed a similar distribution. The adsorbed material could be removed from the polysomes by treatment with deoxycholate or with Triton X-100 in the presence of high concentrations of salt. Upon subsequent reduction of the salt concentration or by removal of deoxycholate, the detached material again became adsorbed to the polysomes. The adsorption of labelled protein to unlabelled polysomes was studied at various salt concentrations in the presence of Triton X-100. The amount of radioactivity that adsorbed to the polysomes decreased with increasing salt concentration. It is shown that essentially pure polysomes can be obtained by isolation procedures including the use of Triton X-100 at a salt concentration of 150 mM KCl. It is suggested that specific membrane proteins with high affinity to ribosomes may be involved in the binding of polysomes to membranes in the cell.