USE OF RADIOACTIVE CHOLINE AS A LABEL FOR MICROSOMAL MEMBRANES .2. STUDIES ON MEMBRANE-RIBOSOME INTERACTION SUBFRACTIONATION AND FUNCTION

1. (1) The specificity of radioactive, methyl-labelled choline for membrane lecithin in rat liver microsomes enabled its use as a marker to follow membranes in a number of microsomal subfractionations: (a) Solubilization of membranes by means of the detergents, sodium deoxycholate and Triton X-100. (b) Separation of free ribosomes from microsomal membranes on 5-20% sucrose gradients. (c) Fractionation of microsomal membranes according to their approximate isopyenic densities on 30-60% sucrose gradients. (d) Location of membranous contaminants on 10-30% sucrose gradients during the sedimentation analysis of free polysome preparations and investigation of the interactions between membranous contaminants and polysomes. 2. (2) Tritiated choline served as a membrane marker in double labelling experiments with [14C]glucosamine, in which the latter was used to measure the ribosomal initiation of carbohydrate prosthetic group attachment onto glycoproteins during their biosynthesis. These confirmed previous results in showing that glycoprotein prosthetic group initiation was virtually restricted to endoplasmic reticulum bound ribosomes. 3. (3) Using choline-labelled lecithin as a radioactive substrate for phospholipase C it was easy to follow the hydrolytic breakdown of phospholipids in intact membranes under very gentle conditions at 0°. This facilitated a study of the role of phospholipids in the attachment and stabilizaton of bound ribosomes on the endoplasmic reticulum membranes. © 1969.