Specificity of factors isolated from free polysomes and microsomes on in vitro protein synthesis in plasmacytoma cells

A simple procedure for measuring chain initiation in complete in vitro systems is described. The capacity of free and membrane-bound polysomes prepared by a detergent technique or by nitrogen cavitation to incorporate radioactive amino acids into proteins was compared with the capacity of these polysomes to initiate polypeptide chains.The extent of amino acid incorporation by polysomes prepared by either of these methods did not differ significantly. However, chain initiation determined by measuring the incorporation of radioactive subunits into polysomes showed that polysomes prepared by the detergent technique were less effective than polysomes made by nitrogen cavitation in chain initiation.Crude initiation factors, prepared by washing either free polysomes or microsomes with 0.5 M KCl, stimulated chain initiation and amino acid incorporation by both types of polysomes. Free polysomes were stimulated almost to the same extent by factors isolated from free polysomes or microsomes. Membrane-bound polysomes on the other hand showed a specific requirement for microsomal factors.The extent of stimulation by crude polysomal factors was dependent on the concentration of high speed supernatant in the assay system.