[45] Isolation of mammalian ribosomal subunits active in polypeptide synthesis

Publisher Summary This chapter aims to develop a method for the preparation and purification of active ribosomal subunits from rat and mouse liver. The technique involves incubating purified polysomes with all components for protein synthesis until all competent ribosomes terminate and release their polypeptide chain. Upon treatment such an incubation mixture, all ribosomes are found as 60 S and 40 S subunits. The purified subunits, when recombined, spontaneously associate to 80 S couples in the absence of tRNA, mRNA, or supernatant factors. The purified 60 S subunit contains the enzymatic site for the catalysis of peptide bond formation. Purified mouse liver 60 S subunits are able to catalyze the formation of peptide bonds using substrates puromycin and N-formyl- or N-acetylaminoacyl oligonucleotides from tRNA. A relative comparison of the rates of N-acetylleucyl-puromycin formation catalyzed by E. coli 50 S subunits and mouse liver 60 S subunits shows that the reaction rate with E. coli ribosomes is approximately twice that with liver subunits.