Isolation and Characterization of Active Ribosomal Subunits from Human Placenta

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Abstract
We have developed a method for the large‐scale isolation of active ribosomal subunits from human placenta. The technique involves incubating crude ribosomes for 15 min at 37°C with 0.2 mM puromycin in 50 mM Tris‐HCl buffer, pH 7.6, 500 mM KCl and 3 mM MgCl2 followed by centrifugation at 5 °C in a BXV zonal rotor using an equivolumetric sucrose gradient in the same buffer, upon which 80–90% of all ribosomes are dissociated into subunits. The purified subunits differ in their chemical composition, the 60‐S particle containing no more than 36% protein whereas the 40‐S subunit consists of 43% protein. In poly(U)‐directed protein synthesis, tested in a completely homologous cell‐free system, one recombined couple polymerizes at 37°C 12 to 17 phenylalanine residues at an initial rate of 0.7 residues per minute. However, free 80‐S ribosomes obtained by puromycin treatment of the crude ribosomes and reassociation of the subunits without prior isolation, have an even higher incorporating activity (20–25 mol phenylalanine/mol of ribosome). At least 55% of the subunits were estimated to actively participate in the polyphenylalanine synthesis.