Isolation and characterization of active ribosomal subunits from human placenta.
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 degrees 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 degrees 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 degrees 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.
D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient; D002501:Centrifugation, Zonal; D005260:Female; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010649:Phenylalanine / Q000378:metabolism; D010920:Placenta / Q000032:analysis / Q000378:metabolism / Q000648:ultrastructure*; D011072:Poly U / Q000378:metabolism; D011247:Pregnancy; D014176:Protein Biosynthesis / Q000187:drug effects; D012343:RNA, Transfer / Q000302:isolation & purification; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D013696:Temperature
D011072:Poly U; D010649:Phenylalanine; D012343:RNA, Transfer; D008274:Magnesium