Preparation of active run-off 80S ribosomes and their subunits from mouse liver.
A method is described for the preparation of active "run-off" 80S ribosomes and 40S and 60S subunits of mouse liver. A polysome preparation was incubated at 37 degrees C for 10 min under the condition for protein synthesis (4 mM Mg2+, 100 mM KCL). Puromycin (10 mM)and 2 M KCL were added to a final concentration of 0.1 mM and 500 mM, respectively, and the reaction mixture was further incubated at 37 degrees C for 10 min. This latter treatment destabilized small polysomes and "stuck" 80S particles, which were remaining after the first incubation, leading to complete release of 40S and 60S particles. Thus, the present method minimized variations in yield of subunits due to polysome preparations and preincubation conditions. The subunits were separated by sucrose density-gradient centrifugation or recovered by precipitation following reassociation into 80S particles (run-off 80S). The reformation of 80S particles from the subunits occurred spontaneously at 5 mM Mg2+ and 100mM KCL. The isolated 40S and 60S subunits, separately, showed low phenylalanine-incorporating activity in the presence of poly(U), but when recombined, polymerized up to 10 phenylalanine residues per couple.
D000604:Amino Acyl-tRNA Synthetases / Q000032:analysis; D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D008099:Liver / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure; D051379:Mice; D010445:Peptide Elongation Factors / Q000032:analysis; D010649:Phenylalanine / Q000378:metabolism; D011072:Poly U; D011132:Polyribosomes / Q000187:drug effects; D011189:Potassium Chloride / Q000494:pharmacology; D011691:Puromycin / Q000494:pharmacology; D012343:RNA, Transfer / Q000302:isolation & purification; D012270:Ribosomes / Q000201:enzymology / Q000378:metabolism
D010445:Peptide Elongation Factors; D011072:Poly U; D010649:Phenylalanine; D011691:Puromycin; D011189:Potassium Chloride; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases