| Popis: |
Publisher Summary The chapter discusses the preparation of derived and native ribosomal subunits from rat liver. To prepare ribosomal 40 S and 60 S subunits derived from polysomes (designated d40S and d60S subunits), the microsomes obtained from the postmitochondrial supernatant fraction are extracted to yield ribosomes that are stripped of endogenous peptidyl-tRNA with puromycin and then dissociated into subunits in solutions containing high concentrations of KCl; the subunits are separated by gradient centrifugation. To prepare native subunits (n40S and n60S subunits), the postmicrosomal supernatant is centrifuged to sediment the total subunits fraction, and then resolved by gradient centrifugation. The postmicrosomal supernatant obtained at 100,000 g for 2 hr, (Preparation of Microsomes and Ribosomes), is used for the preparation of free, native subunits. The native 40 S and 60 S subunits are resolved by zonal centrifugation in a linear-with-radius 10-30% sucrose gradient. Incubation of the derived subunits obtained from the zonal gradient in a protein synthesizing system containing radioactive Phe-tRNA, poly(U), elongation factors, and GTP, indicates that the subunits are well resolved from each other and that they are free of elongation factors EF-1 and EF-2. Gel electrophoresis of RNA extracted from individual particles indicates less than 10% contamination with 28 S RNA in 40 S subunits or with 18 S RNA in 60 S subunits. |
