Engagement of cytoplasmic polyribosomes in the synthesis of ribosomal proteins in eukaryotic cells.

ribosomal proteins and @ H-labeled growing chains were mixed and dually counted after fractionation by different techniques. Ehrlich adenocarcinoma ascites cells were taken from mice (Swiss strain) inoculated 6 days earlier. The cells were separated from the ascitic plasma by centrifugation at 500 X g for S mm and washed once with 3 volumes of a medium containing NaC1 (0.12 M), KC1 (13 mM), MgSO4 (0.65 mM), and sodium phosphate buffer, pH 7.4 (10 mM). The washed cells were resuspended in the same medium and a 5-ml sample of cell suspension (containing approximately 500 mg, wet weight, of cells) was pipetted into a conical flask containing 0.5 ml of ascitic plasma; the flask was then shaken in air at 38°. After a 10-mm equilibration, 50 j.iCi of ‘4C-labeled amino acid mixture (uniformly labeled L-amino acid-' 4C mixture; specific activity, 1 mCi/0.32 mg; New England Nuclear Corp., Frankfurt/Main, West Germany) were added, and the incorporation was allowed to proceed for S hr, after which time the label was diluted 2000-fold with unlabeled amino acid mixture. After a further 30-ruin incubation, the cells were sedimented by centrifugation, and the supernatant was discarded. Ribosomes were prepared following the procedure of Wettstein et a!. (13). Structural ribosomal proteins were extracted from purified ribosomes following the procedure of Wailer and Harris (10), dialyzed overnight at 0° against 8 M urea, and centrifuged at 105,000 X g for 2 hr; the sediment was discarded. Proteins labeled with tritium in a cell-free system were obtained as follows. Wet cells, harvested as above, were homogenized in an equal volume of a medium containing Tris buffer, pH 7.8 (50 mM), KC1 (25 mM), sucrose (0.35 M), and @3-mercaptoethanol (1 mM). The homogenate was then centrifuged at †" 1° at 30,000 X g for 10 mm and then a 5-ml sample of supernatant was pipetted into a tube, incubated at 36°for S mm, and supplemented with 1 mg GTP, 3 mg ATP, 15 mg phosphoenolpyruvate, 50 pg phosphoenolpyruvate kinase, and 100 pCi of an @ H-labeled amino acid mixture (generally labeled L-amino acid-3 H mixture; specific activity, 1 mCi/0.049 mg, New England Nuclear). The reaction was allowed to proceed for 15 mm and was then stopped by immersing the tube in cracked ice. The whole reaction mixture was then layered upon a 2-step sucrose gradient (1 ml 0.5 M sucrose-i ml 1 M sucrose in a Spinco rotor 50 …