Radioactive labelling of ribosomal proteins with reductive alkylation and its use in studying ribosome-cytosol interactions.
Mouse brain ribosomes were radioactively labelled by a cell-free reductive alkylation reaction with NaBH4 and [14C]formaldehyde. The radioactivity was largely associated with ribosomal proteins, but little, if any, of the rRNA was radioactive after the alkylation procedure. Both ribosomal structural proteins and loosely associated components were successfully labelled by this procedure. The sedimentation properties of the ribosomes were unaltered and their ability to carry out poly(U)-directed protein synthesis, although decreased, was largely retained. Incubation of 14C-labelled ribosomes with brain cytosol resulted in a 17% loss of radioactivity, although treatment of the ribosomes with 1.0 m-KCl to remove the loosely associated factors rendered the ribonucleoprotein particles resistant to cytosol effects. The ribosome-cytosol interactions did not appear to be related to an exchange process, since the released radioactivity was largely degraded to acid-soluble material. In addition, the incubation of native ribosomes with brain cytosol resulted in an almost complete loss in the ability of the ribosomes to participate in cell-free protein synthesis.
D000478:Alkylation; D000818:Animals; D001894:Borohydrides; D001921:Brain / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D007553:Isotope Labeling / Q000379:methods; D051379:Mice; D014176:Protein Biosynthesis; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism
D001894:Borohydrides; D012269:Ribosomal Proteins