Partial reconstitution of active eukaryotic ribosomes following dissociation with dimethylmaleic anhydride.
Modification of yeast ribosomes with dimethylmaleic anhydride, a reagent for protein amino groups, is accompanied by loss of polypeptide-synthesizing activity. This activity can be recovered by incubation at pH 6, which produces regeneration of the modified amino groups. Dimethylmaleic anhydride modification also causes the dissociation of proteins from the ribosomes. Protein-deficient ribosomal particles are prepared from 80 S ribosomes or 60 S subunits by treatment with a molar excess of reagent relative to ribosomal particles equal to 6300 or 4800, respectively. The core particles from 80 S ribosomes lack 18% and those from 60 S subunits 40% of the total protein in the corresponding untreated control. In both cases, there is a selective release of proteins, the protein-deficient particles being able to reconstitute active 60 S subunits upon addition of the corresponding split proteins. The reconstituted ribosomal particles, when assayed in the presence of native 40 S subunits, are active in poly(U)-directed polyphenylalanine synthesis (30-90% of the activity of a dimethylmaleic anhydride-untreated control, as compared to 0-15% when the split proteins are excluded). This procedure could prove useful in the study of the structure and function of the eukaryotic ribosome.
D005663:Furans / Q000494:pharmacology*; D007700:Kinetics; D046911:Macromolecular Substances; D008299:Maleic Anhydrides / Q000494:pharmacology*; D008970:Molecular Weight; D014176:Protein Biosynthesis / Q000187:drug effects; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D005663:Furans; D046911:Macromolecular Substances; D008299:Maleic Anhydrides; D012269:Ribosomal Proteins; C007474:2,3-dimethylmaleic anhydride