Isolation of the contact regions of the neighboring mammalian ribosomal proteins L6 and L29. Cyanogen bromide cleavage of the disulfide complex after preparative electrophoresis in non-oxidative polyacrylamide gels.
Proteins L6 and L29 in the 60-S subparticle of mammalian ribosomes interact in situ under gentle conditions by intermolecular disulfide bond formation. For identifying the contact regions of the two proteins the disulfide complex was isolated from whole ribosomes by preparative polyacrylamide gel electrophoresis and subjected to cyanogen bromide cleavage. For effective cleavage non-oxidizing conditions had to be maintained throughout the preparation. A simple and generally applicable method of high-efficiency gel pre-electrophoresis with anionic and cationic thiols was developed. Under these conditions reversibly cross-linked CNBr fragments of L6 and L29 could be isolated in high yield (Mr approx. 13 000 and 7000, respectively). After [14C]carboxymethylation of the reduced disulfide links smaller contact sequences were obtained by pepsin digestion and characterized by two-dimensional peptide mapping. These smaller contact peptides were contained within the corresponding CNBr fragments. Both contact peptides were hydrophilic and relatively basic.
D000818:Animals; D001665:Binding Sites; D002855:Chromatography, Thin Layer; D003488:Cyanogen Bromide; D004220:Disulfides / Q000032:analysis*; D004586:Electrophoresis / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D008099:Liver / Q000378:metabolism; D010446:Peptide Fragments; D051381:Rats; D012269:Ribosomal Proteins / Q000302:isolation & purification*
D004220:Disulfides; D010446:Peptide Fragments; D012269:Ribosomal Proteins; C035078:ribosomal protein L29; C031249:ribosomal protein L6; D003488:Cyanogen Bromide