An analysis of alterations in ribosomal conformation using reductive methylation.
Optimal conditions for reductive alkylation of ribosomal proteins in their native and denatured states were examined. The relative accessibility of rat liver ribosomal proteins to reductive alkylation was then examined. Intact ribosomes were firs labeled with [14C]formaldehyde and NaBH4. The proteins were then separated from RNA, denatured in 6 M guanidine, and labeled again using formaldehyde and NaB3H4. The relative accessibility of individual proteins to labeling in the intact state could thus be determined from their 3H/14C ratios following separation by two-dimensional electrophoresis. The results suggest that proteins S6, S11, S26, L3, and L35 are less accessible to labeling while proteins S1, S15, L11, L12, L16, and L24 appear relatively more accessible. The accessibility of individual proteins in ribosomes in different conformational states were then compared. The results indicated that S3, L7, and L36 are likely to be involved in a structural difference when normal polysomes and normal monomers are compared. Also, that S26 and L35, and probably S3, S20, L7, L8, L24, L27, L28 and L34 appear to be involved in a ribosomal conformation change induced by ethionine intoxication.
D000818:Animals; D005557:Formaldehyde; D007700:Kinetics; D008099:Liver / Q000648:ultrastructure*; D008297:Male; D008745:Methylation; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011132:Polyribosomes / Q000648:ultrastructure; D011487:Protein Conformation; D051381:Rats; D012269:Ribosomal Proteins / Q000032:analysis; D012270:Ribosomes / Q000648:ultrastructure*
D012269:Ribosomal Proteins; D005557:Formaldehyde