Identification of Neighboring Protein Pairs in Rat Liver 60S Ribosomal Subunits Cross-Linked with Dimethyl Suberimidate or Dimethyl 3, 3'-Dithiobispropionimidate

Abstract

Protein-protein neighbors in rat liver 60S ribosomal subunits were investigated by using two bifunctional imidoesters; dimethyl suberimidate (DMS) and dimethyl 3, 3'-dithiobispropionimidate (DTP).
1. Complexes cross-linked with DMS were separated by two-dimensional acrylamide/urea gel electrophoresis. Each complex in the gel was labeled with ¹²⁵I (1) and was cleaved into the original monomeric protein constituents by ammonolysis. The products were analyzed by two-dimensional acrylamide/urea gel electrophoresis, followed by radioautography of the stained gel. Eight protein pairs are proposed according to our numbering system (2): L1-L3 (L3-L5), L2-L21 (L4-L26), L5-L13 (L7/L7a-Ll5), L5-L34 (L7/L7a-L36), L6-L34 (L8-L36), L6-L32 (L8-L35), L12-L32 (L13/LI3a-L35), and L18-L27 (LI8a-L27/L27a). The designations according to the proposed uniform nomenclature (3) are given in parentheses.
2. Complexes cross-linked with DTP were analyzed by acrylamide/SDS diagonal gel electrophoresis (4), and the molecular weights of the complexes and their components were determined. The monomer components of cross-linked pairs were labeled with ¹²⁵I in the gel, and identified by two-dimensional acrylamide/urea gel electrophoresis followed by radioautography. Six pairs are proposed; L1-L5 (L3-L7/L7a), L1-L6 (L3-L8), L5-L19 (L7/L7a-L21/L23/L23a), L13-L15 (L15-L14), L13-L16 (L15-L19), and L23-L27 (L30-L27/L27a).
3. Two pairs which were formed by protein-protein interaction without the use of crosslinking reagents were identified as L2-L4 (L4-L6) and L4-L30 (L6-L29).