Extraction of proteins from Saccharomyces cerevisiae ribosomes under nondenaturing conditions.
The differential sensitivity of ribosomal proteins to removal by salts has been studied. Proteins were extracted from the large and small subunits of cytoplasmic ribosomes from Saccharomyces cerevisiae by washing the individual subunits with a series of solutions containing increasing concentrations of NH4Cl (0.74-3.56 M) for a defined time (20 min) at 0 degrees C. The molar ratio of magnesium to ammonium ions of 1:40 was maintained to protect the ribosomal subparticles from complete disassembly. Proteins extracted under each salt condition were analyzed for composition by two-dimensional polyacrylamide gel electrophoresis. The relative quantity of each protein was determined. Most proteins were not removed from the ribosomal particle completely by any one condition, but were preferentially enriched in a single fraction. Whereas most proteins could be solubilized, several proteins remained predominantly or exclusively with the final core particle. The kinetics of protein release from both subunits at a single NH4Cl concentration (0.74 M) were also studied. Release of protein was time dependent, i.e., longer extraction generally removed more of the same proteins. However, prolonged treatment (240 min) of subunits, even at the same salt concentration, resulted in removal of additional species of proteins in varying amounts. Among the ribosomal RNA species, only the 5 S RNA species was released from the ribosomal particles upon treatment.
D000643:Ammonium Chloride; D055598:Chemical Phenomena; D002621:Chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005656:Fungal Proteins / Q000302:isolation & purification*; D011489:Protein Denaturation; D012331:RNA, Fungal / Q000302:isolation & purification; D012269:Ribosomal Proteins / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000032:analysis*; D012995:Solubility
D005656:Fungal Proteins; D012331:RNA, Fungal; D012269:Ribosomal Proteins; D000643:Ammonium Chloride