Two-step purification of the major phosphorylated protein in reticulocyte 40S ribosomal subunits

Abstract

In reticulocytes, a single ribosomal protein, S13, has been shown to be phosphorylated by the cAMP-regulated protein kinases. The 40S ribosomal subunits were phosphorylated in vitro with [gamma-32P]ATP to facilitate the identification of S13 during the two-step purification procedure. Total ribosomal protein from the 40S subunit was fractionated by phosphocellulose chromatography in urea, and S13 was purified to homogeneity by gel filtration on Sephadex G-100. The protein was identified by the radioactive phosphate, by molecular weight, and by the migration characteristics in a two-dimensional polyacrylamide gel electrophoresis system. Thin-layer electrophoresis of partial acid hydrolysates of S13 showed that more than one phosphorylated residue was present in the same oligopeptide, indicating at least some of the phosphoryl groups were clustered in the protein molecule.