[54] Isotopic Labeling and Analysis of Phosphoproteins from Mammalian Ribosomes

This chapter describes the methods that are found most useful for the isotopic labeling and characterization of phosphoproteins from mammalian ribosomes. The chapter describes methods for removing contaminating phosphoproteins and for establishing the reality of this ribosomal modification. These same methods should be applicable to analysis of other phosphoproteins, especially those which occur in complex subcellular organelles. The chapter shows that at least five polypeptide chains from ribosomes of rabbit reticulocytes, rat liver cells and mouse sarcoma 180 tumor cells are phosphoproteins rather than simple proteins. The phosphoryl groups in these proteins are present in o-phosphoseryl and in o-phosphothreonyl residues. Because these phosphoryl groups turn over intracellularly, the proteins become radioactive when the cells are incubated with [³²P]orthophosphate.