Characterization of the acidic phosphorprotein of eukaryotic ribosomes using a new system of two-dimensional gel-electrophoresis.

1. A modified method of two-dimensional gel electrophoresis has been developed for detecting acidic eukaryotic ribosomal proteins. 2. Using this method it has been demonstrated that the major phosphoprotein (Lgamma) of mouse ascites and hamster fibroblast 60-S ribosomal subunits is an acidic protein, apparently analagous to L7/12 of Escherichia coli, and not a neutral protein as previously thought. Electrophoretic resolution of phosphorylated and non-phosphorylated forms of Lgamma has enabled the stoichiometric nature of the phosphorylation (previously deduced from measurement of the specific radioactivity of the ATP pool) to be confirmed. 3. When ascites cells were incubated for 3 h there appeared an altered form of Lgamma which is most likely produced by proteolytic cleavage of the original form. The extent of phosphorylation of Lgamma was decreased by one-half or more in these circumstances. 4. Phosphoprotein Lgamma was found to be almost completely phosphorylated in both polyribosomes and monoribosomes isolated from hamster fibroblasts. Thus the function of the phosphorylation of Lgamma appears not to be concerned with the inactivation of ribosomes.