Phosphodipeptide analysis of nonhistone nuclear proteins from Novikoff hepatoma ascites cells.

To study the sites of phosphorylation in nuclear proteins a simple method was developed for the isolation and analysis of phosphodipeptides. Partial acid hydrolysates of unfractionated nonhistone nuclear proteins were subjected to Dowex-1 column chromatography followed by paper electrophoresis at pH 1.8. Phosphoserine- and phosphothreonine-containing dipeptides each had characteristic mobilities in the latter system. By subtractive Edman degradation these peptides were identified as having the general structure, X-PSer or X-PThr, where X is a nonphosphorylated amino acid. The two groups of phosphodipeptides were further purified into unique peptides by two-dimensional paper electrophoresis at pH 3.6 and 6.5. Amino acid analysis, and thus nearest neighbor analysis, of phosphodipeptides from nonhistone nuclear proteins revealed that a heterogeneous group of amino acids was on the amino terminal side of phosphoserine residues. In contrast, phosphothreonine residues were predominantly preceded by proline.