Identification of specific phosphoproteins in nuclease-digested chromatin subunits.

Rat liver chromatin subunits (nucleosomes), which were isolated from nuclease-digested chromatin either treated or not treated with 0.5% sodium deoxycholate and 5% Triton X-100, were found to be similar with regard to sedimentation at 11 S and appeared as spherical particles with a diameter of about 100 A (1 A = 0.1 nm) in electron micrographs. The ratio of histone to nonhistone chromatin proteins (NHCP) in these subunits was 1:0.1 in the absence of detergents and 1:0.25 in the presence of detergents. After ultracentrifugation of the nuclease-digested chromatin in a 5--30% sucrose density gradient two groups of phenol-soluble NHCP were identified, one being released in the top of the gradient (fA) and the other still bound to the chromatin subunits after the nuclease digestion (fB). Two specific phosphoproteins were found in fA, one (A4) with molecular weight (MW) 39 000, pI 5.3--6.0, and the other (A5) with MW 31 000, pI 5.1--5.8. These proteins were not present in fB. Another protein in fB (B2) was highly phosphorylated, with MW 68 000, PI 6.5--8.2, and this was not found in fA. These phosphoproteins were further characterized and found to contain phosphoserine. The presence of specific phosphorylated protein(s) in fB suggests that the interaction of phosphoproteins with DNA in the eukaryotic genome is more than a random process.