Phosphorylation of acid-soluble proteins in isolated nucleoli of Novikoff hepatoma ascites cells. Effects of divalent cations.

Isolated nucleoli from rat Novikoff hepatoma ascites cells incorporated 32P of [T-~~P]ATP into acid-soluble nucleolar proteins in vitro in systems containing 0.25 M sucrose, 5 mM MgCL, 12.5 mM NaCl, and 0.05 M Tris-HCl buffer (pH 7.3) at 37”. Although the total uptake of 32P reached a maximum after 15 min of incubation, the uptake into some protein bands increased with longer incubation times. As shown by twodimensional gel electrophoresis and autoradiography, all the proteins labeled in uitro, including the high molecular weight proteins designated as C23-24, B27, and B13 were shown previously to be labeled in uivo (OLSON, M. 0. J., ORRICK, L. R., JONES, C., AND BUSCH, H. J. Biol. Chem. 249, 2823-2827). Divalent cations were necessary for phosphorylation in vitro but different profiles of phosphoryiation were observed when Mg2f was replaced with Co2+, Znzi, or Mn2+. The presence of Zn2+ produced the highest 32P uptake associated with a marked increase in phosphorylation in proteins A17-19 and B23-25 compared to Mg2f which produced high labeling in spots B13, and C23-24. These data indicate that the nucleolus contains phosphoprotein kinase(s) which exhibit different specificities in the presence of different divalent cations.