Isolation and Characterization of a High‐Molecular‐Weight Acid‐Soluble Nuclear Protein from Mouse Ascites‐Sarcoma Cells

A high‐molecular‐weight acid‐soluble nuclear protein (110000‐ M r protein) was isolated from mouse ascites sarcoma cells (SR‐C3H/He cells) by extraction of nuclei with 0.2 M H 2 SO 4 , chromatography on Sephacryl S‐200, CM‐cellulose, and DEAE‐Sephadex. The molecular weight of this protein was estimated to be 110000 by sodium dodecylsulfate/polyacrylamide gel electrophoresis. The concentration of the 110000‐ M r protein in SR‐C3H/He nuclei was about 4% of total histone. A similar protein exists in other cell types, but at much lower levels relative to SR‐C3H/He cells. Antibody directed against the 110000‐ M r protein of the SR‐C3H/He cells recognized homologous proteins in 0.2 M H 2 SO 4 extracts of several other cell nuclei. The amino acid composition of the protein was rich in glutamic acid, alanine, lysine and aspartic acid, and the acidic: basic amino acid ratio was 1.8. This protein eluted as a single peak from CM‐cellulose and was separated into two distinct forms which eluted at different salt concentrations on DEAE‐Sephadex columns. Both forms showed the same mobility in sodium dodecylsulfate/polyacrylamide gel electrophoresis. When cells were labelled in vivo with [ 32 P]orthophosphate, a high level of 32 P incorporation into the 110000‐ M r protein was observed. Treatment of the phosphorylated protein with alkaline phosphatase released approximately 40% of the incorporated 32 P. The observed amino acid composition, molecular weight and phosphorylation of the 110000‐ M r protein strongly suggest that it is similar to the nucleolar phosphoprotein C23 which has been characterized recently [Mamrack et al. (1979) Biochemistry, 18 , 3381–3386; Lischwe et al. (1979) Life Sci. 25 , 701–708]. In addition, the presence of homologous proteins in several cell types suggests that 110000‐ M r protein plays a fundamental role in nuclear metabolism or structure.