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

Authors

  • Ken TSUTSUI,

    Corresponding author
    1. McArdle Laboratory for Cancer Research, University of Wisconsin, 450 North-Randall Avenue, Madison, Wisconsin, U.S.A. 537060
      *To whom correspondence should be addressed.
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  • Kimiko TSUTSUI,

    1. McArdle Laboratory for Cancer Research, University of Wisconsin, 450 North-Randall Avenue, Madison, Wisconsin, U.S.A. 537060
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  • Takuzo ODA

    1. Department of Biochemistry, Cancer Institute, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama-shi, Japan 700
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*To whom correspondence should be addressed.

Abstract

A high-molecular-weight acid-soluble nuclear protein (110000-Mr protein) was isolated from mouse ascites sarcoma cells (SR-C3H/He cells) by extraction of nuclei with 0.2 M H2SO4, 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-Mr 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-Mr protein of the SR-C3H/He cells recognized homologous proteins in 0.2 M H2SO4 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 [32P]orthophosphate, a high level of 32P incorporation into the 110000-Mr protein was observed. Treatment of the phosphorylated protein with alkaline phosphatase released approximately 40% of the incorporated 32P.

The observed amino acid composition, molecular weight and phosphorylation of the 110000-Mr 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-Mr protein plays a fundamental role in nuclear metabolism or structure.

Enzymes
 

Alkaline phosphate (EC 3.1.3.3)

Ancillary