PURIFICATION AND CHARACTERIZATION OF A NUCLEAR DNA-BINDING PHOSPHOPROTEIN IN FETAL AND TUMOR-TISSUES

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  • 1 January 1981
    • journal article
    • research article
    • Vol. 41  (2) , 537-545
Abstract
The basic nonhistone phosphoprotein 110/8.4 (MW .times. 10-3/pl) was found in 0.35 M NaCl nuclear extracts of 4 tumor tissues, i.e., fast-growing Novikoff [rat] hepatoma, Morris [rat] hepatoma 3924A, HeLa [human cervical carcinoma] cells and Namalwa [human lymphoblastoid cancer] cells; it was also found in fetal rat liver. This protein was not detected in normal or regenerating liver and may represent an oncofetal protein of potential interest as a cancer marker. Protein 110/8.4 was purified .apprx. 4000- to 5000-fold under nondenaturing condition from 0.35 M NaCl nuclear extracts of Novikoff hepatoma cells or Namalwa cells by ammonium sulfate fractionation, calcium phosphate gel treatment and phosphocellulose chromatography. Sodium dodecyl sulfate:polyacrylamide gel electrophoresis of the purified native protein revealed a single polypeptide chain with an MW of .apprx. 110,000. The pl [isoelectric point] of the protein was estimated to be 8.4 by nonequilibrium pH gradient electrophoresis in 9 M urea; accordingly, this protein was designated 110/8.4. Protein 110/8.4 had an acidic:basic amino acid ratio of 1.25 and a high lysine and serine content; .apprx. 20% of the serine residues were phosphorylated. Hydrazinolysis indicated that the carboxyl-terminal amino acid was serine; the amino terminus appeared to be blocked. Binding of protein 110/8.4 to DNA was studied by the nitrocellulose filter assay. High-affinity binding occurred at ionic strength equal to or below 0.15 M.