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Purification and Characterization of a Nuclear DNA-binding Phosphoprotein in Fetal and Tumor Tissues¹

Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030

The basic nonhistone phosphoprotein 110/8.4 (M.W. x 10^-3/pl) was found in 0.35 M NaCl nuclear extracts of four tumor tissues, i.e., fast-growing Novikoff hepatoma, Morris hepatoma 3924A, HeLa cells, and Namalwa cells; it was also found in fetal rat liver. This protein was not detected in normal or regenerating liver and thus may represent an "oncofetal" protein of potential interest as a cancer "marker." Protein 110/8.4 was purified approximately 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 phospho-cellulose chromatography. Sodium dodecyl sulfate:polyacrylamide gel electrophoresis of the purified native protein revealed a single polypeptide chain with a molecular weight of approximately 110,000. The pl 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. Amino acid analysis showed that Protein 110/8.4 had an acidic:basic amino acid ratio of 1.25 and a high lysine and serine content; approximately 20% of the serine residues were found to be 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.

¹ These studies were supported by Cancer Research Grant CA 10893, P. 1, the Taub Foundation, the DeBakey Foundation, and the Pauline Sterne Wolff Memorial Foundation.

² Present address: Chemistry Department, Roberts Wesleyan College, Rochester, N. Y. 14524.

Received 5/23/80. Accepted 10/20/80.