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Identification of Polysomes Synthesizing Novikoff Hepatoma Nucleolar Antigens¹

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

Antibodies to Novikoff hepatoma nucleolar proteins were purified by preabsorption with liver nucleolar proteins and chromatography on Sepharose 4B affinity columns containing normal rat liver or tumor nucleolar proteins. ¹²⁵I-labeled immunoglobulin G (IgG) to tumor nucleolar proteins bound to tumor and regenerating rat liver polysomes but not to rat liver polysomes. The binding of antibody to polysomes was prevented by inclusion of unlabeled antisera or pretreatment of polysomes with puromycin.

Novikoff hepatoma nuclear extracts containing both nucleolar and nucleoplasmic proteins completely competed with tumor polysomes for the ¹²⁵I-labeled IgG. This ability of the nuclear extracts to specifically compete with polysomes for labeled IgG was used as an assay for cellular localization of the immunoreactive polysomal nascent chains. Tumor nucleoli were successively extracted with (a) 0.075 M NaCl/0.025 M ethylenediaminetetraacetate (EDTA), (b) 10 mM tris(hydroxymethyl)aminomethane, (c) 0.6 M NaCl, and (d) 2 M NaCl/7 M urea. Nucleolar extracts a, c, and d partially competed with tumor polysomes for iodinated anti-tumor nucleolar IgG. Mixing experiments demonstrated that the immunologically reactive components of the tumor nucleolar 0.6 M NaCl and 2 M NaCl/7 M urea extracts were similar and distinct from the immunologically reactive antigens in the 0.075 M NaCl/0.025 M EDTA extract. Fetal and regenerating liver nuclear extracts only partially competed with the polysomes for antibody. Mixing experiments demonstrated that the competing antigens in the fetal and regenerating liver nuclear extracts were similar, corresponded to the antigens in the tumor nucleolar 0.6 M NaCl and 2 M NaCl/7 M urea fractions, and were distinct from the antigens in the tumor nucleolar NaCl/EDTA extract. The antigens in the Novikoff hepatoma nucleolar NaCl/EDTA extract could not be detected in 8- to 30-fold protein excesses of normal, fetal, and regenerating rat liver nuclear extracts.

¹ These studies were supported by Grant CA-10893-P1 awarded by the National Cancer Institute, Department of Health, Education, and Welfare; the Pauline Sterne Wolff Memorial Foundation; and the Bristol-Myers Fund.

² Present address: Developmental Therapeutics Department, M. D. Anderson Hospital, Houston, Texas 77030.

Received 10/26/79. Accepted 1/23/80.