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Two-Dimensional Gel Electrophoretic Comparison of Proteins of Nuclear Fractions of Normal Liver and Novikoff Hepatoma¹

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

The combination of two-dimensional isoelectric focusing-sodium dodecyl sulfate gel electrophoresis with successive extraction of nuclei of Novikoff hepatoma and normal rat liver with (a) 0.075 M NaCl/0.025 M EDTA, (b) 10 mM Tris, (c) 0.35 M NaCl, (d) 0.6 M NaCl, and (e) 3 M NaCl/7 M urea provides an improved approach to analysis of the number and types of nuclear proteins. Each fraction contained 108 to 200 spots, of which (a) some were present in all of the fractions, (b) some were present in more than one fraction, and (c) others were uniquely found in one fraction. In the Novikoff hepatoma nuclei, 483 different polypeptides were found; 427 polypeptides were found in liver nuclei. The sensitivity of the method was such that spots containing 0.1 µg of protein were readily identified on these gels; such proteins are present in approximately 500 to 600 copies/nucleus.

In the Novikoff hepatoma, 18 protein spots (designated by molecular weight/pl) were found in the various nuclear fractions that were not found in the normal liver nuclei: 170/6.0, 140/6.7, 140/6.8, 132/5.2, 97/8.45, 87/5.7, 84/6.3, 82/5.0, 62/6.2, 61/6.1, 61/7.3, 49/5.3, 48/5.1, 48/7.9, 42/5.3, 38/7.3, 28/5.0, and 27/4.9. Ten of these proteins were found in the 0.6 M NaCl extract. Earlier studies with other techniques showed several protein differences in this fraction between these tissues.

In normal liver nuclei, 12 spots were present in various nuclear fractions that were not found in the Novikoff hepatoma: 160/5.8, 98/5.0, 94/7.8, 94/7.9, 78/7.4, 76/8.4, 72/5.5, 65/5.2, 45/8.2, 41/5.6, 38/5.5, and 28/8.45 (molecular weight/pl). These studies substantiate and extend earlier studies from this laboratory which showed differences in nuclear nonhistone proteins in tumors and other tissues. They provide a basis for more definitive comparisons of specific nuclear proteins in tumors of different growth rates and growing nontumorous tissues.

¹ These studies were supported by Cancer Research Center Grant CA-10893, P.1, awarded by the National Cancer Institute; Department of Health, Education and Welfare, by the Pauline Sterne Wolff Memorial Foundation; by the Bristol-Myers Fund; and by a generous gift from Mrs. Jack Hutchins.

Received 3/27/78. Accepted 10/23/78.