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Nucleic Acids Research, 1985, Vol. 13, No. 18 6621-6634
© 1985

Articles

Binding studies of SV40 T-antigen to SV40 binding site II

Philip Gottlieb, Marc S. Nasoff1, Eric F. Fisher2, Ann M. Walsh and Marvin H. Caruthers*

Department of Chemistry and Biochemistry, University of Colorado Boulder, CO 80309, USA

*To whom reprint requests should be addressed

Received May 13, 1985. Revised August 13, 1985. Accepted August 23, 1985.

SV40 T-Antigen binding site II was synthesized, cloned and analyzed for its ability to bind purified SV40 T-antigen. We report the binding constant of T-antigen for Isolated site II. Using a filter binding assay the calculated binding constant was 6–8 fold less efficient than site I previously reported. Binding constants were calculated using two methods. The first was a direct calculation using a protein titration curve (KD). The second was by the ratio of measured association and dissociation rates. Both methods gave similar constants. Protection studies with SV40 T-antigen on the T-antigen binding sites in the wild-type array demonstrated that the binding constants of site I and site II are similar to those calculated for the individual sites. These results demonstrate that SV40 T-antigen does not bind cooperatively to sites one and two as earlier believed and are in agreement with recent observations emanating from several laboratories.

1Present address: Genetics Institute, 225 Longwood Avenue, Boston, MA 02115, USA

2Present address: AMGEN, 1900 Oak Terrace Lane, Thousand Oaks, CA 93120, USA


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