Binding studies of SV40 T-antigen to SV40 binding site II.
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.
D000952:Antigens, Polyomavirus Transforming; D000957:Antigens, Viral, Tumor / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003850:Deoxyribonuclease I; D007700:Kinetics; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010641:Phenotype; D010957:Plasmids; D011494:Protein Kinases / Q000378:metabolism*; D013539:Simian virus 40 / Q000201:enzymology* / Q000235:genetics
D000952:Antigens, Polyomavirus Transforming; D000957:Antigens, Viral, Tumor; D009856:Oncogene Proteins, Viral; D011494:Protein Kinases; D003850:Deoxyribonuclease I