Purification and characterization of a DNA binding protein in a nuclear scaffold fraction from rat ascites hepatoma cells.
Our most recent work [Hibino et al. (1995) Cancer Lett., 88, 49-55] has shown that the selective binding affinities of highly repetitive DNA components for a nuclear scaffold protein from rat ascites hepatoma cells (P230) depend on the degree of sequence-directed bending of the helix axis. In the present experiment, this protein has been highly purified and isolated by a series of column chromatographic procedures to migrate as a single band to a molecular weight position of 230 kd on a SDS-polyacrylamide gel. A filter binding assay showed that the binding of a repetitive AT-rich component (369 bp XmnI fragment) from the hepatoma nucleus, which has a strongly bent overall structure, to isolated P230 is based on a cooperative mode of interaction. Distamycin A, which binds specifically to AT-rich DNA, removed the bend in the XmnI fragment and inhibited binding to this protein. These results suggest that AT-rich regions in highly repetitive DNA cause bending of the helix axis to be recognized by nuclear scaffold protein(s). Moreover, it has been shown that the nuclear scaffold fraction from rat liver or an actively growing hepatocyte cell line (Ac2F cells) does not contain P230, but does have a repetitive bent DNA binding protein (P130), which has an apparent molecular weight of 130 kd. In addition, the immunoblot analysis showed that mouse anti-P130 antiserum reacts with P230. Thus, the results in the present study imply that there is some difference in the higher order structure of the nuclear DNA attachment region between rat liver or actively growing hepatocytes and the hepatoma, although P230 appears to be immunochemically similar to P130.
Hibino
Y
Y
Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama City, Japan.
Tsukada
S
S
Sugano
N
N
D000818:Animals; D001201:Ascites / Q000378:metabolism* / Q000473:pathology; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D002850:Chromatography, Gel; D004273:DNA, Neoplasm / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008114:Liver Neoplasms, Experimental / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D051381:Rats; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins