Purification and further characterization of three DNA polymerases of rat ascites hepatoma cells
| Autor: | Tyunosin Ukita, Takashi Tsuruo |
|---|---|
| Rok vydání: | 1974 |
| Předmět: |
Carcinoma
Hepatocellular Time Factors DNA polymerase Poly ADP ribose polymerase DNA polymerase II Tritium Biochemistry Genetics and Molecular Biology (miscellaneous) chemistry.chemical_compound Adenosine Triphosphate Cytosol Animals Carbon Radioisotopes Polymerase Klenow fragment Cell Nucleus Membranes DNA clamp Ethanol DNA synthesis biology Liver Neoplasms Neoplasms Experimental Templates Genetic Hydrogen-Ion Concentration Chromatography Ion Exchange Molecular biology Chromatin Rats Isoenzymes Kinetics Biochemistry chemistry DNA Nucleotidyltransferases Chromatography Gel biology.protein Spectrophotometry Ultraviolet Chloromercuribenzoates DNA |
| Zdroj: | Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis. 353:146-159 |
| ISSN: | 0005-2787 |
| Popis: | Three DNA polymerases have been isolated from rat ascites hepatoma cells and the properties of the reactions have been investigated. A polymerase (cytoplasmic polymerase: polymerase C) has been purified 560-fold from the soluble fraction of the cells and two different DNA polymerases (polymerase P-1 and P-2) have been purified about 70- and 60-fold, respectively, from the nuclear membrane chromatin fraction of the cells. Polymerase C has a low level of exonuclease activity but both preparations of P-1 and P-2 have no detectable nuclease activity. ATP stimulates DNA synthesis by nuclei and nuclear membrane chromatin fraction but has no stimulating effect on the purified polymerases. The activities of polymerase C and P-1 are strongly inhibited by 0.8 mM p- chloromercuribenzoate and by 1 mM N- ethylmaleimide , while only 17% and 23% inhibitions are observed with P-2, respectively. Ethanol inhibits the reaction with polymerase C and P-1, but stimulates P-2 reaction. The pH optimum of polymerase C is 7.0 in potassium phosphate buffer and that of P-1 is 6.3 in potassium phosphate, 7.3 in Tris-HCl, and 8.5 in glycine-KOH buffer. The pH optimum of P-2 is 9.8 in glycine-KOH buffer. Native DNA is more effective than heat-denatured DNA as a template for three polymerases, but activated DNA is far more effective than native DNA. Digestion of activated DNA with exonuclease III results in increases of template activity for polymerase C and P-1, but the increase with P-1 is very small. While for P-2 polymerase, no stimulating effect was observed. Poly[d(A-T) · poly[d(T-A)] is more effective than activated DNA for P-2 enzyme. |
| Databáze: | OpenAIRE |
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