Studies on catalytic subunits of mouse myeloma alpha-polymerase.
Activity gel analysis has been used to identify mouse myeloma polypeptides with DNA polymerase activity. Proteins in a crude homogenate of mouse myeloma were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sodium dodecyl sulfate was soaked from the gel, and polypeptides were allowed to renature in situ; then the intact gel was incubated in a DNA polymerase reaction mixture in order to localize DNA polymerases (Spanos, A., Sedgwick, S. G., Yarranton, G. T., Hübscher, U., and Banks, G. R. (1981) Nucleic Acids Res. 9, 1825-1839). This activity gel analysis revealed that the homogenate contains a Mr = 40,000 polypeptide with strong DNA polymerase activity; from its Mr and catalytic properties this enzyme was identified as beta-polymerase. The homogenate also contains two additional DNA polymerase activities giving relatively strong bands at Mr = approximately 76,000 and approximately 120,000, respectively. Results on the catalytic properties of both of these enzymes suggest that they are alpha-polymerases. Further evidence for the existence of a Mr approximately 120,000 alpha-polymerase catalytic polypeptide came from the observation that a purified preparation of one of the recognized species of mouse myeloma alpha-polymerase is composed of a Mr = approximately 120,000 polypeptide, as revealed by Coomassie blue staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 3 M urea. This purified enzyme does not contain polypeptides in the Mr = 40,000 to 70,000 range, and is capable of producing a strong band at Mr = 120,000 in the activity gel assay.
D000818:Animals; D016590:Aphidicolin; D002460:Cell Line; D004257:DNA Polymerase II / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004224:Diterpenes / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008970:Molecular Weight; D009374:Neoplasms, Experimental / Q000201:enzymology; D010954:Plasmacytoma / Q000201:enzymology*
D004224:Diterpenes; D046911:Macromolecular Substances; D016590:Aphidicolin; D004257:DNA Polymerase II; D004259:DNA-Directed DNA Polymerase