Studies on catalytic subunits of mouse myeloma alpha-polymerase

J Biol Chem. 1982 Nov 10;257(21):13129-34.

Abstract

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.

MeSH terms

  • Animals
  • Aphidicolin
  • Cell Line
  • DNA Polymerase II / antagonists & inhibitors
  • DNA Polymerase II / isolation & purification
  • DNA Polymerase II / metabolism*
  • DNA-Directed DNA Polymerase / metabolism*
  • Diterpenes / pharmacology
  • Kinetics
  • Macromolecular Substances
  • Mice
  • Molecular Weight
  • Neoplasms, Experimental / enzymology
  • Plasmacytoma / enzymology*

Substances

  • Diterpenes
  • Macromolecular Substances
  • Aphidicolin
  • DNA Polymerase II
  • DNA-Directed DNA Polymerase