AraUTP-Affi-Gel 10: a novel affinity absorbent for the specific purification of DNA polymerase alpha-primase

Anal Biochem. 1988 Oct;174(1):318-24. doi: 10.1016/0003-2697(88)90552-0.

Abstract

For the specific purification of eukaryotic DNA-dependent DNA polymerase alpha, we prepared two novel affinity resins bearing 5-(E)-(4-aminostyryl) araUTP as a ligand. One of them was araUTP-Sepharose 4B which was coupled directly with the ligand and the other was araUTP-Affi-Gel 10 which was coupled with the ligand through a spacer. No DNA polymerase alpha-primase activity from cherry salmon (Oncorhynchus masou) testes was bound on the araUTP-Sepharose 4B in all cases examined. On the other hand, the araUTP-Affi-Gel 10 retains this enzyme activity when poly(dA) or poly(dA)-oligo(dT)12-18 is present. The retained enzyme activity was sharply eluted around 100-mM KCl concentrations as a single peak, and this fraction showed a specific activity of about 170,000 units/mg as alpha-polymerase activity. The highly purified DNA polymerase alpha-primase isolated using the araUTP-Affi-Gel 10 contained only three polypeptides, which showed Mr values of 120,000, 62,000, and 58,000, respectively, as judged using sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

MeSH terms

  • Animals
  • Chromatography, Affinity / methods*
  • DNA Polymerase II / isolation & purification
  • DNA Primase
  • Male
  • Molecular Weight
  • Peptides / isolation & purification
  • RNA Nucleotidyltransferases / isolation & purification*
  • Salmon
  • Sepharose / analogs & derivatives*
  • Testis / enzymology
  • Uracil Nucleotides* / analogs & derivatives
  • Uridine Triphosphate* / analogs & derivatives

Substances

  • Peptides
  • Uracil Nucleotides
  • ara-UTP-Affi-Gel 10
  • Sepharose
  • DNA Primase
  • RNA Nucleotidyltransferases
  • DNA Polymerase II
  • Uridine Triphosphate