Purification and determination of the NH2-terminal amino acid sequence of uracil-DNA glycosylase from human placenta

Biochemistry. 1989 Jan 24;28(2):780-4. doi: 10.1021/bi00428a055.

Abstract

Uracil-DNA glycosylase has been purified approximately 130,000-fold from extracts of human placenta. Although all of the uracil-DNA glycosylase activity coeluted through six chromatographic steps, at least four distinct peaks of activity were resolved in the final purification on a Mono S column. Each of the peaks containing uracil-DNA glycosylase activity contained two peptides of Mr = 29,000 and Mr = 26,500, respectively, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Experimental evidence indicated that the Mr = 29,000 peptide was the uracil-DNA glycosylase enzyme. The amino-terminal sequence of each peptide was determined after blotting of the peptides from the gel onto Polybrene GF/C paper. The sequences were not related to each other, and neither was any significant homology to other proteins found. Uracil-DNA glycosylase had a molecular turnover number of approximately 600/min and apparent Km value of 2 microM. The enzyme is a basic protein and was stimulated about 10-fold by 60-70 mM NaCl whereas higher concentrations were inhibitory.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Chromatography, Affinity
  • Chromatography, Gel
  • Chromatography, Ion Exchange
  • Chromatography, Liquid
  • DNA Glycosylases*
  • DNA Repair
  • Female
  • Humans
  • Molecular Sequence Data
  • Molecular Weight
  • N-Glycosyl Hydrolases / isolation & purification*
  • Placenta / enzymology*
  • Pregnancy
  • Uracil-DNA Glycosidase

Substances

  • DNA Glycosylases
  • N-Glycosyl Hydrolases
  • Uracil-DNA Glycosidase