Characterization of distinct nuclear and mitochondrial forms of human deoxyuridine triphosphate nucleotidohydrolase

R D Ladner, D E McNulty, G D Roberts, S J Caradonna

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68 Citations (Scopus)

Abstract

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase; EC 3.6.1.23) was purified from HeLa cells by immunoaffinity chromatography. Based on SDS-polyacrylamide gel electrophoresis, two distinct forms of dUTPase were evident in the purified preparation. These proteins were further characterized by a combination of NH2-terminal protein sequencing, mass spectrometry, and mass spectrometry-based protein sequencing. These analyses indicate that the two forms of dUTPase are largely identical, differing only in a short region of their amino-terminal sequences. Despite the structural difference, both forms of dUTPase exhibited identical binding characteristics for dUTP. Each form of dUTPase has a distinct cellular localization. Cellular fractionation and isopycnic density centrifugation indicate that the lower molecular weight form of dUTPase (DUT-N) is associated with the nucleus, while the higher molecular weight species (DUT-M) fractionates with the mitochondria. The DUT-N isoform is approximately 30-fold more abundant in HeLa cells than DUT-M as determined by densitometry. The NH2-terminal protein sequence of both DUT-N and DUT-M did not match previous reports of the predicted amino-terminal sequence for human dUTPase (McIntosh, E.M., Ager, D.D., Gadsden, M.H., and Haynes, R.H. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 8020-8024; Strahler, J.R., Zhu X., Hora, N., Wang, Y.K., Andrews, P.C., Roseman, N.A., Neel, J.V., Turka, L., and Hanash, S.M. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 4991-4995). A cDNA corresponding to the DUT-N isoform was isolated utilizing an oligonucleotide probe based on the determined NH2-terminal sequence. The cDNA contains a 164-amino acid open reading frame, encoding a protein of Mr 17,748. The DUT-N cDNA sequence matches the previously cloned cDNAs with the exception of a few discrepancies in the 5' end. Our data indicate a 69-base pair addition to the 5' end of the previously reported open reading frame.

Original languageEnglish
Pages (from-to)7745-51
Number of pages7
JournalThe Journal of biological chemistry
Volume271
Issue number13
Publication statusPublished - 29 Mar 1996

Keywords

  • Amino Acid Sequence
  • Base Sequence
  • Blotting, Western
  • Cell Fractionation
  • Cell Nucleus
  • Chromatography, Affinity
  • Chromatography, Ion Exchange
  • Cloning, Molecular
  • Electrophoresis, Polyacrylamide Gel
  • Gene Library
  • HeLa Cells
  • Humans
  • Isoenzymes
  • Mass Spectrometry
  • Mitochondria
  • Molecular Sequence Data
  • Molecular Weight
  • Oligodeoxyribonucleotides
  • Peptide Fragments
  • Pyrophosphatases
  • Sequence Homology, Amino Acid
  • T-Lymphocytes
  • Comparative Study
  • Journal Article
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

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