Characterization of distinct nuclear and mitochondrial forms of human deoxyuridine triphosphate nucleotidohydrolase.
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.
Ladner
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RD
Department of Molecular Biology, University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, Stratford, 08084, USA.
McNulty
D E
DE
Carr
S A
SA
Roberts
G D
GD
Caradonna
S J
SJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000201:enzymology*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013058:Mass Spectrometry; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011755:Pyrophosphatases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013601:T-Lymphocytes
D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase