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J. Biol. Chem., Vol. 277, Issue 16, 13771-13777, April 19, 2002

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Molecular Characterization of a Specific Thiamine Triphosphatase Widely Expressed in Mammalian Tissues^*

Bernard Lakaye, Alexander F. Makarchikov^§, Adelio Fernandes Antunes^¶, Willy Zorzi, Bernard Coumans, Edwin De Pauw^¶, Pierre Wins^**, Thierry Grisar, and Lucien Bettendorff^**

From the  Center for Cellular and Molecular Neurobiology and the  Department of Human Histology, University of Liège, 4020 Liège, Belgium, the ^¶ Department of Physical Chemistry, University of Liège, 4000 Liège Sart Tilman, Belgium, and the ^§ Laboratory of Enzymology, Institute of Biochemistry, National Academy of Sciences of Belarus, 230009 Grodno, Belarus

Thiamine triphosphate (ThTP) is found at low concentrations in most animal tissues, and recent data suggest that it may act as a phosphate donor for the phosphorylation of some proteins. In the mammalian brain, ThTP synthesis is rapid, but its steady-state concentration remains low, presumably because of rapid hydrolysis. In this report we purified a soluble thiamine triphosphatase (ThTPase; EC 3.6.1.28) from calf brain. The bovine ThTPase is a 24-kDa monomer, hydrolyzing ThTP with virtually absolute specificity. Partial sequence data obtained from the purified bovine enzyme by tandem mass spectrometry were used to search the GenBank^TM data base. A significant identity was found with only one human sequence, the hypothetical 230-amino acid protein MGC2652. The coding regions from human and bovine brain mRNA were amplified by reverse transcription-PCR, cloned in Escherichia coli, and sequenced. The human open reading frame was expressed in E. coli as a GST fusion protein. Transformed bacteria had a high isopropyl--D-thiogalactopyranoside-inducible ThTPase activity. The recombinant ThTPase had properties similar to those of human brain ThTPase, and it was specific for ThTP. The mRNA was expressed in most human tissues but at relatively low levels. This is the first report of a molecular characterization of a specific ThTPase.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AF432862 (human) and AF432863 (bovine).

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