Molecular characterization of a specific thiamine triphosphatase widely expressed in mammalian tissues.
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 ) 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 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-beta-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.
Lakaye
Bernard
B
Center for Cellular and Molecular Neurobiology, Department of Human Histology, University of Liège, 4020 Liège, Belgium.
Makarchikov
Alexander F
AF
Antunes
Adelio Fernandes
AF
Zorzi
Willy
W
Coumans
Bernard
B
De Pauw
Edwin
E
Wins
Pierre
P
Grisar
Thierry
T
Bettendorff
Lucien
L
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D019992:Databases as Topic; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013837:Thiamin-Triphosphatase / Q000096:biosynthesis* / Q000737:chemistry*; D013997:Time Factors; D014018:Tissue Distribution; D014162:Transfection
D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D007544:Isopropyl Thiogalactoside; D005982:Glutathione Transferase; D013837:Thiamin-Triphosphatase