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• W2063991909 abstract "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; EC3.6.1.28) 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 GenBankTM 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-β-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. 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; EC3.6.1.28) 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 GenBankTM 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-β-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. In most cells, the major form of thiamine (vitamin B1) is thiamine diphosphate (ThDP), 1The abbreviations used are: ThDPthiamine diphosphateGSTglutathione S-transferaseIPTGisopropyl-β-d-thiogalactopyranosideThMPthiamine monophosphateThTPthiamine triphosphateThTPasethiamine triphosphataseHPLChigh performance liquid chromatographyMSmass spectroscopy1The abbreviations used are: ThDPthiamine diphosphateGSTglutathione S-transferaseIPTGisopropyl-β-d-thiogalactopyranosideThMPthiamine monophosphateThTPthiamine triphosphateThTPasethiamine triphosphataseHPLChigh performance liquid chromatographyMSmass spectroscopya cofactor for pyruvate and 2-oxoglutarate dehydrogenases, as well as transketolase. However, most animal tissues also contain free thiamine, thiamine monophosphate (ThMP), and small amounts of thiamine triphosphate (ThTP) (1.Bettendorff L. Wins P. Recent Res. Dev. Neurochem. 1999; 2: 37-62Google Scholar). ThTP is found in most animal cells, as well as in yeast and bacteria, but its physiological importance remains unclear. For several decades, ThTP was thought to play a specific function in excitable tissues (2.Cooper J.R. Pincus J.H. Neurochem. Res. 1979; 4: 223-239Crossref PubMed Scopus (129) Google Scholar, 3.Bettendorff L. Metab. Brain Dis. 1994; 9: 183-209Crossref PubMed Scopus (58) Google Scholar) but until recently, no compelling evidence could be found to support this hypothesis. In inside-out patches of neuroblastoma cells, ThTP activates a high conductance chloride channel, possibly through phosphorylation (4.Bettendorff L. Kolb H.A. Schoffeniels E. J. Membr. Biol. 1993; 136: 281-288Crossref PubMed Scopus (54) Google Scholar), but the role of this so-called maxi-chloride channel remains unknown. In Torpedoelectric organ, [γ-32P]ThTP was found to phosphorylate rapsyn (5.Nghiêm H.O. Bettendorff L. Changeux J.P. FASEB J. 2000; 14: 543-554Crossref PubMed Scopus (68) Google Scholar), a protein required for the clustering of acetylcholine receptors at the neuromuscular junction (6.Gautam M. Noakes P.G. Mudd J. Nichol M. Chu G.C. Sanes J.R. Merlie J.P. Nature. 1995; 377: 232-236Crossref PubMed Scopus (468) Google Scholar). This phosphorylation was highly specific for ThTP compared with ATP, and, surprisingly, ThTP phosphorylated a histidyl residue (5.Nghiêm H.O. Bettendorff L. Changeux J.P. FASEB J. 2000; 14: 543-554Crossref PubMed Scopus (68) Google Scholar). Two or three phosphorylated protein bands were also observed in membranes prepared from rodent brain, but they have not been identified so far. To our knowledge, this is the first description of a protein phosphorylation in mammalian tissues with a phosphate donor other than ATP, and this could be part of a novel signal transduction pathway. It is therefore of interest to study the metabolism of ThTP in animal cells. thiamine diphosphate glutathione S-transferase isopropyl-β-d-thiogalactopyranoside thiamine monophosphate thiamine triphosphate thiamine triphosphatase high performance liquid chromatography mass spectroscopy thiamine diphosphate glutathione S-transferase isopropyl-β-d-thiogalactopyranoside thiamine monophosphate thiamine triphosphate thiamine triphosphatase high performance liquid chromatography mass spectroscopy The enzymatic mechanisms of ThTP synthesis are still poorly understood. Skeletal muscles sometimes contain unusually high amounts of ThTP because its synthesis can be catalyzed by adenylate kinase according to the reaction ADP + ThDP ↔ AMP + ThTP (7.Miyoshi K. Egi Y. Shioda T. Kawasaki T. J. Biochem. (Tokyo). 1990; 108: 267-270Crossref PubMed Scopus (30) Google Scholar). Because ThDP is a very poor substrate for adenylate kinase, this mechanism can be of importance only in cell types where adenylate kinase is very abundant. In most tissues, ThTP is believed to be synthesized from ThDP according to the reaction ThDP + ATP ↔ ThTP + ADP, catalyzed by ThDP kinase, an enzyme that remains poorly characterized. Purification procedures from bovine brain (8.Nishino K. Itokawa Y. Nishino N. Piros K. Cooper J.R. J. Biol. Chem. 1983; 258: 11871-11878Abstract Full Text PDF PubMed Google Scholar), rat liver (9.Voskoboev A.I. Luchko V.S. Vopr. Med. Khim. 1980; 26: 564-568PubMed Google Scholar), and brewer's yeast (10.Chernikevich I.P. Luchko V.S. Voskoboev A.I. Ostrovsky Y.M. Biokhimiya. 1984; 49: 899-907Google Scholar) were described, but in each case, the material obtained had a very low specific activity, and no sequencing of the enzyme was attempted. In contrast, the enzymes catalyzing ThTP hydrolysis have been studied in more detail. Animal tissues contain a membrane-associated as well as a soluble thiamine triphosphatase (ThTPase; EC 3.6.1.28). The membrane-bound ThTPase (11.Barchi R.L. Braun P.E. J. Biol. Chem. 1972; 247: 7668-7673Abstract Full Text PDF PubMed Google Scholar, 12.Bettendorff L. Michel-Cahay C. Grandfils C. De Rycker C. Schoffeniels E. J. Neurochem. 1987; 49: 495-502Crossref PubMed Scopus (55) Google Scholar, 13.Bettendorff L. Grandfils C. Wins P. Schoffeniels E. J. Neurochem. 1989; 53: 738-746Crossref PubMed Scopus (23) Google Scholar) has not been purified, and its specificity for ThTP remains uncertain. The soluble ThTPase first described by Hashitani and Cooper (14.Hashitani Y. Cooper J.R. J. Biol. Chem. 1972; 247: 2117-2119Abstract Full Text PDF PubMed Google Scholar) in rat brain has been purified to homogeneity from bovine brain (15.Makarchikov A.F. Chernikevich I.P. Biochim. Biophys. Acta. 1992; 1117: 326-332Crossref PubMed Scopus (29) Google Scholar) and kidney (16.Makarchikov A.F. J. Biochem. Mol. Biol. Biophys. 2001; 5: 75-82Google Scholar). Bovine ThTPase has an alkaline pH optimum, a relatively low Km(about 35 μm), and a virtually absolute specificity for ThTP (15.Makarchikov A.F. Chernikevich I.P. Biochim. Biophys. Acta. 1992; 1117: 326-332Crossref PubMed Scopus (29) Google Scholar, 16.Makarchikov A.F. J. Biochem. Mol. Biol. Biophys. 2001; 5: 75-82Google Scholar). Soluble ThTPase is found in most mammalian tissues studied so far (17.Penttinen H.K. Uotila L. Med. Biol. 1981; 59: 177-184PubMed Google Scholar). 2A. F. Makarchikov, unpublished results.2A. F. Makarchikov, unpublished results. In this work, we report the purification of the soluble ThTPase from calf brain and its partial sequencing by tandem mass spectrometry. The partial sequence screened against known expressed sequence tags allowed us to obtain the complete bovine and human sequences. Both were cloned by reverse transcription-PCR, the human enzyme was functionally expressed in Escherichia coli, and its distribution in human tissue was investigated. After the high affinity thiamine transporter, whose mutation causes thiamine-responsive megaloblastic anaemia (18.Fleming J.C. Tartaglini E. Steinkamp M.P. Schorderet D.F. Cohen N. Neufeld E.J. Nat. Genet. 1999; 22: 305-308Crossref PubMed Scopus (201) Google Scholar), and thiamine pyrophosphokinase (19.Nosaka K. Onozuka M. Nishino H. Nishimura H. Kawasaki Y. Ueyama H. J. Biol. Chem. 1999; 274: 34129-34133Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), ThTPase is the third protein of thiamine metabolism to be characterized in mammals. Chemicals, if not otherwise stated, were from Sigma or Merck Eurolab (Leuven, Belgium). ThTP was obtained from Wako Chemicals (Osaka, Japan). Trypsin was from Hoffman-La Roche Ltd. (Basel, Switzerland), and acetonitrile was obtained from J. T. Baker (Mallinckrodt Baker Inc., Phillipsburg, NJ). The water used was of Milli-Q grade (Millipore Co., Bedford, MA). The procedure used was derived from methods previously described (14.Hashitani Y. Cooper J.R. J. Biol. Chem. 1972; 247: 2117-2119Abstract Full Text PDF PubMed Google Scholar, 15.Makarchikov A.F. Chernikevich I.P. Biochim. Biophys. Acta. 1992; 1117: 326-332Crossref PubMed Scopus (29) Google Scholar). Briefly, 16 calf forebrains (4.8 kg) from a local slaughterhouse were homogenized (Polytron blender, 25,000 rpm for 5 min, 0 °C) in 2 volumes of Tris-Cl buffer (5 mm, pH 8.2) containing 1 mm Na2EDTA. After gentle stirring at 0–4 °C for 30 min, the homogenate was centrifuged (30 min, 100,000 ×g), and the supernatant (S1) was brought to pH 4.5 with acetic acid (14.Hashitani Y. Cooper J.R. J. Biol. Chem. 1972; 247: 2117-2119Abstract Full Text PDF PubMed Google Scholar). After centrifugation (20,000 × g, 15 min), the acidic supernatant was neutralized to pH 7.8 with NaOH, and ammonium sulfate was added to 50% saturation. After centrifugation (15 min, 15,000 × g), the pellet was suspended in 5% of the initial volume of Tris-EDTA buffer (pH 7.8), dialyzed against the same buffer, and applied on a DEAE-Sephacel resin (Amersham Biosciences). Elution was carried out using a Tris-Cl gradient (20–500 mm, pH 7.8) containing 20% glycerol. The fractions containing ThTPase activity were dialyzed before chromatography on a Toyopearl HW 65F resin (Tosoh Corporation, Tokyo, Japan) as described earlier (15.Makarchikov A.F. Chernikevich I.P. Biochim. Biophys. Acta. 1992; 1117: 326-332Crossref PubMed Scopus (29) Google Scholar). After concentration of the fractions containing the highest specific activity (Centriplus 10, Amicon Inc., Beverly, MA), the enzyme was run on Sephadex G-75 and Blue-Sepharose Cl-4B (15.Makarchikov A.F. Chernikevich I.P. Biochim. Biophys. Acta. 1992; 1117: 326-332Crossref PubMed Scopus (29) Google Scholar). The purity of the preparation was tested by polyacrylamide (12%) gel electrophoresis in the presence of SDS according to Laemmli (20.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar). Protein bands were visualized by silver or Coomassie Blue staining. The purification data are summarized in Table I.Table IPurification of soluble ThTPase from bovine brainFractionTotal activity1-aThe ThTP concentration in the reaction medium was 10 μm.Total proteinSpecific activityPurificationYieldμmol·min−1mgμmol·min−1·mg−1-fold%S1230990000.00231100Acid supernatant170320000.00532.374Ammonium sulfate precipitate14647000.03113.464DEAE-Sephacel561400.4117626Toyopearl HW-65F264.85.4231011Sephadex G-75220.6037159318.5Blue-Sepharose110.11103444744.81-a The ThTP concentration in the reaction medium was 10 μm. Open table in a new tab If not otherwise stated, the reaction medium contained 70 μl of Bis-Tris-propane buffer (50 mm, pH 8.7), 10 μl of MgCl2 (50 mm), 10 μl of ThTP (100 μm), and 10 μl of the enzyme preparation at the appropriate dilution (20–10,000×). After incubation (10 min, 37 °C), the reaction was stopped by the addition of 500 μl of trichloroacetic acid. After extraction with 3 × 1.5 ml of diethyl ether, the ThDP formed was estimated by HPLC (21.Bettendorff L. Peeters M. Jouan C. Wins P. Schoffeniels E. Anal. Biochem. 1991; 198: 52-59Crossref PubMed Scopus (71) Google Scholar). To assess the substrate specificity of either the purified bovine enzyme or the GST-ThTPase, several potential substrates were tested for enzymatic hydrolysis. For 4-nitrophenyl phosphate, the absorbance of the released 4-nitrophenolate was read at 408 nm at pH 10. For ThDP, ThMP, ATP, GTP, CTP, and ITP, the inorganic phosphate released was measured (22.Lanzetta P.A. Alvarez L.J. Reinach P.S. Candia O.A. Anal. Biochem. 1979; 100: 95-97Crossref PubMed Scopus (1794) Google Scholar). In all cases, the incubation was run at 37 °C for 100 min in the presence of 25 mm Tris buffer (pH 8.5), 5 mm MgCl2, 4 mmsubstrate, and an enzyme concentration 20 times higher than for determination of ThTPase activity. One of the purified protein fractions (about 12 μg/ml) obtained after Blue-Sepharose chromatography was concentrated by ultracentrifugation using a Microcon-YM10 centrifugal filter device (Millipore). The initial volume of 1 ml was concentrated to about 80 μl, and the buffer was replaced by digestion buffer (500 mm ammonium acetate, 20 mm CaCl2). The protein was digested by adding 6 μl of a trypsin solution (0.1 μg/μl) reconstituted in 1 mm HCl. Acetonitrile (final concentration, 1% v/v) was added to accelerate digestion, which was performed for 12 h at 37 °C, pH 7.4. The tryptic peptides were fractionated and desalted by elution on a ZipTipC18 pipette tip (Millipore). The elution was carried out using a mixture of water/acetonitrile/acetic acid with successive volume ratios of 93/5/2, 88/10/2, 83/15/2, 73/25/2, 68/30/2, 58/40/2, 48/50/2, and 28/70/2. The fractions obtained were analyzed by nano-electrospray ionization MS/MS using a Q-TOF2 mass spectrometer (Micromass Co., Manchester, UK) as described by Shevchenkoet al. (23.Shevchenko A. Chernushevich I. Wilm M. Mann M. Methods. Mol. Biol. 2000; 146: 1-16PubMed Google Scholar). The selection of the analyzed ions and the adjustment of the collision energy were made manually. The obtained fragmentation data were analyzed using sequencing software, PepSeq (Micromass Co.). Data base search was performed with the sequences obtained to eliminate those resulting from trypsin autodigestion. The peptides were delivered to the mass spectrometer by silica capillaries purchased from Protana (MDS Proteomics, Odense, Denmark). For the sequencing of the N- and C-terminal peptides, the enzyme was partially digested in the absence of acetonitrile for only 1 h at 37 °C. For the prediction of MS/MS fragmentation from peptide sequences and the comparison with mass spectra, BioLynx software (Micromass Co.) was used. One microgram of human or bovine brain poly(A)+ RNA (CLONTECH, Palo Alto, CA) was reverse transcribed into cDNA for 1 h at 37 °C by random priming using Moloney murine leukemia virus reverse transcriptase (Invitrogen) as described by the manufacturer. One-tenth of the reverse transcription reaction medium was submitted to 35 PCR cycles using Pwo polymerase (Roche Molecular Biochemicals). For human ThTPase amplification, primers HumF (5′-TCCTTGGGAACTCAGCAAACGT-3′) and HumR (5′-AGGAGTGGACTCCGTTAGACC-3′) were used. For bovine ThTPase, primers BovF (5′-ATGGCTCAGGGCCTGATTGAAG-3′) and BovR (5′-AGCGAGAGGAGTCACTGTGAG-3′) were used. Each PCR cycle consisted of denaturation at 94 °C for 30 s, hybridization at 63 °C for 30 s, and elongation at 72 °C for 60 s. The resulting PCR products were inserted into pCRII by TOPO cloning (Invitrogen) and sequenced using the T7 DNA sequencing kit (Amersham Biosciences). The human ThTPase open reading frame was amplified from the cloned cDNA using forward (5′-GGATCCCCATGGCCCAGGGCTTGATTGA-3′) and reverse (5′-GCGGCCGCCTAGCCCAGGCAGTGGTCAG-3′) primers. The amplified product was then inserted into BamHI/NotI sites of pGEX-5X-1 to produce a GST-ThTPase fusion protein. The E. coli strain BL 21 (Amersham Biosciences) was transformed with either the native or the recombinant plasmid and grown overnight on LB agar plates (1.5% (w/v) agar in LB broth) containing ampicillin (200 μg/ml). Individual bacterial colonies were grown under aerobial conditions at 37 °C in 2XYT/ampicillin medium at a density of about 5 × 109 cells/ml. Overexpression of GST or GST-ThTPase was induced by dilution of 100 μl of this bacterial culture in 1.6 ml of 2XYT/ampicillin medium in the presence of isopropyl-β-d-thiogalactopyranoside (IPTG) at 1.5 mg/ml. After 0, 1, 2, 3, or 4 h, the bacteria were collected by centrifugation (20,000 × g, 1 min) and suspended in 150 μl of 2XYT medium. Control experiments were made under the same conditions but without IPTG. 50 μl of the bacterial suspension were diluted with 50 μl of loading buffer (2×) and boiled for 1 min, and aliquots of 4 μl were submitted to SDS-PAGE electrophoresis on 12% gels. The rest of the bacterial suspension was incubated in the presence of 10% Triton X-100 for 30 min on ice and diluted 100–1000 times before determination of enzyme activity as described above. The entire cloned human ThTPase cDNA was used as a probe. It was labeled with [α-32P]dCTP (ICN, Costa Mesa, CA) using the Random Primers DNA labeling system (Invitrogen) and then purified on ProbeQuant G50 Micro columns (Amersham Biosciences). The human multiple tissue expression array (CLONTECH) was prehybridized for 1 h at 60 °C in ExpressHyb (CLONTECH). Hybridization was performed for 15 h at 60 °C in the above solution containing 5 × 106 cpm/ml of the heat-denatured probe. The multiple tissue expression array was washed twice in 2× SSC, 0.1% SDS at 60 °C and twice in 0.1× SSC, 0.1% SDS at 55 °C as described by the manufacturer and then exposed in a PhosphorImager (Amersham Biosciences) for 36 h. The enzyme had to be purified about 45,000-fold before a homogenous preparation was obtained, suggesting that it is a relatively rare protein in the bovine brain. Our purification procedure gave 107 μg of ThTPase with a total yield of 4.8% (Table I). Analysis by SDS-PAGE revealed a single band with an apparent molecular mass of 27 kDa (Fig. 1). Mass spectrometry gave a molecular mass of 23,892 Da, a value lower than the one obtained from SDS-PAGE. The difference between the two values might be the consequence of the overall important negative charge of the protein, which can lead to decreased mobility during SDS-PAGE (24.Matagne A. Joris B. Frère J.M. Biochem. J. 1991; 280: 553-556Crossref PubMed Scopus (60) Google Scholar). Chromatography on Sephadex G-75 gave a molecular mass of 25 kDa (not shown), suggesting that the native protein is a monomer, in agreement with previous results (15.Makarchikov A.F. Chernikevich I.P. Biochim. Biophys. Acta. 1992; 1117: 326-332Crossref PubMed Scopus (29) Google Scholar). The purified bovine enzyme obeyed Michaelis-Menten kinetics with aKm of 39 ± 7 μm (substrate concentration, 0.01–1 mm) and a specific activity of 9 ± 2 μmol·s−1·mg−1(Vmax at 37 °C). We can thus calculate that the catalytic constant (kcat) and the catalytic efficiency (kcat/Km) are 240 s−1 and 6 × 106s−1·m−1, respectively. In agreement with previous results (15.Makarchikov A.F. Chernikevich I.P. Biochim. Biophys. Acta. 1992; 1117: 326-332Crossref PubMed Scopus (29) Google Scholar, 16.Makarchikov A.F. J. Biochem. Mol. Biol. Biophys. 2001; 5: 75-82Google Scholar, 17.Penttinen H.K. Uotila L. Med. Biol. 1981; 59: 177-184PubMed Google Scholar), we found that the purified ThTPase was highly substrate-specific. A slight 4-nitrophenyl phosphatase activity was detected, but it was less than 1% of ThTPase activity. With ThDP, ThMP, and nucleoside 5′-triphosphates, no significant enzymatic hydrolysis could be detected by the very sensitive method used (22.Lanzetta P.A. Alvarez L.J. Reinach P.S. Candia O.A. Anal. Biochem. 1979; 100: 95-97Crossref PubMed Scopus (1794) Google Scholar), indicating that if any enzymatic hydrolysis of those substrates occurs, it is less than 0.2% of ThTPase activity. The sequence of several internal peptides (see Fig. 2 for their sequence) was obtained by electrospray ionization MS/MS analysis. Each peptide was compared with the sequences of the GenBankTM data base using the BLAST algorithm, and all gave a nearly perfect match (Fig. 2A) with two newly described hypothetical proteins, one in human called MGC2652 (NM_024328) and another in Macaca fascicularis (AB055296). Using primers designed on the basis of the MGC2652 sequence, we were able to amplify a cDNA of the expected size from human brain poly(A)+ RNA. The human protein is 230 amino acids long and has a predicted molecular mass of 25,550 Da. Its gene is located on the short arm of chromosome 14. The amino acid composition is characterized by a high percentage of negatively charged residues (17.4% of Glu and Asp) and a low content of Ile (1.7%) and Asn (0.4%). The comparison of the sequence of the human cDNA with the bovine expressed sequence tags gave other matches (AW654551,BG690979, BF076311, and BF653287) that allowed the amplification of a cDNA encoding a 219-amino acid protein from bovine brain poly(A)+ RNA. Its sequence perfectly matches the bovine peptide sequences obtained by mass spectrometry, except for the N-terminal methionine residue (see below). The predicted average molecular mass of the bovine protein is 23,983 Da, a value slightly higher than the mass determined by mass spectrometry for the purified bovine enzyme (23,892 Da). Most of the difference can be accounted for if we assume that the N-terminal methionyl residue is cleaved and that the new N-terminal alanine is acetylated. This hypothesis was confirmed by comparing the predicted collision-induced dissociation mass spectra for two N-terminal peptides of the protein (Fig. 3). The C-terminal peptide (209–219, LLEVYGSKEKP) was obtained in a similar manner. In addition, considering that the bovine protein contains only two cysteyl residues, at positions 66 and 88, respectively (Fig. 2), the presence of a disulfide bridge would lead to the loss of two hydrogen atoms, giving a mass of 23,892 Da, exactly as determined. A difference of 2 Da is, however, within the error on the mass determination of the entire protein by mass spectrometry (100 ppm). On the other hand, modifiers of free thiols such as p-chloromercuribenzoate and Ellman's reagent (5,5′dithiobis-2-nitro-benzoic acid) inhibit the activity of the purified bovine ThTP (not shown), suggesting that the presence of free SH groups is essential for catalytic activity. In addition, we found that 2-mercaptoethanol does not change the electrophoretic mobility of the protein in polyacrylamide gels. The presence of a disulfide bridge therefore appears unlikely. At the amino acid level, the bovine ThTPase has 80 and 79% identity with the human and the macaque enzyme, respectively (Fig. 2A). Analysis of the sequences using the PROSITE motif search revealed the presence of several potential phosphorylation sites present in the three sequences, among them two consensus sites (at positions 34 and 123) for protein kinase C and three consensus sites (at positions 34, 38, and 60) for casein kinase 2. The hydrophobicity plot of the human enzyme is typical of a soluble protein (Fig. 2B), with several highly polar or charged regions. Interestingly, no homology, even partial, with any other known vertebrate protein was found. Partial short sequences with significant identity corresponding to hypothetical open reading frames were found in E. coli (AP002564 and AE000387), Caenorhabditis elegans (L23650), Drosophila melanogaster (AE003477 andAE003598), or Saccharomyces cerevisiae (NP_014781), but they do not seem to be related to each other. The human ThTPase cDNA was overproduced in E. coli as a GST fusion protein in the presence of IPTG (Fig. 4). The fusion protein had a molecular mass of 50 kDa, which corresponds to an approximate molecular mass of 25 kDa for the ThTPase moiety, as expected from the amino acid sequence. In E. coli transfected with GST, we found only a relatively low intrinsic ThTPase activity (120 ± 34 pmol·min−1·mg of protein−1,n = 7). In fact, bacteria do not appear to contain a specific ThTPase, but they do contain nonspecific phosphatases able to hydrolyze ThTP to some extent (25.Nishimune T. Hayashi R. J. Nutr. Sci. Vitaminol. (Tokyo). 1987; 33: 113-127Crossref PubMed Scopus (6) Google Scholar).2 As shown in Fig. 5, the activity was increased over 1000-fold in noninduced GST-ThTPase recombinant bacteria, reaching 0.17 μmol·min−1·mg−1. After induction by IPTG, this activity still increased over 10-fold, reaching 2.1 μmol·min−1·mg−1 after 4 h (Fig. 5C). No increase in ThTPase activity was observed after induction in bacteria transfected with GST alone (Fig. 5B). ThTP hydrolysis by recombinant GST-ThTPase resulted in the formation of ThDP only. If an unspecific phosphatase was present, ThDP would have been further hydrolyzed to ThMP, but this was not observed. Furthermore, when ATP (100 μm) replaced ThTP in the incubation medium under the same conditions (Fig. 5, D–F), no hydrolysis of ATP was observed. Although IPTG increased ThTPase activity over 10 times in GST-ThTPase recombinant bacteria, it did not increase to any significant amount the hydrolysis of 4-nitrophenyl phosphate, ThDP, ThMP, or nucleoside 5′-triphosphates. This suggests that the recombinant enzyme, like the native ThTPase, has little or no hydrolytic activity on substrates other than ThTP. Because human ThTPase has not been studied in detail so far, we compared some properties of the GST-ThTPase with genuine ThTPase prepared from human brain. The activity of the GST-ThTPase fusion protein falls more abruptly at alkaline pH than with the enzyme prepared from human brain. Although both the human and the bovine enzyme have about the same pH optimum (around 8.5), the human enzyme has a broader pH spectrum, with a higher activity at neutral pH. ThTPase from human cerebellar cortex had a Km of 126 μm (Table II), a value three to four times higher than in crude extracts from bovine or rat brain. Notice that the Km of the bovine enzyme for ThTP was not significantly different before (32 ± 6 μm) and after (39 ± 7 μm) purification. Human ThTPase was isolated from postmortem tissue, but we have not found any effect of the postmortem delay (≤15 h) on theVmax or the Km of bovine enzyme. Actually, the enzyme was remarkably insensitive to chemical denaturation and proteolytic attack; although it was isolated from calf brain in the absence of protease inhibitors, it remained intact as was shown by sequencing. The properties of the recombinant GST-ThTPase were similar to those of the native human enzyme, with aKm of 220 ± 23 μm(n = 5).Table IIKinetic parameters of ThTPase in the crude supernatant fraction of brain in several speciesVmax2-aThe ThTP concentration varied between 10 and 200 μm. Km andVmax were obtained by fitting the experimental data to the Michaelis-Menten equation using GraphPad Prism (GraphPad Software, San Diego, CA).Km2-aThe ThTP concentration varied between 10 and 200 μm. Km andVmax were obtained by fitting the experimental data to the Michaelis-Menten equation using GraphPad Prism (GraphPad Software, San Diego, CA).nmol·mg−1·min−1μmHuman cerebellar cortex2-bMean ± S.D. for five determinations on the postmortem (4 h postmortem delay) cerebellar cortex of a 59-year-old male deceased from bronchopneumonia (Department of Pathology, University of Liège).8.7 ± 0.9126 ± 5Calf cerebral cortex2-cThis study, means ± S.D. for three determinations on a pool of 16 calf brains.11.5 ± 332 ± 6Rat cerebral cortex2-dMean ± S.D. for three animals.21 ± 423 ± 72-a The ThTP concentration varied between 10 and 200 μm. Km andVmax were obtained by fitting the experimental data to the Michaelis-Menten equation using GraphPad Prism (GraphPad Software, San Diego, CA).2-b Mean ± S.D. for five determinations on the postmortem (4 h postmortem delay) cerebellar cortex of a 59-year-old male deceased from bronchopneumonia (Department of Pathology, University of Liège).2-c This study, means ± S.D. for three determinations on a pool of 16 calf brains.2-d Mean ± S.D. for three animals. Open table in a new tab ThTPase expression was profiled by dot blot hybridization on a mRNA multiple tissue expression array, using the entire cDNA as probe (Table III). The main conclusion to be drawn from this experiment is that ThTPase mRNA appears to be very widely expressed, but only at a low level," @default.
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• W2063991909 date "2002-04-01" @default.
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• W2063991909 title "Molecular Characterization of a Specific Thiamine Triphosphatase Widely Expressed in Mammalian Tissues" @default.
• W2063991909 cites W1483518403 @default.
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