Pig tissues express a catalytically inefficient 25-kDa thiamine triphosphatase: Insight in the catalytic mechanisms of this enzyme
| Autor: | Thierry Grisar, Jan Czerniecki, Pierre Wins, Ilca Margineanu, Lucien Bettendorff, Bernard Coumans, Bernard Lakaye, Piotr Szyniarowski, Alexander F Makarchikov |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
Swine
Molecular Sequence Data Lysine Biophysics Mutagenesis (molecular biology technique) Biology Biochemistry Catalysis chemistry.chemical_compound Thiamine triphosphatase Complementary DNA Escherichia coli Animals Humans Amino Acid Sequence Cloning Molecular Molecular Biology chemistry.chemical_classification Sequence Homology Amino Acid Brain Immunohistochemistry Fusion protein Enzyme assay Molecular Weight Kinetics Enzyme chemistry Thiamin-Triphosphatase Mutagenesis Site-Directed biology.protein Thiamine triphosphate |
| Zdroj: | Biochimica et Biophysica Acta (BBA) - General Subjects. 1725:93-102 |
| ISSN: | 0304-4165 |
| Popis: | Thiamine triphosphate (ThTP) is found in most organisms and may be an intracellular signal molecule produced in response to stress. We have recently cloned the cDNA coding for a highly specific mammalian 25-kDa thiamine triphosphatase. The enzyme was active in all mammalian species studied except pig, although the corresponding mRNA was present. In order to determine whether the very low ThTPase activity in pig tissues is due to the absence of the protein or to a lack of catalytic efficiency, we expressed human and pig ThTPase in E. coli as GST fusion proteins. The purified recombinant pig GST-ThTPase was found to be 2-3 orders of magnitude less active than human GST-ThTPase. Using site-directed mutagenesis, we show that, in particular, the change of Glu85 to lysine is responsible for decreased solubility and catalytic activity of the pig enzyme. Immunohistochemical studies revealed a distribution of the protein in pig brain very similar to the one reported in rodent brain. Thus, our results suggest that a 25-kDa protein homologous to hThTPase but practically devoid of enzyme activity is expressed in pig tissues. This raises the possibility that this protein may play a physiological role other than ThTP hydrolysis. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect