Reference : Pig tissues express a catalytically inefficient 25-kDa thiamine triphosphatase: Insig...
Scientific journals : Article
Life sciences : Biochemistry, biophysics & molecular biology
http://hdl.handle.net/2268/4633
Pig tissues express a catalytically inefficient 25-kDa thiamine triphosphatase: Insight in the catalytic mechanisms of this enzyme
English
Szyniarowski, Piotr [> > > >]
Lakaye, Bernard mailto [Université de Liège - ULg > Département des sciences biomédicales et précliniques > Biochimie et physiologie humaine et pathologique >]
Czerniecki, Jan [Université de Liège - ULg > > Biochimie et physiologie humaine et pathologique >]
Makarchikov, Alexander F [> > > >]
Wins, Pierre [> > > >]
Margineanu, Ilca [> > > >]
Coumans, Bernard mailto [Université de Liège - ULg > > CNCM/ Centre fac. de rech. en neurobiologie cell. et moléc. >]
Grisar, Thierry mailto [Université de Liège - ULg > Département des sciences biomédicales et précliniques > Biochimie et physiologie humaine et pathologique]
Bettendorff, Lucien mailto [Université de Liège - ULg > Département des sciences biomédicales et précliniques > Biochimie et physiologie humaine et pathologique >]
2005
Biochimica et Biophysica Acta - General Subjects
Elsevier Science Bv
1725
1
93-102
Yes (verified by ORBi)
International
0304-4165
Amsterdam
[en] thiamine triphosphate ; thiamine triphosphatase ; site-directed mutagenesis ; pig ; brain ; immunohistochemistry
[en] 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.
http://hdl.handle.net/2268/4633
10.1016/j.bbagen.2005.05.026

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