Metabolism of thiamine triphosphate in rat brain: correlation with chloride permeability.

Our results show that a net synthesis of thiamine triphosphate (TTP) can be demonstrated in vitro using rat brain extracts. The total homogenate was preincubated with thiamine or its diphosphate derivative (TDP), centrifuged, and washed twice. With TDP (1 mM) as substrate, a 10-fold increase in TTP content was observed in this fraction (nuclear fraction, membrane vesicles). A smaller, but significant, increase was observed in the P2 fraction (mitochondrial/synaptosomal fraction). In view of the low TTP content of our fractions, it was carefully assessed that authentic TTP was being formed. Incorporation of radioactivity from [beta-32P]TDP and [gamma-32P]ATP in TTP suggests that these two compounds are its precursors. Furthermore, TTP synthesis was inhibited by ADP and relatively low concentrations of Zn2+. These results suggest that TTP synthesis is catalyzed by an ATP:TDP transphosphorylase rather than by the cytoplasmic adenylate kinase that may be present in the vesicles. After osmotic lysis of the vesicles at alkaline pH, TTP was recovered in protein-bound form. Concomitantly, a soluble thiamine triphosphatase, with alkaline pH optimum, was also released from the vesicles. No net synthesis could be obtained in the cytosolic fraction or in detergent-solubilized systems. Like TTP synthesis, chloride permeability of the vesicles was increased when the homogenate had been incubated with thiamine and particularly with TDP. Our results suggest a regulatory role of TTP on chloride permeability, but the target remains to be characterized.

Phosphate derivatives of thiamine and Na+ channel in conducting membranes.

The results show that thiamine derivatives are copurified with the specific proteins forming the Na+ channel in conducting membranes. Therefore, thiamine derivatives could well play a specific role in the molecular aspects of bioelectrogenesis , an interpretation that could help explain the neurological symptoms observed in human pathology as well as in animals experimentally rendered deficient in vitamin B1.

[Preparative separation of thiamine di- and triphosphates by ion-exchange chromatography]. / Preparatyvne rozdilennia tiamindy- i tryfosfativ metodom Ionoobminnoi khromatohrafii

The conditions were developed for preparative isolation of thiamine triphosphate (TTP) from a mixture of phosphoric acid esters of thiamine (PET) (40-80 mg thiamine). The Dowex 1 x 4 resin (acetate form) and triethylammonium acetate (TEAA), pH 4.5 as an eluent were used. The employment of volatile TEAA instead of acetate buffer facilitates the obtaining of concentrated TTP preparations and their subsequent identification. This method is successfully used for purification of commercial cocarboxylase preparations (TDP) (80 mg by PET). In comparison with electrophoresis and paper chromatography, the preparative isolation of TTP and TDP by the ion-exchange chromagraphic method is less laborious and permits larger amounts of PET to be separated for a single run.