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Journal archive > 2010 > N 1 January-February

EXISTENCE OF TWO DIFFERENT ACTIVE SITES ON THIAMINE BINDING PROTEIN IN SYNAPTIC PLASMA MEMBRANES

Yu. M. Parkhomenko, A. А. Strokina, S.Yu. Pylypchuk, S. P. Stepanenko, L.I.Chekhivska, G. V. Donchenko

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
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The current work is aimed at understanding the structure and functionality of thiamine binding protein (TBP) in neural cells plasma membranes. The influence of thiamine triphosphate on thiamine binding by TBP in synaptic plasma membranes (SPM) isolated from the rat brain was investigated. It was shown that thiamine triphosphate inhibits thiamine binding activity of SPM in concurrent manner (Кi = 1.0 ± 0.3 µМ). At the same time thiamine had no effect on thiamine triphosphatase (ThTPase) activity at the concentration range 0.5–20 µМ. Otherwise, ThTPase activation with the maximum at the concentration about 2.5?µМ was observed.
Further, the influence of classic thiamine antagonists (amprolium, oxythiamine and pyrithiamine) on both biological activities of TBP in SPM was studied. The IC50 value for inhibition of thiamine binding on SPM by amprolium comprised 50?±?4.0 µМ. Still, this antagonist had no effect on ThTPase activity. For the oxythiamine inhibition of both TBP activities was detected. The values of IC50 were 125 ± 28 and 1000 ± 95 µМ for thiamine binding and ThTPase activity, respectively. The values of IC50 for thiamine binding and ThTPase activity inhibition differed by more than one order of magnitude and comprised 2.2 ± 0.2 and 43 ± 9?µМ, respectively.
The obtained data indicate that the active sites on SPM responsible for thiamine binding and ThTPase activity have different sensitivity to thiamine antagonists. Our results allow us to suppose that different active protein sites are responsible for the specific binding and for thiamine phosphates hydrolysis by TBP of synaptic membranes.

Key words: thiamine, thiamine triphosphate, thiamine triphosphatase, synaptic plasma membranes, thiamine-binding protein, thiamine antagonists.

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