Thiamine Intestinal Transport and Phosphorylation: a Study in vitro of Potential Inhibitors of Small Intestinal Thiamine-Pyrophosphokinase Using a Crude Enzymatic Preparation

Abstract

Using as enzymatic source the cytoplasmatic fraction of enterocytes isolated from the rat small intestine, thiamine-pyrophosphokinase activity was studied with a radiometric method using [thiazole-2⁻¹⁴C] thiamine. The K_m value for thiamine was 2.14 × 10⁻⁶ and V 0.87 nmol of thiamine pyrophosphate mg⁻¹ protein h⁻¹. Eleven thiamine structural analogs and derivatives were assayed for their inhibitory action on the small intestine thiamine-pyrophosphokinase activity. Their K₁ values were: pyrithiamine, 2.25 × 10⁻⁶M; thiamine mono- phosphate, 4 × 10⁻⁶ M; 2'-ethylthiamine, 8 × 10⁻⁶ M; 2'-butylthiamine, 6 × 10'6; chloroethylthiamine and dimethalium, 1.5 × 10⁻⁶M; am-prolium, 1.8 × 10⁻⁴M; L-582571, 1.65 × 10⁻⁴; oxythiamine, 4.2 × 10⁻³ M.

Of the miscellaneous compounds tested (toxopyrimidine, Na-pyrophosphate, choline, L-phenylalanine, ethyl-urethane and 5-fluorouracil), none had any inhibitory action on intestinal thiamine-pyrophosphokinase activity, even if used at concentrations hundred times higher than that of labelled thiamine.