Thiamine intestinal transport and phosphorylation : a study in vitro of potential inhibitors of small intestinal thiamine-pyrophosphokinase using a crude enzymatic preparation.
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-(14)C] thiamine. The Km value for thiamine was 2.14 X 10(-6) M 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 Ki values were : pyrithiamine, 2.25 X 10(-6) M; thiamine monophosphate, 4 X 10(-6) M; 2'-ethylthiamine, 8 X 10(-6) M; 2'-butylthiamine, 6 X 10(-6) M; chloroethylthiamine and dimethalium, 1.5 X 10(-5) M; amprolium, 1.8 X 10(-4) M; L-582571, 1.65 X 10(-4) M; oxythiamine, 4.2 X 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.
D000818:Animals; D001692:Biological Transport; D005260:Female; D007421:Intestine, Small / Q000378:metabolism*; D007700:Kinetics; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity; D013836:Thiamin Pyrophosphokinase / Q000378:metabolism*; D013831:Thiamine / Q000031:analogs & derivatives / Q000378:metabolism*
D010770:Phosphotransferases; D013836:Thiamin Pyrophosphokinase; D013831:Thiamine