Thiamine uptake in Escherichia coli: I. General properties of thiamine uptake system in Escherichia coli

Abstract

Properties of a system of 14C-thiamine uptake have been investigated in Escherichia coli. Uptake of 14C-thiamine by the system was found to be an energy- and temperature-dependent process which has an optimal pH at 6.5 and follows Michaelis-Menten kinetics; apparent K m was 8.3 × 10 −7 m and V max was 1.1 × 10 −10 moles per min per mg dry weight of the cells. It was characteristic in this system that 14C-thiamine accumulated as 14C-thiamine pyrophosphate but not as free 14C-thiamine. This was confirmed by paper chromatography of the extract of the cells exposed to 14C-thiamine at 37 °. Intracellular concentration of 14C-thiamine (pyrophosphate) accumulated when incubated with 1 μ m 14C-thiamine was approximately 1.75 × 10 −7 moles per ml cellular fluid at equilibrium. This was 30-fold the initial intracellular thiamine concentration and 175-fold the external thiamine concentration. Both pyrithiamine and oxythiamine, anti-thiamine compounds, competed with 14C-thiamine for the uptake system under conditions in which thiamine kinase (ATP:thiamine pyrophosphotransferase, EC 2.7.6.2.) in the membrane fraction was not inhibited by these analogues. A role of membrane thiamine kinase of E. coli probably participating in a process of the uptake to accumulate 14C-thiamine as thiamine pyrophosphate in the cell was discussed.