Uptake of thiamine propyl disulfide in Escherichia coli.

Abstract

Properties of a system of thiamine propyl disulfide (TPD) uptake have been investigated in Escherichia coli. It was found that the cells incubated with TPD in the presence of glucose at 37° accumulated thiamine pyrophosphate in the cytoplasm with a negligible amount of TPD. TPD in the reaction medium remained practically in the unchanged form after incubation with the cells for 30 minutes at 37°. Thiamine pyrophosphate accumulation in TPD uptake was an energy- and temperature dependent process which follows Michaelis-Menten kinetics; apparent Km was 3.0×10-6 M and Vmax was 3.5×10-5 moles per minute per g dry weight of the cells.Treatment of the cells with N-ethylmaleimide resulted in inhibition of the rate of thiamine pyrophosphate accumulation much stronger in TPD uptake than that in thiamine uptake. A thiamine transport-negative mutant of E. coli which is defective in the “carrier” protein specific for thiamine, did not accumulate thiamine pyrophosphate when incubated with TPD.These results strongly suggest that in E. coli TPD is not directly taken up by the cells but is first reduced to thiamine on the outer surface of the cell membrane probably by a non-enzymatic process. Thiamine thus formed is transported via the uptake system specific for thiamine; the “carrier” protein functions for passage through the membrane and membrane thiamine kinase functions to accumulate thiamine as thiamine pyrophosphate in the cytoplasm.