STUDIES ON THIAMINE KINASE OF ESCHERICHIA COLI : (I) THE PROPERTIES OF THIAMINE KINASE IN THE CYTOPLASMIC MEMBRANE FRACTION

Abstract

In the course of studies on thiamine transport by several strains of Escherichia coli, the thiamine was accumulated by the cells as thiamine diphosphate (TDP) even at the earliest time of thiamine transport. This fact led me to study on the existence and peoperties of thiamine kinase in E. coli which probably participated in the transport of thiamine by phosphorylating thiamine. The thiamine kinase activity was detected in the membrae fraction separated from the sonicated cells. Substitution of ATP by ADP in the reaction mixture of membrane fraction was observed. The enzyme activity in the presence or absence of ADP was markedly decreased by addition of 3.3 mM KCN. This result showed that the enzyme reaction required ATP which was apparently generated from endogenous or exogenous ADP through oxidative phosphorylation associated with the membrane fraction. Furthermore, it was found that the thiamine kinase activity was located in the spheroplast membrane fraction separated by treatment with lysozyme and EDTA. The reaction velocities by the membrane fraction were proportional to the amounts of enzyme proteins substrates and reaction time. The optimum pH was given 7.0 and apparent Km for thiamine was 3.0×10^<-7> M. The activities were inhibited by the addition of thiamine antagonists such as pyrithiamine, oxythiamine and dimethialium, but the degrees of inhibition were far less than that of purified yeast thiamine kinase. Evidence for the regulation of thiamine kinase by TDP in the cells was obtained by using thiazoleless strain of E. coli.