Abstract
The plausible intermediary products derived from the pyrimidine and thiazole moieties of thiamine in thiamine synthesis by the enzymes extracted from bakers' yeast were investigated bioautographically using Escherichia coli mutants, i.e., pyrimidineless(70-17), thiazoleless (26-43) and thiamineless (70-23) strains. The minimal amount of 2-methyl-4-amino-5-hydroxymethylpyrimidine (OMP), OMP-monophosphate (OMP-P) and OMP-diphosphate (OMP-PP) for the growth response of 70-17 strain was 1×10^<-3>, 1 and 1×10^<-1> mμmole respectively, whereas that of 4-methyl-5-β-hydroxyethylthiazole (Th), Th-monophosphate (Th-P) and Th-diphosphate (Th-PP) for the growth response of 26-43 strain was 1×10^<-4>, 1×10^<-3> and 1×10^<-1> mμmole respectively. The detection of OMP-P and OMP-PP in the reaction mixture which had been incubated with the enzyme, OMP (0.1 μmole) and ATP was difficult owing to the contamination of thiamine derivatives contained in the enzyme. However, OMP-PP synthesis was demonstrated bioautographically from the concentrated fraction eluted from a Dowex 1 column. On the contrary, Th-P was detected in the reaction mixture which had been incubated with a partially purified Th-kinase, Th (0.1 μmole) and ATP, whereby the formation of Th-P was roughly estimated to reach 55-60 % of the added Th. Th-PP, however, failed to be recognized.
