SummaryThiamine and its phosphate esters in rat tissues were determined by high-performance liquid chromatography after conversion to thiochrome and its phosphate esters as described previously (Ishii, K. et al. (1979) Anal. Blochem., 97, 191-195). Total thiamine concentrations determined in the brain, liver, heart and kidney were 6.39, 25.2, 15.7, and 14.9nmol/g wet weight, respectively. In all the tissues tested, thiamine, its monophosphate, pyrophosphate, and triphosphate were found to be present at the rates of 1.2-7.7, 5.9-11.0, 85.7-90.0, and 0.7-1.6%, respectively. Although these percentages of thiamine phosphates were similar to those reported previously using different procedures, the percentage of thiamine triphosphate content in rat tissues was found to be fairly lower than that reported. The time required for the whole process of analysis averaged 90min after isolation of the organs from the animal.
The biochemical properties of strains (PT-R101 and PT-R108) of Escherichia coli K12 resistant to growth inhibition by pyrithiamine, and antimetabolite of thiamine, have been studied. Intracellular thiamine pyrophosphate concentration in these resistant strains was slightly but definitely higher than that in the parent strain. Thiamine synthesis from the pyrimidine and thiazole moieties of thiamine by cell suspensions was greater in the resistant strains than the parent strain. The activities of enzymes involved in thiamine biosynthesis in these pyrithiamine-resistant strains were 2-3 times higher than the parent strain (3301), except for thiamine-phosphate kinase, which was indetectable in in vitro assay of the activity. However, other evidence indicates that this enzyme is not defective but is functioning in vivo and, furthermore, that the negligible activity of this enzyme did not affect the growth rate of the mutants. The activities of these enzymes were further enhanced when PT-R101 was grown on 5mM adenine and were reduced almost to zero when the strain was grown on 0.1 muM thiamine in the same way as the parent strain. However, when these resistant strains were grown on a low concentration of thiamine such as 0,05 muM, thiamine synthesis by cell suspensions also decreased, but only to a limited extent compared with the parent strain. These results suggest that PT-R101 and PT-R108 are altered in the mechanisms of regulation of thiamine biosynthesis. Their altered properties might be due to a reduced binding affinity of the repressor protein, which is involved on the regulation of thiamine synthesis, for the corepressor, thiamine pyrophosphate,
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