Metabolism of thiamine phosphates in washed suspensions of kidney particles

Abstract

The behavior of thiamine, thiamine monophosphate, cocarboxylase, thiamine tri-phosphate, oxythiamine, and oxythiamine diphosphate on paper chromatograms is descr.; p-toluenesulfonic acid/tertiary-pentanol/water was the solvent. The substances are located by spraying with potassium bismuth iodide. Of the thiamine derivatives tested at 0.005 [image] (thiamine, thiamine monophosphate, cocarboxylase and thiamine triphosphate), only thiamine triphosphate affected oxidation of pyruvate (40% inhibition) by sheep-kidney particles; none altered the ratio of the O2 uptake/ pyruvate consumed. Cocarboxylase is converted into thiamine monophosphate by washed suspensions of kidney particles. The amt. of cocarboxylase disappearing is less in the presence of pyruvate or alpha-oxoglutarate than in absence of substrate, or in presence of succinate. On incubation of cocarboxylase and P32 labelled orthophosphate with respiring kidney particles, a slow incorporation of radioactivity into the cocarboxylase occurred. This was unrelated to the amount of substrate oxidized. Thiamine monophosphate formed by breakdown of cocarboxylase in the presence of P32-labelled orthophosphate was much more radioactive when the tissue suspension contained oxidizable substrate than in the absence of substrate; alpha-oxoglutarate caused a more rapid incorporation of radioactivity than did succinate. The tissue suspensions were unable to synthesize cocarboxylase from thiamine monophosphate or thiamine, or to synthesize thiamine monophosphate from thiamine; thus it is unlikely that the radioactivity found in thiamine monophosphate was incorporated by rephosphorylation of free thiamine. A mechanism of phosphorylation, assuming that "lipothiamide" is the reactive form of thiamine is put forward. The steady-state exchange of the phosphate of thiamine monophosphate during oxidation of alpha-oxoglutarate was measured as 0.053[mu] mole/min.