Abstract

The enzyme ATP:thiamine pyrophosphotransferase (thiamine pyrophosphokinaseJ was purified from acetone powders which were prepared from rat brains up to the chromatography on alumina C_γ step by a modified procedure that Mano (Y. Mano, J. Biochem., 42, 283 (1960)] developed for the purification of the rat liver thiamine pyrophosphokinase. The enzyme was purified 12 fold over that in the original extract of the acetone powder. A spectrophotometric assay for the brain thiamine pyrophosphokinase was developed utilizing brewer's yeast apotransketolase. The transketolase assay was able to detect less than 10^-11 moles of thiamine diphosphate, which was synthesized by the kinase, in the presence of ATP and thiamine. Contaminating yeast thiamine pyrophosphokinase was removed from the transketolase by chromatography of the transketolase on DEAE-cellulose at pH 6.0 with 0.01 M phosphate buffer. The thiamine pyrophosphokinase from 2.0 g of acetone powder (equivalent to 8 rat brains> could synthesize 276 mumoles of thiamine diphosphate per hour at 37°. The K_m 's for ATP and thiamine were found to be 0.02 M and 1.2 x 10-7 M, respectively. The pH optimum was found to be pH 7.8 with glycylglycine buffer. Pyrithiamine and oxythiamine were found to be competitive Inhibitors of thiamine pyrophosphorylation by the kinase. Pyrithiamine and oxythiamine have K_i 's of 2 x 10^-7 M and 2 x I0^-4 M, respectively. An oxythiamine phosphate compound was synthesized by brain thiamine pyrophosphokinase from ATP and oxythiamine which in turn inhibited the formation of transketolase from apotransketolase and thiamine diphosphate.

Degree

PhD

College and Department

Physical and Mathematical Sciences; Chemistry and Biochemistry

Rights

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