[Purification and several properties of thiamine pyrophosphokinase from rat liver].
Thiamin pyrophosphokinase (TPK) (EC 2.7.6.2) was isolated and purified from rat liver tissue ion in exchange chromatography, rechromatography and gel filtration. The enzyme was purified 3000-fold with yield of 12%, it was homogenous in polyacrylamide gel disc electrophoresis. TPK had two pH optima; Km value for thiamin was equal to 6-10(-6) M. Pyrithiamin was shown to be a competitive inhibitor with K1=3-10(-6) M and hydroxythiamin--an inhibitor of the mixed type with K1=1-10(-2) M. The relationship between the rate of the reaction and the substrate concentration (ATP-Mg2+) at the Mg2+/ATP ratio of 1:1 was described by S-like curve, which acquired a hyprebolic form in presence of an excess of Mg2+. Dependence of the reaction rate on Mg2+ concentration was also described by a sigmoid curve. The data obtained suggest that liver TPK was apparently an allosteric enzyme with quaternary structure.
[Purification and several properties of thiamine pyrophosphokinase from rat liver]. Thiamin pyrophosphokinase (TPK) (EC 2.7.6.2) was isolated and purified from rat liver tissue ion in exchange chromatography, rechromatography and gel filtration. The enzyme was purified 3000-fold with yield of 12%, it was homogenous in polyacrylamide gel disc electrophoresis. TPK had two pH optima; Km value for thiamin was equal to 6-10(-6) M. Pyrithiamin was shown to be a competitive inhibitor with K1=3-10(-6) M and hydroxythiamin--an inhibitor of the mixed type with K1=1-10(-2) M. The relationship between the rate of the reaction and the substrate concentration (ATP-Mg2+) at the Mg2+/ATP ratio of 1:1 was described by S-like curve, which acquired a hyprebolic form in presence of an excess of Mg2+. Dependence of the reaction rate on Mg2+ concentration was also described by a sigmoid curve. The data obtained suggest that liver TPK was apparently an allosteric enzyme with quaternary structure.