ABSTRACT
Radiometric and fluorescent methods for detection of thiamine triphosphate have been used to show the presence of thiamine diphosphate kinase activity in brewer's yeast and to determine optimal conditions for its manifestation. A method of enzyme purification has been developed which involves glycerol-EDTA solution extraction, heat treatment, 2-fold ammonium sulphate fractionation, Sephadex G-200 gel filtration and ion-exchange chromatography on Sephadexes CP-C-50 and QAE-A-25. The protein has been purified 2000-fold in a 19% yield. The isoelectric and isoionic points, the amino acid composition and the molecular weight have been determined. Hydrophobic amino acids and those responsible for alpha-helix formation of the protein globule are predominant. The isoelectric point, as calculated by the amino acid composition and found by the maximum of the changes in fluorescence, is 5.8. The isoionic point value is identical with the isoelectric point. Upon gel filtration thiamine diphosphate kinase is eluted as two protein peaks with molecular weights of 162,000 +/- 8,000 and 81,000 +/- 4,000. After treatment with urea or sodium dodecyl sulphate, the protein dissociates into subunits with molecular weights of 12,500 and 14,000. The purified enzyme has some properties typical of a dissociating enzyme system.
Subject(s)
Phosphotransferases (Phosphate Group Acceptor) , Phosphotransferases/isolation & purification , Saccharomyces cerevisiae/enzymology , Chromatography, Gel , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Enzyme Stability , Hot Temperature , Hydrogen-Ion Concentration , Isoelectric Point , Kinetics , Molecular Weight , Phosphotransferases/metabolismABSTRACT
A radioassay was developed for estimation of the thiamin diphosphate kinase activity (EC 2.7.4.15); 14C-TDP was used as a substrate. After termination of the reaction (formation of TTP), the products obtained were separated using ion exchange chromatography on SP-Sephadex C-25. The method developed was very sensitive and enabled to estimate the enzymatic activity in tissue homogenates. TDP-kinase was isolated from rat liver with a 70-fold purification. Dependence of the reaction rate on pH value, concentrations of the enzyme and thiamin were studied.
