title: Studies on thiamine diphosphate kinase (EC 2.7.4.15) from brewer's yeast: purification and some properties.
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.
mesh_terms: D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D010770:Phosphotransferases / Q000302:isolation & purification* / Q000378:metabolism; D017856:Phosphotransferases (Phosphate Group Acceptor); D012441:Saccharomyces cerevisiae / Q000201:enzymology*
chemical_list: D010770:Phosphotransferases; D017856:Phosphotransferases (Phosphate Group Acceptor); C022733:thiamin-diphosphate kinase