Voskoboyev A I, Chernikevich I P, Luchko V S
Biomed Biochim Acta. 1987;46(1):3-13.
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.
已使用检测三磷酸硫胺素的放射性和荧光方法来显示酿酒酵母中硫胺素二磷酸激酶活性的存在，并确定其表现的最佳条件。已开发出一种酶纯化方法，该方法包括甘油 - 乙二胺四乙酸溶液提取、热处理、两倍硫酸铵分级分离、葡聚糖G - 200凝胶过滤以及在葡聚糖CP - C - 50和QAE - A - 25上进行离子交换色谱。该蛋白质已以19%的产率纯化了2000倍。已确定了等电点和等离子点、氨基酸组成以及分子量。疏水氨基酸和负责蛋白质球状体α - 螺旋形成的氨基酸占主导。根据氨基酸组成计算并通过荧光变化最大值测得的等电点为5.8。等离子点值与等电点相同。在凝胶过滤时，硫胺素二磷酸激酶以两个蛋白质峰的形式洗脱，分子量分别为162,000 ± 8,000和81,000 ± 4,000。用尿素或十二烷基硫酸钠处理后，该蛋白质解离成分子量为12,500和14,000的亚基。纯化后的酶具有一些典型的解离酶系统的特性。
