Makarchikov A F, Chernikevich I P
Laboratory of Applied Enzymology and Biotechnology, Academy of Sciences of Belarus, Grodno.
Biochim Biophys Acta. 1992 Oct 27;1117(3):326-32. doi: 10.1016/0304-4165(92)90032-p.
Soluble thiamine triphosphatase (EC 3.6.1.28) of bovine brain has been purified 68,000-fold to an electrophoretically homogeneous state with an overall recovery of 5.5% by hydrophobic chromatography on Toyopearl HW-60, Sephadex G-75 gel filtration, DEAE-Toyopearl 650M chromatography and Blue Sepharose CL-4B chromatography. The enzyme has an absolute specificity among thiamine and nucleoside phosphate esters for thiamine triphosphate and shows no nonspecific phosphatase activities. Thiamine triphosphatase is composed of a single polypeptide chain with molecular mass of 33,900 kDa as estimated by Sephadex G-100 gel filtration and SDS-polyacrylamide gel electrophoresis. The enzyme has a pH optimum of 8.7 and is dependent on divalent metal ions. Mg2+ has been found to be the most effective among cations tested. A study of the reaction kinetics over a wide range of thiamine triphosphate concentrations has revealed a biphasic saturation curve being described by higher-degree rational polynomials.
牛脑可溶性硫胺素三磷酸酶（EC 3.6.1.28）通过在Toyopearl HW - 60上进行疏水色谱、Sephadex G - 75凝胶过滤、DEAE - Toyopearl 650M色谱和Blue Sepharose CL - 4B色谱，已被纯化68000倍至电泳纯状态，总回收率为5.5%。该酶在硫胺素和核苷磷酸酯中对硫胺素三磷酸具有绝对特异性，且不表现出非特异性磷酸酶活性。通过Sephadex G - 100凝胶过滤和SDS - 聚丙烯酰胺凝胶电泳估计，硫胺素三磷酸酶由一条分子量为33900 kDa的单多肽链组成。该酶的最适pH为8.7，且依赖于二价金属离子。已发现Mg2+在所测试的阳离子中最有效。在广泛的硫胺素三磷酸浓度范围内对反应动力学的研究揭示了一条由高阶有理多项式描述的双相饱和曲线。
