Tate J R, Nixon P F
Anal Biochem. 1987 Jan;160(1):78-87. doi: 10.1016/0003-2697(87)90616-6.
Human erythrocyte transketolase could be resolved from thiamin diphosphate (TDP) by acidification of the ammonium sulfate precipitate to pH 3.5, but not by other tested procedures. Resolution was 98% by chemical measurement of residual thiamin and 95% by residual enzyme activity. Reconstitution of the resolved preparation by incubation with TDP was dependent upon TDP concentration, duration, temperature, and the presence of dithiothreitol. At low TDP concentrations, 1 h was required for maximum activation; kinetic analysis then yielded an apparent Km value for TDP of 65 nM (SD 14 nM) from 100 erythrocyte lysates and similar values for reconstituted resolved preparations previously purified 400-fold and 10,000-fold. Velocity data obtained by transketolase assays in which the TDP was added to resolved preparations simultaneously with substrates yielded an apparent Km value for TDP of 2.3 microM (SD 1.6 microM) from 114 erythrocyte lysates and similar values for purified preparations. The recovery of activity following resolution and reconstitution ranged from 21 to 60% from lysates and 38 to 70% from purified preparations. Residual ammonium sulfate up to 4.9 mM decreased the apparent Km value for TDP, while a concentration of 11.3 mM increased the value in a manner competitive with TDP and with an apparent Ki value of 2.3 mM. The spectrophotometric assay of transketolase activity was greatly affected by storage of frozen solutions of the substrate ribose 5-phosphate.
通过将硫酸铵沉淀酸化至pH 3.5可从硫胺素二磷酸（TDP）中分离出人类红细胞转酮醇酶，但其他测试方法无法做到。通过化学测量残留硫胺素，分离率为98%，通过残留酶活性测定，分离率为95%。通过与TDP孵育对分离后的制剂进行重组，这取决于TDP浓度、孵育时间、温度以及二硫苏糖醇的存在。在低TDP浓度下，需要1小时才能达到最大激活；动力学分析得出，从100份红细胞裂解物中测得TDP的表观Km值为65 nM（标准差14 nM），对于先前分别纯化了400倍和10000倍的重组分离制剂，该值相似。在转酮醇酶测定中，将TDP与底物同时添加到分离制剂中获得的速度数据显示，从114份红细胞裂解物中测得TDP的表观Km值为2.3 μM（标准差1.6 μM），对于纯化制剂，该值相似。分离和重组后的活性回收率在裂解物中为21%至60%，在纯化制剂中为38%至70%。高达4.9 mM的残留硫酸铵会降低TDP的表观Km值，而11.3 mM的浓度会以与TDP竞争的方式提高该值，表观Ki值为2.3 mM。转酮醇酶活性的分光光度测定受底物5-磷酸核糖冷冻溶液储存的影响很大。
