Wang J J, Martin P R, Singleton C K
Department of Molecular Biology, Vanderbilt University, Nashville, TN 37235, USA.
Alcohol Clin Exp Res. 1997 Jun;21(4):576-80.
Thiamine deficiency, a frequent complication of alcoholism, contributes significantly to the development of damage in various organ systems, including the brain. The molecular mechanisms that underlie the differential vulnerabilities to thiamine deficiency of tissue and cell types and among individuals are not understood. Investigations into these mechanisms have examined potential variations in thiamine utilizing enzymes. Transketolase is a homodimeric enzyme containing two molecules of noncovalently bound thiamine pyrophosphate. In the present study, we examined a his-tagged human transketolase that was produced in and purified from Escherichia coli cells. Previous findings demonstrated that purified his-transketolase had a Km app for cofactor and a thiamine pyrophosphate-dependent lag period for attaining steady-state kinetics that was similar to transketolase purified from human tissues. Interestingly, the time of the lag period, which is normally independent of enzyme concentration, was found herein to be dependent on the concentration of the recombinant protein. This atypical behavior was due to production in E. coli. Generation of the normal, enzyme concentration-independent state required a cytosolic factor(s) derived from human cells. Importantly, the required factor(s) was found to be defective in a Wernicke-Korsakoff patient whose cells in culture show an enhanced sensitivity to thiamine deficiency.
硫胺素缺乏是酗酒常见的并发症，在包括大脑在内的各种器官系统损伤的发展过程中起重要作用。组织和细胞类型以及个体之间对硫胺素缺乏的不同易感性背后的分子机制尚不清楚。对这些机制的研究考察了硫胺素利用酶的潜在变异。转酮醇酶是一种同二聚体酶，含有两个非共价结合的硫胺素焦磷酸分子。在本研究中，我们检测了一种在大肠杆菌细胞中产生并纯化的带有组氨酸标签的人转酮醇酶。先前的研究结果表明，纯化的组氨酸-转酮醇酶对辅因子的Km app以及达到稳态动力学的硫胺素焦磷酸依赖性延迟期与从人体组织中纯化的转酮醇酶相似。有趣的是，在此发现通常与酶浓度无关的延迟期时间取决于重组蛋白的浓度。这种非典型行为是由于在大肠杆菌中产生所致。产生正常的、与酶浓度无关的状态需要源自人细胞的胞质因子。重要的是，在一名韦尼克-科尔萨科夫综合征患者中发现所需因子存在缺陷，该患者的培养细胞对硫胺素缺乏表现出增强的敏感性。
