Nishimura H, Sempuku K, Nosaka K, Iwashima A
Department of Biochemistry, Kyoto Prefectural University of Medicine, Japan.
Arch Biochem Biophys. 1988 Oct;266(1):248-53. doi: 10.1016/0003-9861(88)90255-x.
We have synthesized and characterized O-bromoacetylthiamine (BrAcThiamine), a new reagent for inactivating the thiamine transport system in Saccharomyces cerevisiae. A Lineweaver-Burk plot of data from the transport kinetic measurements showed that BrAcThiamine was a competitive inhibitor of thiamine transport in S. cerevisiae with a Ki value of 0.60 microM. Incubating BrAcThiamine with yeast cells at 40 degrees C in 0.05 M potassium phosphate buffer, pH 5.0, caused concentration- and time-dependently a remarkable loss of thiamine transport activity. The inactivating reaction of yeast thiamine transport by BrAcThiamine proceeded most effectively at pH 5.0, coinciding with the optimal pH of the transport activity. Thiamine and thiamine analogs (pyrithiamine and O-acetylthiamine) protected yeast thiamine transport activity against the inactivation by BrAcThiamine. In addition, it was found that a membrane fraction prepared from yeast cells treated with BrAcThiamine had a thiamine-binding activity only 20% of that from control cells without inactivating the binding activity of the soluble fraction. These results suggest that BrAcThiamine inactivates the uptake activity by irreversible binding to the binding site of carrier protein(s) in the thiamine transport system.
我们已经合成并表征了O-溴乙酰硫胺（溴乙酰硫胺），这是一种用于使酿酒酵母中硫胺转运系统失活的新试剂。转运动力学测量数据的Lineweaver-Burk图表明，溴乙酰硫胺是酿酒酵母中硫胺转运的竞争性抑制剂，Ki值为0.60微摩尔。在0.05 M磷酸钾缓冲液（pH 5.0）中于40℃将溴乙酰硫胺与酵母细胞一起孵育，会导致硫胺转运活性浓度和时间依赖性地显著丧失。溴乙酰硫胺使酵母硫胺转运失活的反应在pH 5.0时进行得最有效，这与转运活性的最佳pH一致。硫胺和硫胺类似物（吡硫胺和O-乙酰硫胺）可保护酵母硫胺转运活性免受溴乙酰硫胺的失活作用。此外，发现用溴乙酰硫胺处理的酵母细胞制备的膜部分的硫胺结合活性仅为对照细胞的20%，而可溶性部分的结合活性未失活。这些结果表明，溴乙酰硫胺通过与硫胺转运系统中载体蛋白的结合位点不可逆结合而使摄取活性失活。
