简化ALS活性测定方法的可行性探讨

Feasibility of Simplifying for Assay on the ALS Activity

摘要: 为探讨在离体法测试ALS活性实验中简化提取ALS蛋白步骤和廉价易购的二硫苏糖醇(DTT)替代黄素腺嘌呤二核苷酸(FAD)可行性，以暗培养的玉米幼苗为ALS提取材料，测定了粗蛋白、半精蛋白、精蛋白提取方法提取的ALS蛋白对ALS活性的酶促反应效果；分析了DTT替代FAD辅酶的可行性。试验结果，ALS蛋白提取过程中，可用缓冲液代替液氮碾磨材料，离心速度可降至为10,000 r/min；酶促反应效果粗蛋白酶好于半精蛋白酶、半精蛋白好于精蛋白酶。在离体法测定ALS活性体系中，DTT替代黄素腺嘌呤二核苷酸FAD配制的缓冲液对ALS蛋白的提取及酶促反应效果类似于FAD缓冲液，但由于提取ALS蛋白中携带痕量FAD分子，所以尚未证明在酶促反应中DTT分子完全替代FAD分子起到了ALS辅酶作用。

Abstract: In order to investigate the feasibility of simplifying the steps of extracting ALS protein and replacing Flavin Adenine Dinucleotide (FAD) with cheap and easily available Dithiothreitol (DTT) in the in vitro test of ALS activity, maize yellow seedlings were cultured for 5 days without light at 30˚C as ALS extraction material. The enzymic reaction effect of ALS protein extracted by crude protein, Semispermic protein and pure protein extraction methods on ALS activity was determined and feasibility of DTT to replace FAD-coenzyme was analyzed. The results showed that in the process of ALS protein extraction, the buffering fluid could be used instead of liquid nitrogen milling material, and the centrifugal speed could be reduced to 10,000 r/min. The best enzyme reaction was crude protein ALS, and the worst was pure protein ALS. In the in vitro determination of ALS activity system, the effect of the buffer substituted for FAD by DTT on the extraction of ALS protein and enzymatic reaction was similar to buffer for contains the FAD. However, since the extracted protein carries trace amount of FAD molecule, it has not been proved that DTT molecule completely substituted for FAD molecule in the enzymatic reaction and played the role of ALS coenzyme.