Singh B K, Lumanglas A, Wang B S
Agricultural Research Division, American Cyanamid Company, Princeton, NJ 08543-0400.
Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4572-6. doi: 10.1073/pnas.88.11.4572.
Acetohydroxyacid synthase [AHAS; acetolactate pyruvate-lyase (carboxylating), EC 4.1.3.18], the first enzyme unique to the biosynthesis of valine, leucine, and isoleucine, is a known target for several different chemical classes of herbicides. Antibodies required for immunological characterization of the enzyme have not been generated by the conventional method of antibody production using purified protein. Monoclonal antibodies were raised against AHAS from corn by using as immunogen a synthetic peptide representing this enzyme. This antibody immunoprecipitated the enzyme activity from corn. On a Western blot, a protein band with a molecular weight of 65,000 was detected in crude extracts of corn. Furthermore, a monoclonal antibody immunoaffinity gel was used to isolate a single protein from crude enzyme preparations that migrated at Mr 65,000 in an SDS/polyacrylamide gel. The molecular weight of this protein band is the molecular weight predicted for a plant AHAS from a cloned gene sequence. These results strongly suggest that the Mr 65,000 protein represents AHAS in corn extracts. Interestingly, the monoclonal antibody specifically recognized the enzyme from monocots and did not crossreact with AHAS from any dicot species tested. Identification of this monoclonal antibody that distinguishes monocot and dicot AHAS is significant because of a very high degree of amino acid conservation (85%) between AHAS proteins from different species.
乙酰羟酸合酶[AHAS；乙酰乳酸丙酮酸裂解酶（羧化），EC 4.1.3.18]是缬氨酸、亮氨酸和异亮氨酸生物合成中特有的第一种酶，是几种不同化学类别的除草剂的已知作用靶标。通过使用纯化蛋白的常规抗体制备方法尚未产生用于该酶免疫特性鉴定所需的抗体。通过使用代表该酶的合成肽作为免疫原，制备了针对玉米AHAS的单克隆抗体。该抗体从玉米中免疫沉淀了酶活性。在蛋白质印迹上，在玉米粗提物中检测到一条分子量为65000的蛋白条带。此外，使用单克隆抗体免疫亲和凝胶从粗酶制剂中分离出一种单一蛋白质，该蛋白质在SDS/聚丙烯酰胺凝胶中的迁移率为Mr 65000。该蛋白条带的分子量是根据克隆基因序列预测的植物AHAS的分子量。这些结果强烈表明，Mr 65000的蛋白代表玉米提取物中的AHAS。有趣的是，该单克隆抗体特异性识别单子叶植物中的酶，并且与所测试的任何双子叶植物物种的AHAS均无交叉反应。由于来自不同物种的AHAS蛋白之间具有非常高的氨基酸保守性（85%），因此这种区分单子叶植物和双子叶植物AHAS的单克隆抗体的鉴定具有重要意义。
