Okunuki Haruyo, Akiyama Hiroshi, Teshima Reiko, Hino Akihiro, Goda Yukihiro, Sawada Jun-ichi, Toyoda Masatake, Maitani Tamio
National Institute of Health Sciences: 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
Shokuhin Eiseigaku Zasshi. 2003 Apr;44(2):77-82. doi: 10.3358/shokueishi.44.77.
A liquid chromatography-mass spectrometry (LC/MS) method for determining the enzymatic activity of 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase), an enzyme of the shikimate pathway, was developed. EPSP synthase catalyzes the formation of 5-enolpyruvylshikimate-3-phosphate (EPSP) from shikimate-3-phosphate (S-3-P) and phosphoenolpyruvate (PEP) in microorganisms and plants. The enzymatic activity of EPSP synthase was assessed by the determination of EPSP after a 30-min incubation with S-3-P and PEP using the LC/MS system. EPSP synthase activity is given in terms of the produced EPSP (pmol/min/mg protein). Glyphosate (N-phosphonomethyl glycine)-tolerant EPSP synthase from the Agrobacterium sp. strain CP4 (CP4-EPSP synthase) in genetically modified soybeans (GM-soybeans) was found to have an enzymatic activity of 736 EPSP pmol/min/mg protein in the presence of 3 nmol of S-3-P. In contrast, the enzyme activity of non-GM-soybeans was 21 EPSP pmol/min/mg protein. The EPSP synthase activity was markedly decreased in the non-GM-soybeans by the addition of glyphosate, but the enzyme activity of the GM-soybeans was only slightly decreased with this treatment. This LC/MS system could also be applicable to the measurement of EPSP synthase activity in different plant species and the detection of herbicide-tolerant EPSP synthase in GM foods.
开发了一种用于测定莽草酸途径中的一种酶——5-烯醇丙酮酰莽草酸-3-磷酸合酶（EPSP合酶）酶活性的液相色谱-质谱联用（LC/MS）方法。EPSP合酶在微生物和植物中催化由3-磷酸莽草酸（S-3-P）和磷酸烯醇丙酮酸（PEP）形成5-烯醇丙酮酰莽草酸-3-磷酸（EPSP）。使用LC/MS系统，通过在与S-3-P和PEP孵育30分钟后测定EPSP来评估EPSP合酶的酶活性。EPSP合酶活性以产生的EPSP（皮摩尔/分钟/毫克蛋白质）表示。在转基因大豆（GM大豆）中，来自农杆菌属CP4菌株（CP4-EPSP合酶）的耐草甘膦（N-膦酰甲基甘氨酸）EPSP合酶在存在3纳摩尔S-3-P的情况下，酶活性为736皮摩尔EPSP/分钟/毫克蛋白质。相比之下，非转基因大豆的酶活性为21皮摩尔EPSP/分钟/毫克蛋白质。通过添加草甘膦，非转基因大豆中的EPSP合酶活性显著降低，但这种处理对转基因大豆的酶活性仅略有降低。该LC/MS系统也可适用于测量不同植物物种中的EPSP合酶活性以及检测转基因食品中的耐除草剂EPSP合酶。
