Purification and characterization of an inducible sesquiterpene cyclase from elicitor-treated tobacco cell suspension cultures.
Purification and characterization of an inducible sesquiterpene cyclase from elicitor-treated tobacco cell suspension cultures.
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DOI：
10.1104/pp.93.1.182
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发表时间：
1990-05
期刊：
影响因子：
7.4
通讯作者：
U. Vögeli;James W. Freeman;Joseph Chappell
中科院分区：
文献类型：
--
作者：
U. Vögeli;James W. Freeman;Joseph Chappell
通过疏水相互作用、阴离子交换、羟基磷灰石和色谱聚焦色谱的组合纯化激发子诱导型倍半萜环化酶，该酶催化法尼基二磷酸转化为5-表马兜铃烯（IM Whitehead，DR Threlfall，DF Ewing [1989] Phytochemistry 28：775-779），并且代表烟草中植物抗毒素生物合成途径中的关键步骤。从2公斤诱导的烟草（Nicotiana tabacum）细胞中纯化了约500微克环化酶蛋白，这表明该酶的比活性增加了130倍以上，起始活性恢复了4%。纯化的酶通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）解析为60和62千道尔顿的两个主要多肽。酶活性的生化表征包括对镁的绝对需求，等电点为4.5至4.9，法呢基二磷酸的K（m）为2至5微摩尔。纯化的环化酶蛋白用于产生在体外测定中有效抑制环化酶活性的小鼠多克隆抗体。从激发子处理的细胞或纯化的环化酶的天然聚丙烯酰胺凝胶上的提取物的电泳分离的环化酶酶成四个多肽所示的免疫印迹分析，使用多和单克隆抗体。原卟啉环化酶活性与这些多肽一起迁移。通过免疫印迹分析，在对照细胞的提取物中未检测到环化酶多肽。然而，从激发子处理的细胞使用四个独立的单克隆抗体系和多克隆抗体的蛋白质的免疫印迹分析检测到相同的多肽，无论蛋白质是否通过天然或SDS-PAGE分离。结果表明，诱导多环化酶多肽在激发子处理的细胞中产生的多个基因的表达或多个翻译后加工事件。
An elicitor-inducible sesquiterpene cyclase, which catalyzes the conversion of farnesyl diphosphate to 5-epi-aristolochene (IM Whitehead, DR Threlfall, DF Ewing [1989] Phytochemistry 28:775-779) and representing a committed step in the phytoalexin biosynthetic pathway in tobacco, was purified by a combination of hydrophobic interaction, anion exchange, hydroxylapatite, and chromatofocusing chromatography. From 2 kilograms of elicited tobacco (Nicotiana tabacum) cells, approximately 500 micrograms of cyclase protein was purified, representing greater than a 130-fold increase in the specific activity of the enzyme and a 4% recovery of the starting activity. The purified enzyme resolved as two major polypeptides of 60 and 62 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Biochemical characterization of the enzyme activity included an absolute requirement for magnesium, an isoelectric point of 4.5 to 4.9, and a K(m) for farnesyl diphosphate of 2 to 5 micromolar. The purified cyclase protein was used to generate mouse polyclonal antibodies which efficiently inhibited cyclase activity in an in vitro assay. Electrophoresis of extracts from elicitor-treated cells or purified cyclase enzyme on native polyacrylamide gels separated the cyclase enzyme into four polypeptides as shown by immunoblot analysis using poly- and monoclonal antibodies. Proportionate cyclase enzyme activity comigrated with those polypeptides. No cyclase polypeptides were detectable in extracts of control cells by immunoblot analysis. However, immunoblot analysis of proteins from elicitor-treated cells using four independent monoclonal antibody lines and the polyclonal antibodies detected the same polypeptides, regardless of whether the proteins were separated by native or SDS-PAGE. The results suggest an induction of multiple cyclase polypeptides in elicitor-treated cells resulting from either the expression of multiple genes or multiple post-translational processing events.