Welle R, Grisebach H
Lehrstuhl für Biochemie der Pflanzen, Biologisches Institut II der Universität, Freiburg, Federal Republic of Germany.
Arch Biochem Biophys. 1989 Jul;272(1):97-102. doi: 10.1016/0003-9861(89)90199-9.
Chromatofocusing on Mono P proved to be an efficient purification procedure for the NADPH-dependent reductase from soybean (Glycine max L.) cell cultures which acts together with chalcone synthase in the biosynthesis of 2',4',4-trihydroxychalcone (6'-deoxychalcone). By isoelectric focusing the pI of reductase was determined to be 6.3. Addition of pure soybean reductase to cell-free extracts from stimulated cell cultures of parsley and bean (Phaseolus vulgaris) and from young flowers of Dahlia variabilis caused in each case synthesis of 6'-deoxychalcone. When 4-coumaroyl-CoA was replaced by caffeoyl-CoA in the reductase assay, formation of 2',4',3,4-tetrahydrochalcone (butein) was observed. A polyclonal antireductase antiserum was raised in rabbits and proved to be specific in Ouchterlony diffusion experiments, Western blots and immunotitration. The reductase antiserum showed no cross-reactivity with soybean chalcone synthase (CHS). A biotin/[125I]streptavidin system provided a quantitative Western blot for the reductase. Changes in the activities, amounts of protein, and mRNA activities of reductase and CHS were determined after challenge of soybean cell cultures by elicitor (from Phytophthora megasperma f.sp. glycinea or yeast). For both enzymes a pronounced and parallel increase in activity and amounts of protein was observed after elicitor addition with a maximum at about 16 h after challenge. Parallel increases in mRNA activities occurred earlier. The results indicate a parallel induction of de novo synthesis of reductase and CHS which coact in synthesis of 6'-deoxychalcone.
事实证明，使用Mono P进行色谱聚焦是从大豆（Glycine max L.）细胞培养物中纯化依赖NADPH的还原酶的有效方法，该还原酶在2',4',4-三羟基查耳酮（6'-脱氧查耳酮）的生物合成中与查耳酮合酶共同起作用。通过等电聚焦测定，还原酶的pI为6.3。向来自欧芹和菜豆（Phaseolus vulgaris）受刺激细胞培养物以及大丽花（Dahlia variabilis）幼花的无细胞提取物中添加纯大豆还原酶，在每种情况下都会导致6'-脱氧查耳酮的合成。当在还原酶测定中用咖啡酰辅酶A替代4-香豆酰辅酶A时，观察到2',4',3,4-四羟基查耳酮（紫铆因）的形成。用兔制备了多克隆抗还原酶抗血清，在免疫双扩散试验、蛋白质免疫印迹和免疫滴定中证明具有特异性。还原酶抗血清与大豆查耳酮合酶（CHS）无交叉反应。生物素/[125I]链霉亲和素系统为还原酶提供了定量蛋白质免疫印迹。在用激发子（来自大豆疫霉大豆专化型或酵母）刺激大豆细胞培养物后，测定了还原酶和CHS的活性、蛋白质含量以及mRNA活性的变化。对于这两种酶，添加激发子后观察到活性和蛋白质含量显著且平行增加，在刺激后约16小时达到最大值。mRNA活性的平行增加出现得更早。结果表明，还原酶和CHS的从头合成是平行诱导的，它们在6'-脱氧查耳酮的合成中共同起作用。
