Lumsden J, Coggins J R
Biochem J. 1977 Mar 1;161(3):599-607. doi: 10.1042/bj1610599.
A new procedure for the purification of the arom multienzyme complex from Neurospora crassa is presented. Important factors are the inactivation of proteinases by phenylmethanesulphonyl fluoride and the use of cellulose phosphate as an affinity adsorbent. A homogeneous enzyme, with a specific shikimate dehydrogenase activity of 70 units/mg of protein, is obtained in 25% yield. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, combined with cross-linking studies using dimethyl suberimidate, suggest that the complex is composed of two subunits of molecular weight 165000. Glycerol-density-gradient centrifugation indicates a molecular weight for the intact complex of about 270000. Evidence for the effects of proteolysis, both during the preparation and on storage of the purified complex, is presented, and previous reports in the literature of the occurrence of multiple subunits are discussed in this light.
本文介绍了一种从粗糙脉孢菌中纯化芳香多酶复合物的新方法。重要的因素包括用苯甲基磺酰氟使蛋白酶失活，以及使用磷酸纤维素作为亲和吸附剂。获得了一种均一的酶，其莽草酸脱氢酶的比活性为70单位/毫克蛋白质，产率为25%。在十二烷基硫酸钠存在下的聚丙烯酰胺凝胶电泳，结合使用亚氨基二琥珀酸二甲酯的交联研究表明，该复合物由两个分子量为165000的亚基组成。甘油密度梯度离心表明完整复合物的分子量约为270000。本文提供了在制备过程中以及纯化复合物储存期间蛋白水解作用的证据，并据此讨论了文献中先前关于多个亚基存在的报道。
