Manstein D J, Pai E F
J Biol Chem. 1986 Dec 5;261(34):16169-73.
The bifunctional enzyme FAD synthetase from Brevibacterium ammoniagenes was purified by a method involving ATP-affinity chromatography. The final preparation was more than 95% pure. The apparent molecular weight of the enzyme was determined as 38,000 and the isoelectric point as 4.6. Although previous attempts to separate the enzymatic activities had failed, ATP:riboflavin 5'-phosphotransferase and ATP:FMN-adenylyltransferase activities in B. ammoniagenes were believed to be located on two separate proteins with similar properties, possibly joined in a complex. The following evidence, however, suggests the presence of both activities on a single polypeptide chain. The two activities copurify in the same ratio through the purification scheme as presented. Only a single band could be detected when aliquots from the final purification step were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, nondenaturing gel electrophoresis, and isoelectric focusing. Edman degradation of the protein yielded a single N-terminal sequence.
利用一种涉及ATP亲和层析的方法，对产氨短杆菌中的双功能酶黄素腺嘌呤二核苷酸合成酶进行了纯化。最终制备物的纯度超过95%。该酶的表观分子量测定为38,000，等电点为4.6。尽管之前分离酶活性的尝试均告失败，但产氨短杆菌中的ATP：核黄素5'-磷酸转移酶和ATP：FMN-腺苷酸转移酶活性被认为位于两种性质相似的不同蛋白质上，它们可能以复合物的形式结合在一起。然而，以下证据表明这两种活性存在于一条单一的多肽链上。在所示的纯化方案中，这两种活性以相同的比例共同纯化。当对最终纯化步骤的等分试样进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、非变性凝胶电泳和等电聚焦时，只能检测到一条单一的条带。对该蛋白质进行的埃德曼降解产生了单一的N端序列。
