Watanabe F, Fukui K, Momoi K, Miyake Y
Department of Biochemistry, National Cardiovascular Center Research Institute, Osaka, Japan.
Biochem Biophys Res Commun. 1989 Dec 29;165(3):1422-7. doi: 10.1016/0006-291x(89)92762-9.
Expression plasmids for normal and abnormal porcine D-amino acid oxidases (E.C. 1.4.3.3, DAO) have been constructed from cloned cDNA that encodes the entire protein sequence of DAO, and the enzymes were expressed in Escherichia coli cells on a large scale. The expressed enzymes were purified to apparent homogeneity. The molecular weight of the normal DAO (38 kD) was identical with that of DAO purified from porcine kidney, whereas that of the abnormal DAO was 39 kD, which comprised the normal DAO with an additional decapeptide at its amino terminus. However, the specific activities of the two enzymes were comparable with that of natural DAO. The results indicate that the bulky decapeptide does not affect the structure necessary for the catalytic function of DAO in the amino-terminal region. The use of a GTG triplet in the 5'-untranslated region of DAO cDNA as the initiation codon for the synthesis of the abnormal DAO is suggested.
已从编码D-氨基酸氧化酶（E.C. 1.4.3.3，DAO）完整蛋白质序列的克隆cDNA构建了正常和异常猪D-氨基酸氧化酶的表达质粒，并在大肠杆菌细胞中大规模表达了这些酶。表达的酶被纯化至表观均一性。正常DAO的分子量（38kD）与从猪肝中纯化的DAO相同，而异常DAO的分子量为39kD，它是在正常DAO的氨基末端额外带有一个十肽。然而，这两种酶的比活性与天然DAO的比活性相当。结果表明，这个庞大的十肽不会影响DAO在氨基末端区域催化功能所需的结构。建议将DAO cDNA 5'-非翻译区中的GTG三联体用作合成异常DAO的起始密码子。
