Momoi K, Fukui K, Tada M, Miyake Y
Department of Biochemistry, National Cardiovascular Center Research Institute, Osaka.
J Biochem. 1990 Sep;108(3):406-13. doi: 10.1093/oxfordjournals.jbchem.a123214.
Although D-amino acid oxidase (DAO) [EC 1.4.3.3] activity in rabbit kidney extract was undetectable, protein immunoreactive toward rabbit anti-pig kidney DAO antiserum and RNAs that hybridized with fragments of human and pig DAO cDNAs were detected distinctly in the rabbit kidney. A cDNA clone, RD22, was isolated from the rabbit kidney cDNA library by hybridization with a fragment of human DAO cDNA. Analysis of the nucleotide sequence revealed a 2,018 nucleotide sequence encoding a protein consisted of 347 amino acids. The number of amino acid residues was identical with those of human and pig DAOs, and the amino acid sequence showed 80 and 83% identity with pig and human DAOs, respectively. RNAs that hybridized with RD22 DNA fragment also existed in rabbit kidney, and their sizes were the same as those of the RNAs detected with the human and pig DAO cDNA fragments. RD22-derived protein was hardly synthesized by an in vitro expression system. However, a cDNA fragment lacking most of the 5'-untranslated region and its mutants containing base changes around the initiation codon did direct protein synthesis. Moreover, the protein derived from the partial cDNA fragment containing a large part of the coding region sequence showed immunoreactivity toward anti-pig DAO antiserum. The results suggest that one of the causes of the very poor synthesis of DAO protein in rabbit kidney is translational suppression in the synthetic process.
尽管兔肾提取物中未检测到D-氨基酸氧化酶（DAO）[EC 1.4.3.3]的活性，但在兔肾中可明显检测到与兔抗猪肾DAO抗血清发生免疫反应的蛋白质以及与人和猪DAO cDNA片段杂交的RNA。通过与人DAO cDNA片段杂交，从兔肾cDNA文库中分离出一个cDNA克隆RD22。对核苷酸序列的分析揭示了一个2018个核苷酸的序列，编码一个由347个氨基酸组成的蛋白质。氨基酸残基的数量与人和猪的DAO相同，氨基酸序列分别与猪和人的DAO显示出80%和83%的同一性。与RD22 DNA片段杂交的RNA也存在于兔肾中，其大小与用人和猪DAO cDNA片段检测到的RNA相同。RD22衍生的蛋白质很难通过体外表达系统合成。然而，一个缺少大部分5'-非翻译区的cDNA片段及其在起始密码子周围含有碱基变化的突变体确实指导了蛋白质的合成。此外，来自包含大部分编码区序列的部分cDNA片段的蛋白质显示出对抗猪DAO抗血清的免疫反应性。结果表明，兔肾中DAO蛋白合成非常差的原因之一是合成过程中的翻译抑制。
