Motoyoshi Naomi, Kobayashi Hiroko, Itagaki Tadashi, Inokuchi Norio
School of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funahashi, Chiba 274-8555, Japan.
School of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funahashi, Chiba 274-8555, Japan
J Biochem. 2016 Sep;160(3):131-9. doi: 10.1093/jb/mvw017. Epub 2016 Feb 26.
The aim of this study was to phylogenetically characterize the location of the RNase T2 enzyme in the starfish (Asterias amurensis). We isolated an RNase T2 ribonuclease (RNase Aa) from the ovaries of starfish and determined its amino acid sequence by protein chemistry and cloning cDNA encoding RNase Aa. The isolated protein had 231 amino acid residues, a predicted molecular mass of 25,906 Da, and an optimal pH of 5.0. RNase Aa preferentially released guanylic acid from the RNA. The catalytic sites of the RNase T2 family are conserved in RNase Aa; furthermore, the distribution of the cysteine residues in RNase Aa is similar to that in other animal and plant T2 RNases. RNase Aa is cleaved at two points: 21 residues from the N-terminus and 29 residues from the C-terminus; however, both fragments may remain attached to the protein via disulfide bridges, leading to the maintenance of its conformation, as suggested by circular dichroism spectrum analysis. The phylogenetic analysis revealed that starfish RNase Aa is evolutionarily an intermediate between protozoan and oyster RNases.
本研究的目的是从系统发育角度表征核糖核酸酶T2（RNase T2）酶在海星（多棘海盘车）中的位置。我们从海星卵巢中分离出一种RNase T2核糖核酸酶（RNase Aa），并通过蛋白质化学方法和克隆编码RNase Aa的cDNA来确定其氨基酸序列。分离出的蛋白质含有231个氨基酸残基，预测分子量为25,906道尔顿，最适pH值为5.0。RNase Aa优先从RNA中释放鸟苷酸。RNase T2家族的催化位点在RNase Aa中保守；此外，RNase Aa中半胱氨酸残基的分布与其他动植物T2核糖核酸酶中的相似。RNase Aa在两个位点被切割：从N端起21个残基处和从C端起29个残基处；然而，如圆二色光谱分析所示，两个片段可能通过二硫键仍与蛋白质相连，从而维持其构象。系统发育分析表明，海星RNase Aa在进化上是原生动物和牡蛎核糖核酸酶之间的中间体。
