CAJ | 학술논문

背景：前期研究从赤子爱胜蚓组织中获得一组核酸酶样蛋白，对该组核酸酶样蛋白的一级结构进行研究，有利于揭示其结构的基本性质，为进一步结构与功能的研究奠定基础。 　　目的：对核酸酶样蛋白EWD1和EWD2进行一级结构的测定。 　　方法：采用Edman降解法测定EWD1和EWD2的N末端氨基酸序列，采用酸水解法测定其氨基酸组成， LC-MS/MS测定蛋白质内部分氨基酸的序列，MALDI-TOF-MS测定蛋白的含糖量和二硫键数目。 　　结果与结论：核酸酶样蛋白EWD1和EWD2的氨基酸组成中，天冬氨酸和天冬酰胺之和的含量最高，都达到近10%，疏水氨基酸亮氨酸的含量也较高，半胱氨酸含量较少，氨基酸组成比较显示，该2种酶与其它核酸酶较相似。Edman降解法测定，EWD1蛋白大亚基的N末端6个氨基酸序列为：D、E、W、V、Y、P，EWD2蛋白的N末端9个氨基酸序列为：L、L、G、P、Y、K、P、K、C；串，联质谱的结果提示2种核酸酶为新蛋白；MALDI-TOF-MS法显示1号核酸酶中含有8个半胱氨酸，形成4对二硫键，2号核酸酶中有6个半胱氨酸，形成3对二硫键。EWD1，2均为糖蛋白，EWD1中的含糖量为17.3%，EWD2蛋白中的含糖量为15.6%。
배경：전기연구종적자애성인조직중획득일조핵산매양단백，대해조핵산매양단백적일급결구진행연구，유리우게시기결구적기본성질，위진일보결구여공능적연구전정기출。 　　목적：대핵산매양단백EWD1화EWD2진행일급결구적측정。 　　방법：채용Edman강해법측정EWD1화EWD2적N말단안기산서렬，채용산수해법측정기안기산조성， LC-MS/MS측정단백질내부분안기산적서렬，MALDI-TOF-MS측정단백적함당량화이류건수목。 　　결과여결론：핵산매양단백EWD1화EWD2적안기산조성중，천동안산화천동선알지화적함량최고，도체도근10%，소수안기산량안산적함량야교고，반광안산함량교소，안기산조성비교현시，해2충매여기타핵산매교상사。Edman강해법측정，EWD1단백대아기적N말단6개안기산서렬위：D、E、W、V、Y、P，EWD2단백적N말단9개안기산서렬위：L、L、G、P、Y、K、P、K、C；천，련질보적결과제시2충핵산매위신단백；MALDI-TOF-MS법현시1호핵산매중함유8개반광안산，형성4대이류건，2호핵산매중유6개반광안산，형성3대이류건。EWD1，2균위당단백，EWD1중적함당량위17.3%，EWD2단백중적함당량위15.6%。
BACKGROUND:A group of nuclease-like proteins were previously purified from Eisenia foetida tissues, exploring primary structures of these proteins wil help to uncover basic structure characteristics of them and provide foundations for the study addressing the relationship of their structures and functions. OBJECTIVE:To explore primary structures of nuclease-like proteins EWD1 and EWD2. METHODS:Edman degradation method was used to sequence the N-terminal amino acids of EWD1 and EWD2, acid hydrolisis method was used to analyze amino acid compositions of EWD1 and EWD2, LC-MS/MS was used to analyze some peptide sequences within the proteins, and MALDI-TOF-MS was used to calculate the number of the disulfide bonds and the contents of polysaccharides. RESULTS AND CONCLUSION:Among the amino acid compositions in EWD1 and EWD2, the sum contents of aspartate and asparagines were the highest (al nearly 10%), the contents of hydrophobic amino acids were also high, and the contents of cysteine was low. The EWD1 and EWD2 had similar amino acid compositions with other nucleases. Edman degradation results showed that, the N-terminal sequences of the large subunit of EWD1 were in turn as fol ows:D, E, W, V, Y, P;the N-terminal sequences of EWD2 were as fol ows:L, L, G, P, Y, K, P, K, C. The results of LC-MS/MS indicated the two proteins were novel proteins;MALDI-TOF-MS results showed that 8 cysteine residues formed 4 disulfide bonds in EWD1, 6 cysteine residues formed 3 disulfide bonds in EWD2. EWD1 and EWD2 were al glycoproteins, the content of polysaccharides was 17.3%in EWD1 and 15.6%in EWD2.