Sequence analysis of bovine lens aldose reductase.
Sequence analysis of bovine lens aldose reductase.
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DOI：
10.1016/s0021-9258(19)39639-5
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发表时间：
1990-03
期刊：
影响因子：
--
通讯作者：
S. Schade;S. Early;T. R. Williams;F. Kézdy;R. Heinrikson;C. Grimshaw;C. C. Doughty-C.
中科院分区：
文献类型：
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作者：
S. Schade;S. Early;T. R. Williams;F. Kézdy;R. Heinrikson;C. Grimshaw;C. C. Doughty-C.
通过对同质酶的特异性和化学裂解产生的多肽进行序列分析，确定了牛晶状体醛糖还原酶(Aditol-NADP+氧化还原酶，EC 1.1.1.21)的共价结构。用反相高效液相色谱分离纯化的多肽，用气相自动Edman降解仪进行组成分析和测序。醛糖还原酶由315个氨基酸残基组成。该酶在氨基末端封闭，用质谱仪鉴定封闭的乙酰基，并对氨基末端的胰蛋白酶多肽进行测序。尽管醛糖还原酶含有N-连接糖基化的共同序列--天冬氨酸-赖氨酸-苏氨酸，但该酶不含碳水化合物。比较序列分析和预测二级结构和核苷酸结合域的算法的应用与醛糖还原酶是具有β-α-β二级结构组织的双域蛋白的观点是一致的。NADPH结合位点似乎与酶的氨基末端半部分有关。基于二氢叶酸和谷胱甘肽还原酶三级结构的建模研究表明，NADPH结合位点从Lys-11开始，并继续核苷酸结合蛋白的β-α-β折叠特征。
The covalent structure of bovine lens aldose reductase (alditol-NADP+ oxidoreductase, EC 1.1.1.21) was determined by sequence analysis of peptides generated by specific and chemical cleavage of the homogeneous apoenzyme. Peptides, purified by reverse-phase high performance liquid chromatography were subjected to compositional analysis and sequencing by gas-phase automated Edman degradation. Aldose reductase was found to contain 315 amino acid residues. The enzyme is blocked at the amino terminus, and mass spectrometry was employed to identify the blocking acetyl group and to sequence the amino-terminal tryptic peptide. The aldose reductase was shown to contain no carbohydrate despite the fact that the enzyme contains the consensus sequence -Asn-Lys-Thr- for N-linked glycosylation. Comparative sequence analysis and application of algorithms for prediction of secondary structure and nucleotide binding domains are consistent with the view that aldose reductase is a double-domain protein with a beta-alpha-beta secondary structural organization. The NADPH binding site appears to be associated with the amino-terminal half of the enzyme. Modeling studies based on the tertiary structures of dihydrofolate and glutathione reductases indicate that the NADPH binding site begins at Lys-11 and continues with a beta-alpha-beta fold characteristic of nucleotide binding proteins.