Murine aspartoacylase: cloning, expression and comparison with the human enzyme

被引:23
作者
Namboodiri, MAA [1 ]
Corigliano-Murphy, A
Jiang, G
Rollag, M
Provencio, I
机构
[1] NICHD, Dev Neurobiol Lab, Sect Neuroendocrinol, Bethesda, MD 20892 USA
[2] NIH, Cell Biol Lab, NHLBI, Bethesda, MD 20892 USA
[3] Uniformed Serv Univ Hlth Sci, Dept Physiol, Bethesda, MD 20814 USA
[4] Uniformed Serv Univ Hlth Sci, Dept Anat & Cell Biol, Bethesda, MD 20814 USA
来源
MOLECULAR BRAIN RESEARCH | 2000年 / 77卷 / 02期
关键词
Canavan disease; N-acetylaspartate; amide hydrolysis; human aspartoacylase; acetate deficiency hypothesis; acetate supplementation therapy;
D O I
10.1016/S0169-328X(00)00068-1
中图分类号
Q189 [神经科学];
学科分类号
071006 ;
摘要
Canavan disease is caused by mutations in aspartoacylase, the enzyme that degrades N-acetylaspartate (NAA) into acetate and aspartate, Murine aspartoacylase (mASPA) was cloned using sequence information from mouse expressed sequence tags homologous to the human cDNA. The open reading frame was cloned into a thioredoxin fusion vector, overexpressed in bacteria, and the protein was purified using affinity chromatography to near homogeneity. Recombinant human ASPA (hASPA) was prepared by a similar method. Both recombinant enzymes were highly specific to NAA, with about 10% of the NAA activity toward N-acetylasparagine. More interestingly, the product of N-acetylasparagine was aspartate but not asparagine, indicating that ASPA catalyzed deacetylation as well as hydrolysis of the beta acid amide. Our success in preparing the recombinant ASPA in high purity should permit multiple lines of investigations to understand the pathogenic mechanisms of Canavan disease and the functional roles of NAA. (C) 2000 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:285 / 289
页数:5
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