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Purification and Characterization of a Rat Brain Aldehyde Dehydrogenase Able to Metabolize gamma-Amino Butyraldehyde to gamma-Aminobutyric Acid

能够将γ-氨基丁醛代谢为γ-氨基丁酸的大鼠脑醛脱氢酶的纯化和表征

基本信息

批准号：
01570167
负责人：
OHKAWA Kiyoshi
金额：
$ 1.34万
依托单位：
Jikei University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

An enzyme which catalyses dehydrogenation of gamma-aminobutyraldehyde (ABAL) to gamma-aminobutyric acid (GABA) was purified to homogeneity from rat brain tissues by using DEAE cellulose and affinity chromatography on 5'-AMP-Sepharose and Blue agarose, followed by gel filtration. Such an enzyme was first purified from mammalian brain tissues, and was identified as isoenzyme of aldehyde dehydrogenase. It has an 210 kDa determined by PAEG, and appeared to be composed of subunits of 50 kDa. Substrate specificity of the enzyme toward ABAL was higher than those of aldehyde dehydrogenases from human liver (E2), and was lower than those of ABAL dehydrogenase from human liver (E3). The molecular weight and relative amino acid composition of the enzyme are also similar to those of E1 and E2. Polyclonal and monoclonal antibodies against the purified enzyme were generated. Both antibodies reacted weakly but significantly with a molecule of 50 kDa band of the enzyme by SDS-PAGE, western blot analysis. By immunohistochemical analysis of the rat brains, antibodies showed positive, homogenous staining in the cytoplasms of the nerve cells without any special localization or tissue distribution of the reactive products. The existence of this enzyme in mammalian brain seems to be related to a glutamate decarboxylase-independent pathway for GABA synthesis from putrescine.

用DEAE纤维素、5 ′-AMP-琼脂糖和Blue琼脂糖亲和层析、凝胶过滤等方法，从大鼠脑组织中纯化了一种催化γ-氨基丁醛（ABAL）脱氢生成γ-氨基丁酸（GABA）的酶。这种酶首先从哺乳动物脑组织中纯化，并被鉴定为醛脱氢酶同工酶。PAEG测定其分子量为210 kDa，似乎由50 kDa的亚基组成。该酶对ABAL的底物特异性高于人肝醛脱氢酶（E2），低于人肝ABAL脱氢酶（E3）。该酶的分子量和相对氨基酸组成也与E1和E2相似。针对纯化的酶产生多克隆和单克隆抗体。经SDS-PAGE、Western blot分析，两种抗体与酶的50 kDa条带的分子反应微弱但显著。通过对大鼠脑的免疫组织化学分析，抗体在神经细胞的胞浆中显示出阳性的、均匀的染色，而反应产物没有任何特殊的定位或组织分布。这种酶在哺乳动物脑中的存在似乎与从腐胺合成GABA的谷氨酸脱羧酶非依赖性途径有关。