Branched chain aminotransferase isoenzymes. Purification and characterization of the rat brain isoenzyme.
Branched chain aminotransferase isoenzymes. Purification and characterization of the rat brain isoenzyme.
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DOI：
10.1016/s0021-9258(18)53663-2
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发表时间：
1993-02
期刊：
影响因子：
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通讯作者：
T. Hall;R. Wallin;G. Reinhart;S. Hutson
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文献类型：
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作者：
T. Hall;R. Wallin;G. Reinhart;S. Hutson
本文首次从大鼠脑细胞质（BCATc）中完整纯化了支链转氨酶（EC 2.6.1.42）。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，酶为单带，分子量为47 kDa；然而，凝胶排斥色谱法表明BCATc是二聚体。BCATc的色氨酸肽图与该酶的线粒体形式（BCATm）的比较表明它们是不同的蛋白质。蛋白质标记试剂，特别是巯基标记试剂的实验也表明，BCATc和BCATm可能存在一些明显的结构差异。然而，BCATc和BCATm对氨基酸和α -酮酸底物表现出相似的特异性。这两种酶都能使支链氨基酸及其直链类似物l -异亮氨酸和谷氨酸转胺。比氨基酸更广泛的α -酮酸被接受作为底物，包括α -酮丁酸和α -酮酸的蛋氨酸。两种酶均表现出乒乓动力学，亮氨酸和异亮氨酸对缬氨酸的表观Km值分别约为1和5 mM。根据氨基酸底物的不同，α -酮戊二酸的Km值约为0.6至3mm。在家兔中制备了针对纯化BCATc的多克隆抗体。BCATc抗血清中和大鼠脑细胞质中支链转氨酶的活性，但不影响心脏线粒体提取物的活性。然而，免疫印迹显示BCATc和BCATm确实具有共同的表位，因为BCATm抗血清在免疫印迹上识别了BCATc。采用BCATc和BCATm抗血清检测BCATc的组织分布。这些数据表明，BCATc存在于成年和胎鼠大脑、胎鼠大脑皮层、卵巢和胎盘的培养细胞中。大脑的活性最高，其次是卵巢、胎儿大脑和胎盘。BCATc未在胎儿肝脏、成年大鼠肝脏或大鼠肝癌细胞系中发现。这些数据提供了明确的证据，表明BCATc与BCATm不同，仅限于几种高度特化的组织。
This paper presents the first complete purification of the branched chain aminotransferase (EC 2.6.1.42) from rat brain cytosol (BCATc). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the enzyme appeared as a single band with a molecular mass of 47 kDa; however, gel exclusion chromatography suggested that BCATc is a dimer. Comparison of tryptic peptide maps of BCATc and the mitochondrial form of the enzyme (BCATm) indicated that they are different proteins. Experiments with protein labeling reagents, in particular sulfhydryl reagents, also suggested that there may be some distinct structural differences in BCATc and BCATm. Nevertheless, BCATc and BCATm showed similar specificities for amino acid and alpha-keto acid substrates. Both enzymes transaminated branched chain amino acids, their straight chain analogs, L-alloisoleucine and glutamate. A broader range of alpha-keto acids than amino acids was accepted as substrate including alpha-ketobutyrate and the alpha-keto acid of methionine. Both enzymes exhibited ping-pong kinetics with apparent Km values for leucine and isoleucine of about 1 and 5 mM for valine, respectively. Km values for alpha-ketoglutarate ranged from about 0.6 to 3 mM depending on the amino acid substrate. Polyclonal antibodies were raised in rabbits against purified BCATc. BCATc antiserum neutralized branched chain aminotransferase activity in rat brain cytosol but did not affect the activity in a heart mitochondrial extract. However, immunoblotting showed that BCATc and BCATm do share common epitopes since BCATm antiserum recognized BCATc on the immunoblots. The tissue distribution of BCATc was examined using BCATc and BCATm antisera. These data showed that BCATc was found in adult and fetal rat brain, cultured cells from fetal rat brain cortex, ovary, and placenta. Brain had the highest activity followed by ovary, fetal brain, and placenta. BCATc was not found in fetal liver, adult rat liver, or a rat hepatoma cell line. These data provide clear evidence that BCATc, unlike BCATm, is restricted to several highly specialized tissues.