Purification and characterization of a thyrotropin-releasing hormone deamidase from rat brain.
Purification and characterization of a thyrotropin-releasing hormone deamidase from rat brain.
复制标题
大鼠脑中促甲状腺素释放激素脱酰胺酶的纯化和表征。
DOI：
10.1021/bi00574a014
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发表时间：
1979
期刊：
影响因子：
2.9
通讯作者：
J. Dixon
中科院分区：
文献类型：
--
作者：
J. Rupnow;W. Taylor;J. Dixon
本报告描述了一种大鼠脑促甲状腺激素释放激素(TRH)脱胺酶的纯化，使其表面均一。纯度标准包括十二烷基硫酸钠和圆盘凝胶电泳法，以及等电聚焦(等电点=4.5)。利用底物L-pyroglutamyl-Nim-benzylhistidyl-L-prolyl-beta-naphthylamide，建立了一种快速、灵敏的连续测定方法，从而促进了酶的纯化，该方法在萘胺水解后产生荧光产物β-萘胺(β-NA)。在此底物下，均相酶的比活为14.5摩尔的βNA min-1 mg-1。形成的唯一多肽产物为L-焦谷氨酰亚氨基-苄基组氨酸-L-脯氨酸。将[L-脯氨基-2，3-3H]TRH水解，得到唯一的放射性标记产物L-焦谷氨基-L-组氨酸-L-脯氨酸。用凝胶过滤层析和十二烷基硫酸钠凝胶电泳法对该酶进行了鉴定，结果表明该酶是由一条相对分子质量分别为70,000和73,500的多肽链组成。以L-pyroglutamyl-Nim-benzylhistidyl-L-prolyl-beta-naphthylamide为底物，大鼠脑TRH脱酰胺酶的表观Km为34微米，最适pH为7~8。在此底物上，TRH被证明是一种竞争性抑制剂，其表观KI为120+/-20微米。
This report describes the purification of a rat brain thyrotropin-releasing hormone (TRH) deamidating enzyme to apparent homogeneity. Criteria for purity include sodium dodecyl sulfate and disc gel electrophoresis, as well as isoelectric focusing (pI = 4.5). Enzyme purification was facilitated by development of a rapid and sensitive continuous assay using the substrate L-pyroglutamyl-Nim-benzylhistidyl-L-prolyl-beta-naphthylamide, which, upon hydrolysis of the naphthylamide, results in the appearance of the fluorescent product, beta-naphthylamine (beta NA). With this substrate the homogeneous enzyme had a specific activity of 14.5 mumol of beta NA min-1 mg-1. The only peptide product formed was shown to be L-pyroglutamyl-Nim-benzylhistidyl-L-proline. Hydrolysis of [L-prolyl-2,3-3H]TRH was shown to yield L-pyro-glutamyl-L-histidyl-L-proline as the only radiolabeled product. Characterization of the brain deamidase by gel filtration chromatography and sodium dodecyl sulfate gel electrophoresis indicated that the enzyme consists of a single polypeptide chain having molecular weights of 70,000 and 73,500, respectively. Rat brain TRH deamidase has an apparent Km of 34 micron, and a pH optimum between 7 and 8 using L-pyroglutamyl-Nim-benzylhistidyl-L-prolyl-beta-naphthylamide as a substrate. With this substrate, TRH was shown to be a competitive inhibitor with an apparent Ki of 120 +/- 20 micron.