Partial purification and characterization of dipeptidyl peptidase II (DPP II) from guinea pig testes
Partial purification and characterization of dipeptidyl peptidase II (DPP II) from guinea pig testes
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豚鼠睾丸二肽基肽酶 II (DPP II) 的部分纯化和表征
DOI：
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发表时间：
1986
期刊：
影响因子：
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通讯作者：
E. Dudenhausen
中科院分区：
文献类型：
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作者：
G. Dicarlantonio;P. Talbot;E. Dudenhausen
通过硫酸铵沉淀、Con A-Sepharose 4 B层析和Sephadex G-200层析，从豚鼠睾丸中部分纯化了脱肽基肽酶（DPP II），比活性为27.4 μmol Ala 3水解min−1 mg−1蛋白。在经校准的G-200柱上层析，得到该酶的分子量为135，000道尔顿。十二烷基硫酸钠聚丙烯酰胺电泳显示在64- 66，000道尔顿处的宽双峰富集。该酶在pH 4.5时对L-丙氨酰-丙氨酰-丙氨酸的水解具有最佳活性，并对大小增加的阳离子具有敏感性，其中Tris产生的抑制作用最大。丝氨酸蛋白酶抑制剂可中度抑制该酶。薄层色谱法揭示了二肽酶性质的酶的活性三肽和二肽基芳基酰胺。当（NH 4）2SO 4馏分的非变性凝胶电泳的硝酸纤维素电印迹与特定的DPP II底物赖氨酰-丙氨酰-4-甲氧基-2-萘酰胺反应时，发生双重活性。G-200组分的分析等电聚焦，然后使用浸渍有特定DPP II底物赖氨酰-丙氨酰-7-氨基-4-三氟甲基香豆素的三乙酸纤维素覆盖膜进行荧光酶活性检测，显示出多个异构体聚焦在pI = 4.8-5.6。两个突出的条带集中在pI = 4.9和pI = 5.1处。将豚鼠睾丸DPP II的性质与来自其他来源的类似二肽基肽酶进行比较和对比。
Depeptidyl peptidase (DPP II) was partially purified from guinea pig testes by (NH4)2SO4 precipitation, Con A-Sepharose 4B chromatography, and Sephadex G-200 chromatography to a specific activity of 27.4 μmol Ala3 hydrolyzed min−1 mg−1 protein. Chromatography on a calibrated G-200 column yielded a molecular weight of 135,000 daltons for the enzyme. Sodium dodecyl sulfate polyacrylamide electrophoresis showed an enrichment of a broad doublet at 64–66,000 daltons. The enzyme had optimal activity toward hydrolysis of L-alanyl-alanyl-alanine at pH 4.5 and showed sensitivity to cations of increasing size with Tris producing the most inhibition of those tested. The enzyme was moderately inhibited by serine proteinase inhibitors. Thin-layer chromatography revealed the dipeptidase nature of the enzyme's activity on tripeptides and dipeptidyl arylamides. A doublet of activity occurred when nitrocellulose electroblots of nondenaturing gel electrophoresis of the (NH4)2SO4 fraction were reacted with the specific DPP II substrate, lysyl-alanyl-4-methoxy-2-napthylamide. Analytical isoelectric focusing of the G-200 fraction followed by fluorescent enzyme activity detection that used cellulose triacetate overlay membranes impregnated with the specific DPP II substrate, lysyl-alanyl-7-amino-4-trifluoromethylcou-marin, revealed multiple isoforms focusing at pI = 4.8–5.6. Two prominent bands focused at pI = 4.9 and pI = 5.1. The properties of guinea pig testicular DPP II are compared and contrasted with similar dipeptidyl peptidases from other sources.