Purification and characterization of peptidylarginine deiminase from rabbit skeletal muscle.
Purification and characterization of peptidylarginine deiminase from rabbit skeletal muscle.
复制标题
兔骨骼肌肽基精氨酸脱亚胺酶的纯化和表征。
DOI：
10.1093/oxfordjournals.jbchem.a134548
复制
发表时间：
1983
影响因子：
2.7
通讯作者：
K. Sugawara
中科院分区：
文献类型：
--
作者：
H. Takahara;Y. Oikawa;K. Sugawara
前文介绍了兔骨骼肌、肾脏、脑和肺中催化蛋白质中精氨基残基脱亚胺的肽基精氨酸脱亚胺酶的鉴定和部分性质。在本工作中，我们从兔骨骼肌经7步纯化，获得了16%的多肽精氨酸脱氨酶。纯化过程包括DEAE-Sephacel离子交换层析、Bio-Gel A-0.5M凝胶过滤、大豆胰酶抑制剂Sepharose4B和氨基己基Sepharose4B亲和层析。纯化的酶在加入和不加入十二烷基硫酸钠的聚丙烯酰胺凝胶电泳法上均一。十二烷基硫酸钠聚丙烯酰胺凝胶电泳法测得该酶的相对分子质量约为83,000，Sephadex G-200凝胶过滤测得该酶的相对分子质量约为130,000-140,000。测得等电点为5.3，并测定了氨基酸组成。该酶较好地催化了氨基和羧基均被取代的精氨酸衍生物生成瓜氨酸衍生物，对BZ-L-Arg-O-ET的催化活性最高。BZ-L-精氨酸-O-ET的Km值为0.50×10~(-3)M。该酶对天然蛋白底物如硫酸鱼精蛋白、大豆胰蛋白酶抑制剂、组蛋白和牛血清白蛋白也有明显的活性。
The preceding paper described the identification and some properties of peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, from rabbit skeletal muscle, kidney, brain, and lung. In the present work we purified peptidylarginine deiminase from rabbit skeletal muscle with a 16% yield by 7 steps. The purification involved ion-exchange chromatography on DEAE-Sephacel, gel filtration on Bio-Gel A-0.5 m, and affinity chromatography on soybean trypsin inhibitor-Sepharose 4B and aminohexyl-Sepharose 4B. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate. The molecular weight of the enzyme was estimated to be about 83,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 130,000-140,000 by gel filtration on Sephadex G-200. The isoelectric point was 5.3 and the amino acid composition was also determined. The enzyme preferably catalyzed the formation of citrulline derivatives from arginine derivatives in which both the amino and carboxyl groups were substituted and showed the highest activity towards Bz-L-Arg-O-Et among the arginine derivatives tested. The Km value for Bz-L-Arg-O-Et was found to be 0.50 X 10(-3) M. The enzyme also showed marked activities towards native protein substrates, such as protamine sulfate, soybean trypsin inhibitor, histone and bovine serum albumin.