Purification and characterization of arylamidase from monkey brain.
Purification and characterization of arylamidase from monkey brain.
复制标题
猴脑芳基酰胺酶的纯化和表征。
DOI：
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发表时间：
1977
期刊：
影响因子：
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通讯作者：
Kiyoshi Oshima
中科院分区：
文献类型：
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作者：
Motoharu Hayashi;Kiyoshi Oshima
从猴脑提取物中分离出芳基酰胺酶[EC 3.4.11.2]，并通过六步程序纯化约2100倍，产率约为11%，该六步程序包括从猴脑匀浆中提取、硫酸铵分级分离、第一次羟基磷灰石层析、DEAE-纤维素层析、Sephadex G-200凝胶过滤和第二次羟基磷灰石层析。该酶在聚丙烯酰胺圆盘电泳上显示出单一条带，并且由单一多肽链组成，如在十二烷基硫酸钠存在下通过圆盘电泳所判断的。该酶被PCMB、TPCK和嘌呤霉素强烈抑制。嘌呤霉素竞争性抑制酶，Ii值约为5 × 10（-7）M。用EDTA处理导致酶活性的损失。添加Zn ~（2+）、Co ~（2+）、Mn ~（2+）可使酶活力恢复。在各种氨基酸β-萘酰胺中，L-丙氨酸β-萘酰胺水解最快，N-苄氧羰基-L-亮氨酸β-萘酰胺不被该酶制剂水解。用Sephadex G-200凝胶过滤法测得酶的分子量为92，000。
Arylamidase [EC3.4.11.2] was isolated from monkey brain extract and purified about 2100-fold in approximately 11% yield by a six-step procedure comprising extraction from monkey brain homogenate, ammonium sulfate fractionation, first hydroxylapatite chromatography, DEAE-cellulose chromatography, Sephadex G-200 gell filtration and second hydroxylapatite chromatography. The enzyme showed a single band on polyacrylamide disc electrophoresis and consisted of a single polypeptide chain, as judged by disc electrophoresis in the presence of sodium dodecyl sulfate. The enzyme was strongly inhibited by PCMB, TPCK, and puromycin. Puromycin competitively inhibited the enzyme and the Ii value was about 5 x 10(-7)M. Treatment with EDTA resulted in a loss of enzyme activity. The enzyme activity was restored by addition of Zn2+, Co2+, Mn2+. Among various amino acid beta-naphthylamides, L-alanine beta-naphthylamide was most rapidly hydrolyzed and N-carbobenzoxyl-L-leucine beta-naphthylamide was not hydrolyzed by this enzyme preparation. The molecular weight of the enzyme was 92,000 as determined by gel filtration on Sephadex G-200.