猴脑芳基酰胺酶的纯化与特性分析

Purification and characterization of arylamidase from monkey brain.

作者信息

Hayashi M, Oshima K

出版信息

J Biochem. 1977 Mar;81(3):631-9. doi: 10.1093/oxfordjournals.jbchem.a131498.

DOI:10.1093/oxfordjournals.jbchem.a131498

Abstract

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.

摘要

芳基酰胺酶[EC3.4.11.2]从猴脑提取物中分离出来，并通过包括从猴脑匀浆中提取、硫酸铵分级分离、第一次羟基磷灰石色谱、DEAE - 纤维素色谱、Sephadex G - 200凝胶过滤和第二次羟基磷灰石色谱的六步程序以约11%的产率纯化了约2100倍。该酶在聚丙烯酰胺圆盘电泳上显示出一条带，并且由一条多肽链组成，这是在十二烷基硫酸钠存在下通过圆盘电泳判断的。该酶受到对氯汞苯甲酸（PCMB）、对甲苯磺酰-L-苯丙氨酸氯甲基酮（TPCK）和嘌呤霉素的强烈抑制。嘌呤霉素竞争性抑制该酶，其抑制常数（Ki）值约为5×10⁻⁷M。用乙二胺四乙酸（EDTA）处理导致酶活性丧失。通过添加锌离子（Zn²⁺）、钴离子（Co²⁺）、锰离子（Mn²⁺）可恢复酶活性。在各种氨基酸β-萘酰胺中，L-丙氨酸β-萘酰胺被水解得最快，而N-苄氧羰基-L-亮氨酸β-萘酰胺不被该酶制剂水解。通过在Sephadex G - 200上进行凝胶过滤测定，该酶的分子量为92,000。