Oettgen H C, Taylor A
Anal Biochem. 1985 Apr;146(1):238-45. doi: 10.1016/0003-2697(85)90421-x.
Leucine aminopeptidase (LAP) was purified from hog lenses by application of the Himmelhoch procedure for isolation of hog kidney LAP [S. R. Himmelhoch (1970) in Methods in Enzymology (Perlmann, G. E., and Lorand, L., eds.), Vol. 19, pp. 508-513, Academic Press, New York.] This involved treating crude hog lens homogenates with hexadecyltrimethylammonium bromide, DEAE-cellulose adsorption and elution, ammonium sulfate fractionation (53-84% of saturation), and gel filtration on a Bio-Gel A-1.5m column. Purifications ranging from 2080- to 4700-fold with activity yields from 28 to 100% were achieved. The hog lens LAP appeared homogeneous by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE). Bio-Gel chromatography of the native enzyme and SDS-PAGE of dimethylsuberimidate-crosslinked LAP indicated a molecular weight of 326,000. SDS-PAGE of untreated LAP showed a subunit weight of 54,000, consistent with a hexameric enzyme structure. By immunodiffusion, LAP from hog lens and kidney were identical while hog lens and beef lens enzymes demonstrated only partial identity. Electrophoresis of the native enzymes showed a slightly lower mobility for the hog lens LAP than for beef LAP at pH 8.7.
通过应用分离猪肾亮氨酸氨基肽酶（LAP）的希默尔霍赫方法[S. R. 希默尔霍赫（1970年），见《酶学方法》（珀尔曼，G. E. 和洛兰德，L. 编），第19卷，第508 - 513页，学术出版社，纽约]，从猪晶状体中纯化了亮氨酸氨基肽酶（LAP）。这包括用十六烷基三甲基溴化铵处理猪晶状体粗匀浆、DEAE - 纤维素吸附和洗脱、硫酸铵分级分离（饱和度53 - 84%）以及在Bio - Gel A - 1.5m柱上进行凝胶过滤。实现了2080至4700倍的纯化，活性产率为28%至100%。通过天然和十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（PAGE），猪晶状体LAP呈现出均一性。天然酶的Bio - Gel色谱分析和二甲基辛二亚酰胺交联LAP的SDS - PAGE表明分子量为326,000。未处理LAP的SDS - PAGE显示亚基分子量为54,000，与六聚体酶结构一致。通过免疫扩散，猪晶状体和肾的LAP相同，而猪晶状体和牛肉晶状体的酶仅显示部分同一性。在pH 8.7时，天然酶的电泳显示猪晶状体LAP的迁移率略低于牛肉LAP。
