家蚕滞育生物钟蛋白质Ease A4的纯化及其分子结构分析

Purification and Molecular Structure Analysis of the Time interval Protein Esterase A4 from Diapausing Eggs of Silkworm, Bombyx mori

ＥａｓｅＡ４是家蚕卵的一种滞育生物钟蛋白质．产下后２ｄ的家蚕Ｃ１０８品种滞育性卵，经过丙酮脱脂、８５℃热处理、硫酸铵沉淀和ＳｅｐｈａｄｅｘＧ－２５凝胶过滤层析初步分离，进一步经过Ｓｅｐ－ＰａｋＣ１８脱盐浓缩，ＨＰＬＣ（柱为ＹＭＣ－ＰａｃｋＰｒｏｔｅｉｎ－ＲＰ）分离，通过ＳＤＳ－ＰＡＧＥ和ＭＡＬＤＩＭＳ方法鉴定，纯化得到ＥａｓｅＡ４蛋白质．从１０ｇ蚕卵最终得到了１１．８μｇＥａｓｅＡ４．ＥａｓｅＡ４由从Ｈｉｓ到Ｔｙｒ的１５５个氨基酸残基构成，蛋白质部分的分子量为１６６０１．其２２位氨基酸残基Ａｓｎ处有一个Ａｓｎ－Ｘ－Ｔｈｒ糖基化场所，并有糖基结合在该部位，糖基的分子量约为７６０．ＥａｓｅＡ４的６１位和１５０位的两个Ｃｙｓ氨基酸残基之间存在二硫键．糖基和二硫键的存在不仅有利于酶蛋白的分离。

The Esterase A4(Ease A4) of Bombyx silkworm eggs is a kind of time interval measuring enzyme for resumption of embryonic development in it.The silkworm diapausing eggs of C108 were degreased with acetone,heat treatment at 85℃ and gel filtration through Sephadex G 25,then concentrated and separated with Centricon 10 and Sep Pak C18.At last,they were separated 2 times with HPLC on a YMC Protein RP column.After that,11 8 μg Ease A4 protein was obtained from 10 g silkworm eggs,with high purity appraised by SDS PAGE and matrix assisted laser desorption ionization mass spectrometry (MALDIMS).The amino acid sequence of Ease A4 was analyzed useing the purified enzyme protein from C108 Bombyx silkworm eggs 2 days after oviposition.It is composed of 155 amino acid residues from His to Tyr,and the Mr is 16 601.A glycoside combining at 22 nd amino acid Asn(Asn X Thr)was found in the protein,and the Mr was 740.The bond of S S between the 61 st amino acid residue Cys and 150 th Cys was confirmed.The glycoside and the bond of S S in Ease A4 related to purification and activation of the protein.