蓖麻蚕卵天冬氨酸蛋白酶纯化及性质

PURIFICATION AND CHARACTERIZATION OF AN ASPARTIC PROTEINASE IN THE EGGS OF PHILOSAMIA CYNTHIA RICINI

采用硫酸铵沉淀、离子交换层析和离子交换凝胶过滤法，从蓖麻蚕卵母细胞中纯化出一种蛋白酶。该蛋白酶活性能被天冬氨酸蛋白酶特异性抑制剂抑胃肽抑制，初步鉴定该酶为天冬氨酸蛋白酶。经ＳＤＳ聚丙烯酰胺凝胶电泳测得分子量为９０ｋＤ，由凝胶过滤估计分子量在３６０ｋＤ，推测该酶由四亚基组成。该蛋白酶在ｐＨ３～５均可水解牛血红蛋白，最适ｐＨ为４。这是首次报道昆虫卵内存在天冬氨酸蛋白酶。

Egg proteinases play important roles in the degradation of yolk proteins during embryogenesis. Up to now, cysteine proteinases and serine proteinases from insect eggs have been reported. However, there is no report on the egg aspartic proteinase. In our previous studies on the egg cysteine proteinase from Philosamia cynthia ricini , we observed that the activities of the endogenous proteinases in the oocytes could be inhibited by pepstatin, a specific inhibitor of aspartic proteinase. This result suggested that an aspartic proteinase might exist in the oocytes. In this paper we reported that an aspartic proteinase existing in the eggs of Philosamia cynthia ricini was purified by ammonium sulfate fractionation and chromatography. The purified proteinase appeared to be homogeneous on the gel of sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS PAGE). The molecular mass was estimated as 90kD by SDS PAGE. The native molecular mass was estimated as 360kD by gel filtration. Therefore, the proteinase might be a tetramer. The optimum pH of the proteinase for hydrolyzing bovine hemoglobin was pH 4. However, the proteinase could hydrolyze bovine hemoglobin at a wide pH range of 3 to 5. The activity of the proteinase could be inhibited by pepstatin. The proteinase could hydrolyze the vitellin, in vitro. This fact suggested that the proteinase might be involved in the degradation of vitellin during embryogenesis. Previously, we reported an egg cysteine proteinase from Philosamia cynthia ricini. Both of the cysteine proteinase and the aspartic proteinase from this insect prefer acidic pH condition for their activity. The molecular masses of these proteinases were quite different. Moreover, the inhibitors of these two proteinases were also different. Besides, we also found that the antiserum to the aspartic proteinase from Philosamia cynthia ricini could recognize the antigens from Antheraea pernyi and Hilicoverpa armigera (in press). This result suggested that aspartic proteinase might exist in the eggs of some Lepidopterous insects.