Biochemical analyses for the sclerotization of in sect cuticle

昆虫角质层是一种由蛋白质、甲壳素和脂类组成的细胞外基质，具有外骨骼的功能。其硬度是角质层功能的重要性质。一般认为，蜕皮后角质层成分的交联会导致角质层的僵硬。这种硬化过程被称为苯醌鞣制，它是由漆酶类型的酚氧化酶(漆酶)催化的。我们从家蚕幼虫蜕皮后的蚕蛹角质层中分离纯化了活性漆酶。用胰酶消化角质层后，结合柱层析分离溶解的蛋白质。最后，漆酶在FPLC上以单峰形式从Mono Q柱上洗脱出来。经SDS-PAGE分析，该酶的相对分子质量约为70k。与其他角质层成分结合在一起。我们可以利用分离的蚕蛹角质层蛋白和多巴胺在体外重现苯醌的鞣制。结果表明，漆酶在角质层组分的交联中起着重要作用。由纯化的漆酶制备的部分多肽的氨基酸序列与Manduca漆酶样蛋白有很高的相似性。此外，数据库搜索表明，Manduca漆酶样蛋白的核苷酸序列与B.mori EST克隆ce2359相似。该克隆的核苷酸序列编码764个氨基酸残基的蛋白质。根据该核苷酸序列推导出家蚕漆酶的部分氨基酸序列的一级结构。Norten印迹分析表明，在幼虫蜕皮过程中，m RNA开始出现在表皮细胞中。

Insect cuticle is an extracellular matrix composed of proteins, chitin and lipids, and functions as an exoskeleton. Its stiffness is important properties for the function of cuticle. It is thought that the cross-linking of cuticular components after the ecdysis brings the stiffness of the cuticle. This hardening process is called quinone tanning, which is catalyzed by laccase-type phenoloxidase (laccase). We purified activated laccase from pupal cuticles of silkworm, Bombyx mori, just after larval-pupal ecdysis. After digestion of the cuticle with trypsin, solubilized proteins were fractionated by a combination of column chromatography. Finally, laccase was eluted from Mono Q column as a single peak on FPLC. Molecular weight of the typsin-activated laccase was estimated about 70k by SDS-PAGE. Western bound with other cuticle components. We could reproduce quinone tanning using isolated pupal cuticle proteins and dopamin in vitro. The result proved that laccase plays an important role in cross-linking of cuticular components. The amino acid sequences of some peptides prepared from purified laccase indicated high similarity to Manduca laccase like protein. Further, a database search showed that the nucleotide sequence of cDNA for Manduca laccase like protein is similar to a B.mori EST lone, ce2359. Nucleotide sequence of the clone encodes a protein of 764 amino acid residues. The primary structure deduced from the nucleotide sequence the partial amino acid sequences of B.mori laccase. Northen blot analysis revealed that m RNA begins to appear in epidermal cells during larval-pupal molting.