Re-examination of a α-chymotrypsin-solubilized laccase in the pupal cuticle of the silkworm, Bombyx mori: Insights into the regulation system for laccase activation during the ecdysis process
Re-examination of a α-chymotrypsin-solubilized laccase in the pupal cuticle of the silkworm, Bombyx mori: Insights into the regulation system for laccase activation during the ecdysis process
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DOI：
10.1016/j.ibmb.2014.10.004
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发表时间：
2014-12-01
影响因子：
3.8
通讯作者：
Chosa, Naoyuki
中科院分区：
文献类型：
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作者：
Asano, Tsunaki;Taoka, Masato;Chosa, Naoyuki
家蚕（Bombyx mori）的蛹角质层中的漆酶被认为是作为一种无活性的前体积累起来的，它可以根据不同的阶段被激活。在本研究中，我们从家蚕表皮提取物中分离到一个81kda的漆酶，该漆酶是用α -胰凝乳蛋白酶消化蛹表皮得到的。质谱分析表明，该蛋白为家蚕漆酶2基因的产物。纯化的81 kda漆膜酶（α -chymotrypsin-solubilized Bombyx laccase2: Bm-clac2）的RNPADS n端序列对应于推导出的Bmlaccase2蛋白序列的Arg（146）至Ser(151)，表明Bm-clac2缺少残基Arg（146）上游的n端部分。Bm-clac2表现出酶活性，但经过胰蛋白酶处理后，其比活性增加了约17倍，胰蛋白酶涉及c端肽键的切割。我们还发现，在异丙醇的存在下，Bm-clac2的活性增加。在以前的报道中，蛋白质水解过程被假设为体内漆酶激活的一种系统，但目前的结果表明，这种类型的加工并不是将Bm-clac2转化为高活性酶的唯一途径。(C) 2014 Elsevier Ltd.版权所有。
The laccase in the pupal cuticle of the silkworm, Bombyx mori, is thought to accumulate as an inactive precursor that can be activated stage-dependently. In this study we isolated an 81-kDa laccase from cuticular extract of B. mori that was prepared by digestion of the pupal cuticles with alpha-chymotrypsin. The mass spectrometric analysis of the purified protein indicates that this 81-kDa laccase is a product of the Bombyx laccase2 gene. The purified 81-kDa laccase (alpha-chymotrypsin-solubilized Bombyx laccase2: Bm-clac2) has an N-terminal sequence of RNPADS that corresponds to Arg(146) to Ser(151) of the deduced protein sequence of Bmlaccase2 cDNA, indicating that Bm-clac2 lacks the N-terminal part upstream from residue Arg(146). Bm-clac2 shows enzymatic activity, but its specific activity is increased around 17-fold after treatment with trypsin, which involves cleavage of peptide bonds at the C-terminal region. We also found that the activity of Bm-clac2 is increased in the presence of isopropanol. In previous reports, proteolytic processing has been hypothesized as a system for laccase activation in vivo, but the present result implies that this type of processing is not the only way to convert Bm-clac2 to the high-activity enzyme. (C) 2014 Elsevier Ltd. All rights reserved.