Biochemical analyses for the sclerotization of in sect cuticle
| Project/Area Number |
14540632
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
動物生理・代謝
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| Research Institution | Tokyo Metropolitan University |
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Principal Investigator |
IZUMI Susumu Tokyo Metropolitan University, Graduate School of Science, Associate professor, 理学研究科, 助教授 (10145659)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2003: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2002: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | insect / cuticle / sclerotization / phenoloxidase / laccase / silkworm / cDNA / expression / 構造解析 / 表皮細胞 / 精製 |
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| Research Abstract |
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.
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Report
(3 results)
Research Products
(5 results)
