Silk fibroin of Bombyx mori is secreted, assembling a high molecular mass elementary unit consisting of H-chain, L-chain, and P25, with a 6:6:1 molar ratio
Silk fibroin of Bombyx mori is secreted, assembling a high molecular mass elementary unit consisting of H-chain, L-chain, and P25, with a 6:6:1 molar ratio
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DOI：
10.1074/jbc.m006897200
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发表时间：
2000-12-22
影响因子：
4.8
通讯作者：
Mizuno, S
中科院分区：
文献类型：
--
作者：
Inoue, S;Tanaka, K;Mizuno, S
家蚕 Bombyx mori 产生的丝素蛋白由重链、轻链和糖蛋白 P25 组成。重链和轻链通过二硫键连接，P25 通过非共价相互作用与二硫键连接的重链和轻链缔合。定量酶联免疫吸附测定显示，茧中和分泌到后丝腺腔内的丝素中重链、轻链和P25的摩尔比均为6:6:1。三种家蚕“裸蛹”突变体产生的微量丝心蛋白缺乏轻链，但重链与P25的摩尔比也为6:1。凝胶过滤色谱和沉降平衡分析表明，后丝腺细胞中存在约2.3 MDa的大蛋白质复合物，指定为丝心蛋白的基本单位，其重链、轻链和P25的摩尔比为6:6:1。生物素化的刀豆球蛋白 A 无法接近天然基本单元，并且在与过量的 N-糖苷酶 F 或糖苷内切酶 H 一起孵育后，基本单元部分解离，表明 P25 的单分子位于内部，并在维持复合物的完整性方面发挥着重要作用。
Silk fibroin produced by the silkworm Bombyx mori consists of a heavy chain, a light chain, and a glycoprotein, P25. The heavy and light chains are linked by a disulfide bond, and P25 associates with disulfide-linked heavy and light chains by noncovalent interactions. Quantitative enzyme-linked immunosorbent assay revealed that molar ratios of the heavy chain, light chain, and P25 were 6:6:1, both in cocoons and in fibroin secreted into the lumen of posterior silk gland. Trace amounts of fibroin produced by three "naked pupa" mutants of B. mori lacked the light chain, but the molar ratio of heavy chain and P25 was also 6:1. Gel filtration chromatography and sedimentation equilibrium analysis demonstrated that a large protein complex of approximately 2.3 MDa, designated an elementary unit of fibroin having 6:6:1 molar ratios of the heavy chain, light chain, and P25, existed in posterior silk gland cells. Inaccessibility of biotinylated concanavalin A to the native elementary unit and partial dissociation of the elementary unit after incubation with excess N-glycosidase F or endoglycosidase H suggest that a single molecule of P25 is located internally and plays an important role in maintaining integrity of the complex.