Kurahashi Hitoshi, Atiwetin Panida, Nagaoka Sumiharu, Miyata Seiji, Kitajima Sakihito, Sugimura Yukio
Graduate School of Science and Technology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585, Japan.
J Insect Physiol. 2005 Sep;51(9):1055-61. doi: 10.1016/j.jinsphys.2005.05.007. Epub 2005 Jul 6.
Mulberry leaves are the sole diet of the silkworm, Bombyx mori. The host urease is incorporated into the larval hemolymph and involved in nitrogen metabolism in the insect. To investigate the selective absorption of the host urease to the larvae, crude urease was prepared from mulberry leaves and roots. Root urease was identical to leaf urease on the basis of electrophoretic analyses: (1) the urease activity appeared in the same migration position in a native gel; (2) There was no difference in molecular mass of the subunit. The root urease was orally injected to the fifth instar larvae of the silkworm. Just before spinning, the larvae absorbed intact urease from the midgut lumen to the hemolymph without the loss of activity. The capacity to absorb urease occurred only at the specific stage. Localization of host urease in midgut tissue was observed using confocal laser scanning microscopy and transmission electron microscopy. Based on spatial distribution of immunofluorescent signals and immunogold particles, host urease specifically attached to the surfaces of microvilli existing in the apical side of columnar cells and appeared in the cytoplasm of the cells for transport to the hemolymph. The incorporation efficiency of root urease into the hemolymph was significantly higher than for ureases from jack bean seeds and Bacillus pasteurii. The urease that was transported to the hemolymph was electrophoretically altered, compared with the host urease extracted.
桑叶是家蚕(Bombyx mori)的唯一食物。宿主脲酶被纳入幼虫血淋巴并参与昆虫的氮代谢。为了研究宿主脲酶对幼虫的选择性吸收,从桑叶和桑根中制备了粗脲酶。基于电泳分析,根脲酶与叶脲酶相同:(1)在天然凝胶中脲酶活性出现在相同的迁移位置;(2)亚基的分子量没有差异。将根脲酶经口注射到家蚕的五龄幼虫中。就在吐丝前,幼虫将完整的脲酶从中肠腔吸收到血淋巴中,且活性没有损失。吸收脲酶的能力仅在特定阶段出现。使用共聚焦激光扫描显微镜和透射电子显微镜观察宿主脲酶在中肠组织中的定位。基于免疫荧光信号和免疫金颗粒的空间分布,宿主脲酶特异性地附着在柱状细胞顶端侧存在的微绒毛表面,并出现在细胞的细胞质中以便运输到血淋巴。根脲酶进入血淋巴的掺入效率显著高于来自刀豆种子和巴氏芽孢杆菌的脲酶。与提取的宿主脲酶相比,运输到血淋巴中的脲酶在电泳上发生了改变。