Molecular mechanism of digestion and absorption in the silkworm, Bombyx mori

为了阐明家蚕（Bombyx mori）消化吸收的分子机制，研究了蔗糖在中肠中的水解和吸收，特别是蔗糖酶在中肠组织中的作用。测定5龄幼虫和成虫中肠蔗糖酶的最佳条件不同。在此基础上，选择了各发育阶段的最佳检测条件。从四龄到成虫羽化，测定其活动。最大的活动出现在第五星的中间。幼虫和成虫发育过程中可溶性蔗糖酶的电泳图谱存在差异。转变为幼虫-蛹蜕化。中肠蔗糖酶90%的活性位于细胞质中，10%位于5龄和pharate成虫中肠的膜结合部分。利用分离的中肠囊进行的体外实验表明，膜结合的蔗糖酶参与了中肠中蔗糖的水解，而周围营养膜促进了蔗糖水解产物穿过中肠组织进入血流。采用硫酸铵分馏，以离子交换纤维素、羟基磷灰石和丝phacryl为色谱介质，柱层析纯化可溶性蔗糖酶，使其接近均匀性。该酶的动力学性质表明该酶为-果糖呋喃苷酶。建立了膜结合蔗糖酶与洗涤剂的增溶过程，分离得到的蔗糖酶表现出与可溶性β -果糖呋喃糖苷酶不同的特性。

To elucidate molecular mechanism of digestion and absorption in the silkworm, Bombyx mori, hydrolysis and absorption of sucrose was studied in the midgut, and in particular, sucrase in the midgut tissue was investigated.Optimum conditions for assay of midgut sucrase in the fifth instar larvae and in pharate adults were different. Based on this result, optimum assay conditions for each developmental stage was selected. From the fourth instar until adult emergence, activity was determined. Maximum activity was obseved in the middle of the fifth instar. Electrophoretic pattern of soluble sucrase during development showed difference between larvae and pharate adults. The transition was at larval-pupal ecdysis. Ninety % of activity of midgut sucrase localizes in the cytosol and 10 % in the membrane-bound fraction in the fifth instar and pharate adult midgut. In vitro experiments using isolated midgut sac, it was demonstrated that membrane-bound sucrase is involved in hydrolysis of sucrose in the midgut and that peritrophic membrane enhances transport of hydrolysis products of sucrose accross midgut tissue into the haemocoel. Soluble sucrase was purified to near homogeneity using ammonium sulfate fractionation, and column chromatographies of ion exchange cellulose, hydroxylapatite, and Sephacryl as a chromatomedia. The enzyme was disclosed beta-fructofuranosidase from its kinetic properties. Solubilization procedure of membrane-bound sucrase with detergent was established and isolated sucrase showed properties distinct from soluble beta-fructofuranosidase.