DEVELOPMENT AND CONTROL OF SYNTHESIS AND SECRETION

本申请中描述的研究旨在继续 阐明参与生物合成、加工和生产的早期事件。 肠道糖蛋白酶的细胞内转运 插入到微绒毛膜，以及控制微绒毛膜的因素 这些机制的个体发育。将详细研究两种酶和 比较：蔗糖酶-异麦芽糖酶(E.C.3.2.1.48-E.C.3.2.1.10)和 乳糖酶-根皮苷水解酶(E.C.3.2.1.23-E.C.3.2.1.62)。vbl.使用 免疫化学和细胞分离技术，标记模式 新合成的酶将在亚细胞组分中进行检测 (微粒体膜、高尔基体膜、微绒毛膜) 发育和成熟期间的时间。亚基组成和 将确定潜在的微生物体生物合成前体的组装 并用双向聚丙烯酰胺凝胶电泳法进行了表征 和荧光照相。微生物体和微生物体一级结构的同源性 这些酶的微绒毛形式将由溴化氰定义 切割；核心糖基化将通过直接标记实验进行研究 在没有和存在衣霉素的情况下，微粒体的治疗 内切-β-N-乙酰氨基葡萄糖苷酶-H前体的分析 释放出的糖分。糖脂中间体在糖脂代谢中的作用分析 蔗糖酶-异麦芽糖酶和乳糖酶-根皮苷的早期糖基化 将进行水解酶，以及多醇在此过程中的作用 调查过了。肠道组织中的二羟基乙醇含量将通过 高效液相色谱及其合成方法 标有醋酸盐和甲氧戊酸作为前体。二羟苯二醇键的作用 膜蛋白在胎儿和出生后发育过程中的糖基化 也将分析，如果可能，紧凑素对这一点的影响 过程已阐明。此外，胆固醇和糖的影响 将研究以杜仲醇为原料的合成方法。详细分析了 控制这些酶生物合成的早期细胞内步骤 不仅应该让人们了解他们的发育控制，还应该让他们了解 负责它们转运到肠道细胞的机制 浮出水面。此外，还阐明了控制细菌生长的机制 断奶后乳糖酶活性的下降可能会对人类 成人起病的乳糖不耐受。

The studies described in this application are designed to continue to elucidate the early events involved in the biosynthesis, processing and intracellular transport of intestinal glycoprotein enzymes destined for insertion into the microvillous membrane, and the factors controlling the ontogeny of these mechanisms. Two enzymes will be studied in detail and compared: sucrase-isomaltase (E.C. 3.2.1.48 - E.C. 3.2.1.10) and lactase-phlorizin hydrolase (E.C. 3.2.1.23 - E.C. 3.2.1.62). Using immunochemical and cell fractionation techniques, the labeling patterns of newly synthesized enzymes will be examined in subcellular fractions (microsomes, Golgi, microvillous membranes) of rat intestine at various times during development and maturation. The subunit composition and assembly of potential microsomal biosynthetic precursors will be identified and characterized using two dimensional polyacrylamide gel electrophoresis and fluorography. Homologies in primary structure between microsomal and microvillous forms of these enzymes will be defined by cyanogen bromide cleavage; core glycosylation will be studied by direct labeling experiments in the absence and presence of tunicamycin, and treatment of microsomal precursors with endo-Beta-N-acetylglucosaminidase-H, followed by analysis of sugars released. Analysis of the role of sugar-lipid intermediates in the early glycosylation of sucrase-isomaltase and lactase-phlorizin hydrolase will be undertaken, and thr role of dolichol in this process investigated. Dolichol content of intestinal tissue will be quantified by high performance liquid chromatography, and its synthesis delineated using labelled acetate and mevalonate as precursors. The role of dolichol-linked glycosylation of membrane proteins during fetal and post-natal development will also be analyzed, and if possible, the effects of compactin on this process elucidated. In addition, the impact of cholesterol and sugar feeding on dolichol synthesis will be studied. Detailed analysis of the early intracellular steps governing the biosynthesis of these enzymes should allow understanding not only of their developmental control but also the mechanisms responsible for their transport to the intestinal cell surface. In addition, elucidation of mechanisms controlling the post-weaning decline in lactase activity may yield insights into human adult-onset lactose intolerance.