REGULATION OF CARBOHYDRATE DIGESTION

碳水化合物消化的调节

• 批准号: 3224753
• 负责人: GARY M. GRAY
• 金额: $ 1.58万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-05-01 至 1991-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3224753
• 关键词: beta fructofuranosidase; beta galactosidase; brush border membrane; carbohydrate metabolism; castanospermine; dietary carbohydrates; digestion; endoplasmic reticulum; enzyme mechanism; enzyme model; gastrointestinal nutrient absorption; glycoproteins; human tissue; hybrid enzyme; immunochemistry; inborn carbohydrate metabolism disorder; inborn metabolism disorder diagnosis; intestinal villi; laboratory rat; malabsorption; membrane activity; membrane structure; molecular pathology; nutrition related tag; postmortem; protein biosynthesis; puromycin; radioimmunoassay; radiotracer; sucrose; tritium; tunicamycin; ultracentrifugation

Carbohydrate digestion at the small intestinal surface depends upon maintenance of appropriate levels of constituent oligosaccharides at the brush border membrane and information on the intracellular synthesis, assembly and transfer of these glycoprotein hydrolases in intact mammalian intestine under physiological conditions is beginning to emerge. The rat model will be used to examine the mechanism of induction of sucrase-Alpha-dextrinase synthesis at the molecular level. Paired intestinal segments from animals fed a carbohydrate-free diet for 7 days will be perfused with either sucrose plus saline or saline alone and then given a 5 minute intraintestinal pulse of 35S-methionine followed by a non-radioactive methionine chase of 15-180 minutes. Subcellular organelles (Er, Golgi, laterobasal membrane, brush border membrane) will be isolated and their proteins solubilized and sucrase-Alpha-dextrinase specifically immunoprecipitated. Newly synthesized sucrase-dextrinase will be quantified by scintillation counting and the molecular species analyzed by SDS-electrophoresis-autoradiography. In addition to sucrose, glucose plus fructose, lactulose and acarbose, a sucrase inhibitor, will be studied as putative inducers of de novo synthesis; the role of insulin, VIP, glucagon and secretin given intravenously with or without glucose will also be assessed. After the qualitative and quantitative effects on induction of synthesis, assembly and membrane insertion have been established, mRNA specific for sucrase-Alpha-dextrinase will be isolated from polysomes whose sucrase-dextrinase (S-D) nascent chains bind specifically to anti-S-D. The S-D mRNA will be translated in the rabbit reticulocyte system and will be used as a template for 32P-labeled S-D cDNA in the presence of reverse transcriptase. The 32P-cDNA probe will then be used in hybridization assays to estimate both mRNA and specific S-D DNA in normal rats and those whose sucrase-dextrinase has been induced by carbohydrate. The regulation of sucrase-dextrinase assembly will also be examined in the HT29 and CACO-2 colon cancer cell lines that synthesize sucrase-dextrinase under conditions favoring differentiation. Experiments analogous to those in the intact rat will be carried out, and, in particular, the role of glycosylation in the ER and Golgi on synthesis and assembly will be examined by the use of inhibitors of glycosylation such as tunicamycin, castanospermine and swainsonine. In the aggregate, these experiments should provide new information at the molecular level on the regulation of sucrase-dextrinase assembly by luminal carbohydrate.

小肠表面的碳水化合物消化取决于 维持适当水平的组成低聚糖， 刷状缘膜和细胞内合成的信息， 这些糖蛋白水解酶在完整哺乳动物中的组装和转移 肠道在生理条件下开始出现。 大鼠 模型将被用来研究诱导的机制， 蔗糖酶-α-糊精酶在分子水平上的合成。 配对 来自喂食无碳水化合物饮食7天的动物的肠段 将用蔗糖加盐水或仅用盐水灌注，然后 给予5分钟的35 S-蛋氨酸肠内脉冲，随后给予 非放射性蛋氨酸追踪15-180分钟。 亚细胞器 (Er、高尔基体、侧基底膜、刷状缘膜）将被分离 其蛋白质溶解，蔗糖酶-α-糊精酶特异性 免疫沉淀。 新合成的蔗糖酶-糊精酶， 通过闪烁计数进行定量，并通过 SDS-电泳-放射自显影。 除了蔗糖，葡萄糖加 果糖，乳果糖和阿卡波糖，一种蔗糖酶抑制剂，将作为 推定的从头合成诱导剂;胰岛素、VIP、胰高血糖素的作用 和分泌素静脉注射与或不与葡萄糖也将 评估。 在对诱导的定性和定量影响之后， mRNA的合成、组装和膜插入已经建立， 蔗糖酶-α-糊精酶特异性将从多聚核糖体中分离， 蔗糖酶-糊精酶（S-D）新生链与抗S-D特异性结合。 的 S-D mRNA将在兔网织红细胞系统中翻译， 作为模板，用于在存在反向引物的情况下32 P标记的S-D cDNA。 录酶 然后将32 P-cDNA探针用于杂交 检测正常大鼠和正常大鼠的mRNA和特异性S-D DNA， 其蔗糖酶-糊精酶已被碳水化合物诱导。 蔗糖酶-糊精酶组装的调节也将在 合成蔗糖酶-糊精酶的HT 29和CACO-2结肠癌细胞系 在有利于分化的条件下。 类似的实验 在完整的大鼠将进行，特别是，作用 糖基化在内质网和高尔基体的合成和组装将是 通过使用糖基化抑制剂如衣霉素进行检测， 栗精胺和苦马豆素。 总的来说，这些实验 应该在分子水平上提供新的信息， 蔗糖酶-糊精酶组装。