MEMBRANE TOPOLOGY AND BIOSYNTHESIS OF GLYCOSAMINOGLYCANS

膜拓扑结构和糖胺聚糖的生物合成

• 批准号: 3285297
• 负责人: CARLOS Benjamin HIRSCHBERG
• 金额: $ 20.15万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1989-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3285297
• 关键词: Golgi apparatus; affinity chromatography; affinity labeling; antibody; antibody formation; autoradiography; carbohydrate biosynthesis; cell adhesion; cell cell interaction; cell differentiation; complementary DNA; endoplasmic reticulum; gel electrophoresis; genetic library; hamsters; high performance liquid chromatography; laboratory rabbit; laboratory rat; liposomes; membrane activity; membrane permeability; membrane proteins; mitochondrial membrane; mucopolysaccharides; nucleic acid sequence; peptide chemical synthesis; protein biosynthesis; protein reconstitution; protein sequence; radiotracer; spectrometry; tissue /cell culture; transport proteins; uridine diphosphate glucuronate; xylose

The long term goals of this research proposal are (1) to understand the role of the membranes of the endoplasmic reticulum and Golgi apparatus (GA) in mechanisms of biosynthesis of glycosaminoglycans, (2) to establish whether the putative translocator protein for Adenosine 3'-phosphate 5' phosphosulfate (PAPS) located in the GA membrane is coded (at least partially) by DNA sequences in common with the ADP/ATP translocator of the mitochondrial membrane and (3) to understand the relationship between glycosaminoglycans to ascribed functions in vivo such as intercellular recognition, cell adhesion and morphogenesis. Towards achieving these goals the following specific aims will be pursued: Continuation with studies on the mechanism(s) of sulfation in the GA. Attempts will be made to identify, purify and reconstitute into liposomes the putative translocator for PAPS. For this purpose advantage will be taken of the apparent structural similarities between the putative PAPS translocator in the GA membrane and the ADP/ATP translocator in mitochondria; the approaches to be used have been successfully applied for similar aims with this latter translocator. Another aim will be the determination of whether the putative PAPS translocator of the GA membrane and the ADP/ATP translocator of mitochondria have common amino acid sequence regions. A series of studies in vitro are proposed to determine whether translocator activities for UDP-Glucuronic acid and UDP-Xylose can be detected in membranes of rat liver rough endoplasmic reticulum and GA; if successful, attempts will be made to identify, purify, and reconstitute the corresponding translocator proteins with liposomes. Concurrent to the above described studies in vitro on sulfation, attempts will be made to isolate mutant Chinese hamster ovary cells deficient in translocation of PAPS into GA in vivo. For this purpose, radiation suicide of cells with 35S-sulfate will be done, followed by screening of mutants via replica-plating autoradiography, and biochemical analyses.

该研究建议的长期目标是（1）了解 内质网和高尔基体（GA）的膜的作用 在糖胺聚糖的生物合成机制中，（2）建立 是否适用于3'-磷酸腺苷的易位蛋白5' 位于GA膜中的磷酸硫酸盐（PAPS）被编码（至少至少 部分）通过与ADP/ATP转运剂共同的DNA序列 线粒体膜和（3）了解 糖胺聚糖在体内归因于诸如细胞间的归因于赋予功能 识别，细胞粘附和形态发生。 要实现这些 目标将实现以下特定目标：继续 研究GA中硫酸化机制的研究。 将尝试 识别，净化并重新构成脂质体 PAPS的转运器。 为此目的的优势将对 推定的PAP转运器之间的明显结构相似性 线粒体中的GA膜和ADP/ATP转运剂；这 已成功应用的方法已成功应用于类似的目标 后一个转运器。 另一个目的是确定是否 GA膜和ADP/ATP的推定PAPS转换器 线粒体的转运剂具有常见的氨基酸序列区域。 一个 提出了一系列体外研究，以确定转运剂是否 在 大鼠肝粗糙内质网和GA的膜；如果成功， 将尝试识别，净化和重构 与脂质体的相应转运蛋白。 并发 以上描述的在体外研究的硫酸研究，将尝试 分离的突变体中国仓鼠卵巢细胞不足 paps进入体内GA。 为此，细胞的辐射自杀 将完成35s硫酸盐，然后通过 复制放射自显影和生化分析。