MEMBRANE TOPOLOGY AND BIOSYNTHESIS OF GLYCOSAMINOGLYCANS

本研究的长期目标是：（1）了解 内质网和高尔基体（GA）膜的作用 在糖胺聚糖的生物合成机制中，（2）建立 是否假定的腺苷3 '-磷酸5'转运蛋白 位于GA膜上的磷酸硫酸盐（PAPS）被编码（至少 部分）通过与ADP/ATP转运蛋白共同的DNA序列， 线粒体膜和（3）了解之间的关系 糖胺聚糖在体内的功能，如细胞间 识别、细胞粘附和形态发生。 为实现这些 将努力实现以下具体目标： GA硫酸化机理的研究。 将尝试 鉴定、纯化和重组脂质体， PAPS的转运子。 为此，将利用 PAPS转运蛋白与 GA膜和线粒体中的ADP/ATP转运体; 所使用的方法已成功地应用于类似的目的， 这后一个易位。 另一个目标将是确定是否 GA膜和ADP/ATP的假定PAPS转运蛋白 线粒体的转运蛋白具有共同的氨基酸序列区域。 一 一系列的体外研究被提出，以确定是否转运蛋白 UDP-葡糖醛酸和UDP-木糖的活性可以在 大鼠肝粗面内质网和GA的膜;如果成功， 将尝试识别，纯化和重组 相应的转运蛋白与脂质体。 同时， 在上述关于硫酸化的体外研究中，将尝试 易位缺陷分离突变中国仓鼠卵巢细胞 体内PAPS转化为GA。 为此目的，用放射性自杀细胞， 将进行35S-硫酸盐，然后通过 复制平板放射自显影和生化分析。

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.