CHEMISTRY AND METABOLISM OF SPHINGOLIPIDS & GLYCOPROTEIN

要求提供资金，继续执行关于《公约》的项目方案。 鞘糖脂的生物化学和一些参与的酶 他们的新陈代谢。 神经节苷脂生物合成调控的研究进展 代谢将集中在GM 3和GD 3的唾液酸转移酶上 生物合成，其催化唾液酸从CMP-唾液酸 酸分别形成α 2 -3和α 2 -8糖苷键。 这些 酶将从大鼠肝高尔基体囊泡中纯化， 色谱法，并测定其动力学特性。 的 将通过免疫化学方法检测它们的活性调节。 方法，重点是评估转录控制和 翻译后共价修饰。 Hakomori和他的同事提出质膜GM 3 神经节苷脂通过抑制神经细胞的生长来调节培养细胞的生长。 将深入研究对生长因子的促有丝分裂反应。 使用 用于周转研究和唾液酸分析的双标记GM 3， 培养基中的唾液酸酶，一种新的GM 3合成和代谢模型， 细胞周期以及稀疏和汇合培养物中的细胞周期将在实验上进行测定。 测试. 增加细胞外基质分泌的药物的作用 蛋白酶将被确定，和细胞外生长抑制剂， 将使用致密培养物的条件培养基来检验假设 质膜中GM 3转化为乳糖神经酰胺 在高密度培养物中被抑制。 人肝溶酶体α-半乳糖苷酶A的特性和 将继续使用α-N-乙酰氨基半乳糖苷酶（α-Gal B）。 针对α-Gal B的单特异性多克隆抗体将具有亲和力， 纯化并用于筛选人肝癌cDNA表达文库， Lambdagt 11. 对α-半乳糖B一级序列的了解将允许 实验设计以确定天冬酰胺连接的 这些酶上的碳水化合物链，并评估酶活性 微生物和病毒/哺乳动物表达中的翻译产物 系统. 一个有趣的新描述的α-半乳糖苷酶， 西瓜将被详细研究，因为它存在于单体中， 形成并在鞘糖脂的水解中具有催化活性 在法布里病中积累 将开发一种分析人血清的方法 鞘糖脂，并用于评估的存在和结构性质， 在各种肿瘤中释放鞘糖脂。

Funds are requested for the continuation of a program of projects on the biochemistry of glycosphingolipids and some of the enzymes involved in their metabolism. Research on the regulation of ganglioside biosynthesis and metabolism will be focused on the sialyltransferases of GM3 and GD3 biosynthesis, which catalyze the transfer of sialic acid from CMP-sialic acid to form Alpha2-3 and Alpha2-8 glycosidic bonds, respectively. These enzymes will be purified from rat liver Golgi vesicles by affinity chromatography and their kinetic characteristics determined. The regulation of their activities will be examined by an immunochemical approach, with an emphasis on an evaluation of transcriptional control and post-translational covalent modifications. The proposal by Hakomori and his co-workers that plasma membrane GM3 ganglioside modulates growth of cultured cells by inhibition of the mitogenic response to growth factors will be studied in depth. Using doubly labeled GM3 for turnover studies and analyses of sialic acid and sialidase in the medium, a new model of GM3 synthesis and metabolism during the cell cycle and in sparse and confluent cultures will be experimentally tested. The effects of agents that increase the secretion of extracellular proteases will be determined, and an extracellular inhibitor of growth from conditioned medium of dense cultures will be used to test the hypothesis that turnover of GM3 in plasma membrane to lactosylceramide will become inhibited in dense cultures. Characterization of human liver lysosomal Alpha-galactosidase A and Alpha-N-acetylgalactosaminidase (Alpha-Gal B) will be continued. Monospecific, polyclonal antibodies against Alpha-Gal B will be affinity purified and used to screen a human hepatoma cDNA expression library in Lambdagt11. Knowledge of the primary sequences of Alpha-Gal B will permit the design of experiments to determine the locations of asparagine-linked carbohydrate chains on these enzymes and to assess the enzymatic activities of translation products in microbial and viral/mammalian expression systems. An interesting newly described Alpha-galactosidase from watermelon will be studied in some detail because it exists in a monomeric form and has catalytic activity in the hydrolysis of the glycosphingolipid that accumulates in Fabry's disease. A method will be developed for the analysis of human serum glycosphingolipids and used to assess the presence and structural nature of shed glycosphingolipids in various neoplasias.