STUDIES OF THE CARBOHYDRATE MOIETIES OF A-L-FUCOSIDASE

越来越清楚的是， 糖蛋白具有结构和功能意义。 不过小 对于绝大多数哺乳动物来说， 包括溶酶体糖基化酶在内的糖蛋白。 的总目标 建议的研究是确定糖肽的组成， 模型人溶酶体糖苷酶，α-L-岩藻糖苷酶，并确定 这种酶的某些特性是否（以及在多大程度上）受到影响 由于碳水化合物残留物的存在。 α-L-岩藻糖苷酶已被 之所以选择它进行研究，主要是因为它的碳水化合物组成是已知的， 它由多种等电形式组成，至少部分与 唾液酸残留物，纯化程序简单 大量的这种酶。 将纯化α-L-岩藻糖苷酶， 通过简便、高产的亲和性从人肝脏中获得明显的同质性 色谱法， 琼脂糖-表氨基己酰基岩藻糖胺。 糖肽和/或 将通过以下步骤从纯化的α-L-岩藻糖苷酶制备低聚糖： 化学和/或酶方法，并通过几种技术进行分级 包括凝胶过滤、离子交换和凝集素-琼脂糖柱， 高效液相色谱法 一旦糖肽和/或 低聚糖已纯化至表观均一性，其氨基 酸和碳水化合物的组成将由高性能 液相色谱和/或自动氨基酸分析和气液色谱 层析 每种糖肽和/或 将通过500 MHz H-NMR结合 几种常规方法包括Smith降解、甲基化 分析、肼解-亚硝酸脱氨和酶降解 使用纯化的外切糖苷酶。 在化学（例如，高碘酸盐，HF， 肼）和/或酶（例如，外切和内切糖苷酶）方法具有 已经研究出使α-L-岩藻糖苷酶去糖基化而不改变其 蛋白质骨架，碳水化合物的功能意义， 酶将在体外进行研究，通过比较表征 某些性质（动力学、水溶性、对 蛋白水解）。 拟议 研究将提供结构和功能信息， 重要的溶酶体糖苷酶的碳水化合物部分， 最终应该证明有助于更好地了解这些酶， 正常和病理状态。

It is becoming increasingly clear that the carbohydrate moieties of glycoproteins have structural and functional significance. However, little is known about this significance for the vast majority of mammalian glycoproteins including the lysosomal glycosidases. The overall goals of the proposed research are to determine the glycopeptide composition of a model human lysosomal glycosidase, Alpha-L-fucosidase, and to determine whether (and to what extent) certain properties of this enzyme are affected by the presence of carbohydrate residues. Alpha-L-Fucosidase has been chosen for study primarily because its carbohydrate composition is known, it consists of multiple isoelectric forms related at least in part by sialic acid residues and a simple procedure is available for purifying large amounts of this enzyme. Alpha-L-Fucosidase will be purified to apparent homogeneity from human liver by a facile, high-yield affinity chromatographic procedure utilizing agarose-Epsilon-aminocaproylfucosamine. Glycopeptides and/or oligosaccharides will be prepared from the purified Alpha-L-fucosidase by chemical and/or enzymatic methods and fractionated by several techniques including gel filtration, ion exchange and lectin-sepharose columns and high performance liquid chromatography. Once the glycopeptides and/or oligosaccharides have been purified to apparent homogeneity, their amino acid and carbohydrate compositions will be determined by high performance liquid chromatography and/or automated amino acid analysis and gas liquid chromatography. Structural analysis of each glycopeptide and/or oligosaccharide will be accomplished by 500 MHz H-NMR in conjunction with several conventional procedures including Smith degradation, methylation analysis, hydrazinolysis-nitrous acid deamination and enzyme degradation using purified exoglycosidases. After chemical (e.g., periodate, HF, hydrazine) and/or enzymatic (e.g., exo- and endoglycosidases) methods have been worked out to deglycosylate Alpha-L-fucosidase without altering its protein backbone, the functional significance of carbohydrate on this enzyme will be investigated in vitro by comparatively characterizing certain properties (kinetics, aqueous solubility, susceptibility to proteolysis) of the normal and deglycosylated enzyme. The proposed research will provide structural and functional information on the carbohydrate portion(s) of an important lysosomal glycosidase which eventually should prove useful for better understanding of these enzymes in normal and pathological conditions.