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STUDIES OF THE CARBOHYDRATE MOIETIES OF A-L-FUCOSIDASE

A-L-岩藻糖苷酶碳水化合物部分的研究

基本信息

批准号：
3231936
负责人：
JACK A ALHADEFF
金额：
$ 11.01万
依托单位：
LEHIGH UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1984
资助国家：
美国
起止时间：
1984-09-30 至 1989-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3231936
关键词：
O glycosidase affinity chromatography analytical method carbohydrate structure fucose gel filtration chromatography glycopeptides high performance liquid chromatography human tissue ion exchange chromatography liver lysosomes nuclear magnetic resonance spectroscopy oligosaccharides proteolysis

项目摘要

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.

越来越清楚的是，碳水化合物部分