Poretz R D, Yang R S, Canas B, Lackland H, Stein S, Manowitz P
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08855.
Biochem J. 1992 Nov 1;287 ( Pt 3)(Pt 3):979-83. doi: 10.1042/bj2870979.
Human platelet arylsulphatase A (ASA) exhibits a multiple banding pattern when examined by PAGE. The isoform pattern (IVa) of the enzyme obtained from normal subjects differs from variants (IIIa, IIIb, IVb) which are primarily found in alcoholic patients. Alkaline phosphatase and endo-N-acetylglucosaminidase H treatments, as well as ion-exchange chromatography, demonstrate that the isoforms of ASA arise because of charge heterogeneity caused by phosphoglycan moieties. The isoform with the slowest mobility in PAGE lacks phosphate substituents. Based upon mannose-6-phosphate-receptor affinity chromatography it can be concluded that most, if not all, of the enzyme-linked phosphate is in the form of 6-O-phospho-D-mannosyl units. Lectin affinity chromatography and peptide-N-glycosidase F treatment followed by SDS/PAGE and Western-blot analysis indicate that normal platelet ASA contains two oligomannose and/or hybrid glycan moieties of which one, or both, possess a 6-O-L-fucosyl substituent on the asparagine-linked N-acetylglucosamine residue. Comparative analysis indicates that the variant IIIa and IIIb types of ASA differ from the IVa ASA with regard to the level of glycan phosphorylation and glycan structure, as well as the polypeptide structure.
人血小板芳基硫酸酯酶A（ASA）经聚丙烯酰胺凝胶电泳（PAGE）检测时呈现多条带模式。从正常受试者获得的该酶同工型模式（IVa）与主要在酒精性患者中发现的变体（IIIa、IIIb、IVb）不同。碱性磷酸酶和内切N - 乙酰葡糖胺酶H处理以及离子交换色谱表明，ASA同工型的产生是由于磷酸聚糖部分引起的电荷异质性。在PAGE中迁移率最慢的同工型缺乏磷酸取代基。基于甘露糖 - 6 - 磷酸受体亲和色谱可以得出结论，大部分（如果不是全部）酶连接的磷酸是以6 - O - 磷酸 - D - 甘露糖基单元的形式存在。凝集素亲和色谱和肽 - N - 糖苷酶F处理，随后进行SDS / PAGE和蛋白质印迹分析表明，正常血小板ASA含有两个寡甘露糖和/或杂合聚糖部分，其中一个或两个在天冬酰胺连接的N - 乙酰葡糖胺残基上具有6 - O - L - 岩藻糖基取代基。比较分析表明，ASA的变体IIIa和IIIb类型在聚糖磷酸化水平、聚糖结构以及多肽结构方面与IVa ASA不同。
