Johnson S W, Piesecki S, Wang R F, Damjanov I, Alhadeff J A
Department of Chemistry, Lehigh University, Bethlehem, PA 18015.
Biochem J. 1992 Mar 15;282 ( Pt 3)(Pt 3):829-34. doi: 10.1042/bj2820829.
Western-blot analysis [with lectins, polyclonal antibodies (pAbs) and four monoclonal antibodies (mAbs)] was employed to investigate the structural relationship between the separated isoforms and subunits of purified human liver alpha-L-fucosidase. SDS/PAGE and Western-blot analysis indicated the presence of two protein bands of 51 kDa and 56 kDa that were recognized by the pAbs. Polyacrylamide-gel isoelectric focusing (PAG-IEF) followed by blotting indicated that the pAbs and mAbs recognized at least five fucosidase isoforms (pI values 3.6-6.0). Lectin blotting indicated an enrichment of sialic acid residues in the more acidic isoforms. Western-blot analysis indicated that four mAbs recognized the 51 kDa subunit and at least two mAbs recognized the 56 kDa subunit. The subunit composition of the isoforms (separated by PAG-IEF) of human liver alpha-L-fucosidase was investigated by SDS/PAGE. One or two closely spaced bands were found for each isoform with a trend of increasing relative amounts of the high-molecular-mass band in the more acidic isoforms relative to the more neutral isoforms. Neuraminidase treatment of alpha-L-fucosidase resulted in a decrease in the amount of the high-molecular-mass subunit and an increase in the amount of the low-molecular-mass subunit, suggesting that these subunits are related at least in part by sialic acid residues. In addition, blotting with lectins indicated the presence of sialic acid residues only in the high-molecular-mass subunit. N-Glycanase treatment led to the disappearance of the glycosylated 56 kDa and 51 kDa protein bands and the appearance of non-glycosylated protein bands at 48 kDa and 45 kDa. The overall results indicate that (1) N-glycosylation contributes to, but does not account completely for, structural differences in the fucosidase subunits and (2) the more acidic isoforms of fucosidase contain enriched relative amounts of the sialylated high-molecular-mass subunit.
采用蛋白质免疫印迹分析（使用凝集素、多克隆抗体（pAbs）和四种单克隆抗体（mAbs））来研究纯化的人肝α-L-岩藻糖苷酶分离的同工型和亚基之间的结构关系。十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（SDS/PAGE）和蛋白质免疫印迹分析表明存在两条分别为51 kDa和56 kDa的蛋白条带，它们可被多克隆抗体识别。聚丙烯酰胺凝胶等电聚焦（PAG-IEF）后进行印迹分析表明，多克隆抗体和单克隆抗体识别至少五种岩藻糖苷酶同工型（等电点值为3.6 - 6.0）。凝集素印迹表明在酸性更强的同工型中唾液酸残基富集。蛋白质免疫印迹分析表明，四种单克隆抗体识别51 kDa亚基，至少两种单克隆抗体识别56 kDa亚基。通过SDS/PAGE研究了人肝α-L-岩藻糖苷酶同工型（通过PAG-IEF分离）的亚基组成。每种同工型发现有一条或两条紧密相邻的条带，且酸性更强的同工型相对于中性更强的同工型，高分子质量条带的相对含量有增加的趋势。用神经氨酸酶处理α-L-岩藻糖苷酶导致高分子质量亚基的量减少，低分子质量亚基的量增加，这表明这些亚基至少部分通过唾液酸残基相关联。此外，用凝集素进行印迹表明仅在高分子质量亚基中存在唾液酸残基。N-糖苷酶处理导致糖基化的56 kDa和51 kDa蛋白条带消失，在48 kDa和45 kDa出现非糖基化蛋白条带。总体结果表明：（1）N-糖基化对岩藻糖苷酶亚基的结构差异有贡献，但不能完全解释这种差异；（2）酸性更强的岩藻糖苷酶同工型含有相对富集的唾液酸化高分子质量亚基。
