小鼠肝脏α-L-岩藻糖苷酶的亚基、同工型及底物特异性分析

Analysis of the subunits, isoforms and substrate specificity of mouse liver alpha-L-fucosidase.

Shoarinejad F, Johnson S W, Alhadeff J A

Department of Chemistry, Lehigh University, Bethlehem, PA 18015.

Comp Biochem Physiol B. 1993 May;105(1):129-37. doi: 10.1016/0305-0491(93)90179-9.

Abstract

SDS-PAGE indicates the presence of two major protein bands (57 and 62 kDa) for mouse fucosidase and Western blotting indicates that both bands are immunoreactive with polyclonal antibodies (PAbs) and/or monoclonal antibodies (MAbs) raised against human liver fucosidase. The lectins SNA and GNA recognized both mouse protein bands, indicating that both subunits are glycosylated and contain sialic acid residues. 2. Polyacrylamide gel-isoelectric focusing (PAG-IEF) indicated that mouse liver fucosidase contains at least seven isoforms, with three isoforms above pI 6.0, which were not detected in human liver fucosidase. Blotting indicates that the PAbs recognized seven mouse fucosidase isoforms (pIs 3.6-6.8) whereas the four MAbs did not appear to recognize any of the mouse isoforms. 3. The subunit composition of the separated isoforms of mouse alpha-L-fucosidase was investigated by SDS-PAGE. One-to-two closely-spaced protein bands are found in each isoform with a trend of increasing relative amounts of the high-M(r) band in the more acidic isoforms relative to the more neutral isoforms. 4. Human and mouse liver alpha-L-fucosidases hydrolyze L-Fuc from oligosaccharides and glycolipids at comparable rates, with the exception of ganglioside Fuc-GMI which was hydrolyzed by human, but not by mouse, alpha-L-fucosidase.

摘要

SDS - 聚丙烯酰胺凝胶电泳表明小鼠岩藻糖苷酶存在两条主要蛋白带（57和62 kDa），蛋白质印迹法表明这两条带均与针对人肝岩藻糖苷酶产生的多克隆抗体（PAbs）和/或单克隆抗体（MAbs）发生免疫反应。凝集素SNA和GNA识别这两条小鼠蛋白带，表明两个亚基均被糖基化且含有唾液酸残基。2. 聚丙烯酰胺凝胶等电聚焦（PAG - IEF）表明小鼠肝岩藻糖苷酶至少含有七种同工型，其中三种同工型的等电点高于6.0，而在人肝岩藻糖苷酶中未检测到。印迹表明PAbs识别七种小鼠岩藻糖苷酶同工型（等电点3.6 - 6.8），而四种MAbs似乎未识别任何小鼠同工型。3. 通过SDS - 聚丙烯酰胺凝胶电泳研究了小鼠α - L - 岩藻糖苷酶分离同工型的亚基组成。在每种同工型中发现一到两条紧密间隔的蛋白带，且相对于中性更强的同工型，在酸性更强的同工型中高相对分子质量带的相对含量有增加趋势。4. 人和小鼠肝α - L - 岩藻糖苷酶以相当的速率从寡糖和糖脂中水解L - 岩藻糖，但神经节苷脂Fuc - GMI除外，它可被人α - L - 岩藻糖苷酶水解，而不能被小鼠α - L - 岩藻糖苷酶水解。