Sarnesto A, Köhlin T, Hindsgaul O, Thurin J, Blaszczyk-Thurin M
Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104-4268.
J Biol Chem. 1992 Feb 5;267(4):2737-44.
The secretor-type beta-galactoside alpha 1----2-fucosyltransferase from human serum was purified by hydrophobic chromatography on phenyl-Sepharose, ion-exchange chromatography on sulfopropyl-Sepharose, and affinity chromatography on GDP-hexanolamine-Sepharose. Final purification of the enzyme was achieved by high pressure liquid chromatography gel filtration and resulted in a homogeneous protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the radiolabeled protein. The native enzyme appears as a molecule of apparent Mr 150,000 as determined by gel filtration high pressure liquid chromatography. The apparent Mr of the enzyme resolved in the presence of beta-mercaptoethanol by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was determined to be 50,000, indicating a multisubunit structure of the enzyme. Secretor-type alpha 1----2-fucosyltransferase is a glycoprotein as determined by WGA binding properties. A comparison of the Mr of the native blood group H gene encoded with the secretor-type beta-galactoside alpha 1----2-fucosyltransferases as well as comparison of subunit Mr for both enzymes suggests structural similarity. The alpha 1----2 linkage formed between alpha-L-fucose and terminal beta-D-galactose by the purified H- and secretor-type alpha 1----2-fucosyltransferases was determined by 1H NMR homonuclear cross-irradiation analysis of the oligosaccharide products. The substrate specificity and Km values calculated from the initial rate using various oligosaccharide acceptors showed that purified enzymes differ primarily in affinity for phenyl-beta-D-galactopyranoside and GDP-fucose as well as type 1 (Gal beta 1----3GlcNAc), 2 (Gal beta 1----4GlcNAc), and 3 (Gal beta 1----3GalNAc) oligosaccharide acceptors. The secretor-type alpha 1----2-fucosyltransferase shows significantly lower affinity than the H enzyme for phenyl-beta-D-galactopyranoside and GDP-fucose as well as for type 2 oligosaccharide acceptors. On the contrary, type 1 and 3 oligosaccharide acceptors are preferentially utilized by the secretor-type enzyme as compared with the H enzyme. The enzymes also differ in several physicochemical properties, implying nonidentity of the two enzymes (Sarnesto, A., Köhlin, T., Thurin, J., and Blaszczyk-Thurin, M. (1990) J. Biol. Chem. 265, 15067-15075).
人血清中的分泌型β-半乳糖苷α1----2-岩藻糖基转移酶通过在苯基-琼脂糖上进行疏水层析、在磺丙基-琼脂糖上进行离子交换层析以及在GDP-己醇胺-琼脂糖上进行亲和层析来纯化。通过高压液相色谱凝胶过滤实现了该酶的最终纯化，放射性标记蛋白经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定表明得到了一种均一的蛋白质。通过凝胶过滤高压液相色谱测定，天然酶呈现为表观相对分子质量为150,000的分子。在β-巯基乙醇存在下经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳解析得到的该酶的表观相对分子质量为50,000，表明该酶具有多亚基结构。根据WGA结合特性确定分泌型α1----2-岩藻糖基转移酶是一种糖蛋白。对血型H基因编码的天然蛋白与分泌型β-半乳糖苷α1----2-岩藻糖基转移酶的相对分子质量进行比较，以及对这两种酶的亚基相对分子质量进行比较，结果表明它们在结构上具有相似性。通过对寡糖产物进行1H NMR同核交叉照射分析，确定了纯化的H型和分泌型α1----2-岩藻糖基转移酶在α-L-岩藻糖与末端β-D-半乳糖之间形成的α1----2连接。使用各种寡糖受体根据初始速率计算得到的底物特异性和Km值表明，纯化后的酶主要在对苯基-β-D-吡喃半乳糖苷和GDP-岩藻糖以及1型（Galβ1----3GlcNAc）、2型（Galβ1----4GlcNAc）和3型（Galβ1----3GalNAc）寡糖受体的亲和力方面存在差异。分泌型α1----2-岩藻糖基转移酶对苯基-β-D-吡喃半乳糖苷、GDP-岩藻糖以及2型寡糖受体的亲和力明显低于H型酶。相反，与H型酶相比，分泌型酶优先利用1型和3型寡糖受体。这两种酶在一些物理化学性质上也存在差异，这意味着它们并非同一种酶（萨内斯托，A.，克林，T.，图林，J.，以及布拉斯奇克-图林，M.（1990年）《生物化学杂志》265卷，第15067 - 15075页）。
