Rao A K, Garver F, Mendicino J
Biochemistry. 1976 Nov 16;15(23):5001-9. doi: 10.1021/bi00668a009.
Galactosyltransferase (UDPgalactose:glycoprotein galactosyltransferase EC 2.4.1.22) was isolated from swine mesentary lymhromatography on Sepharose 4B colums containing covalently bound p-aminophenyl-beta-D-N-acetylglucosamine. The homogenous enzyme showed a single band on disc gel electrophoresis and had a specific activity of 35 nmol min-1 (mg of protein)-1 at 37 degrees C. A molecular weight of 57 000 was obtained by exclusion chromatography, sucrose density centrifugation, and sodium dodecyl sulfate-gel electrophoresis. The same molecular weight was obtained after reduction and alkylation which indicates that the enzyme is composed of only a single polypeptide chain. The enzyme catalyzed the formation of beta1 leads to 4 bonds between galactose and free terminal N-acetylglucosaminyl residues of soluble preparations of porcine IgG immunoglobulin heavy chain, fetuin, ovalbumin, and ovomucoid. An endogenous glycoprotein, present in particulate subcellular preparations, was also a very good substrate for the enzyme, and it was identified as incomplete IgG immunoglobulin heavy chain. The Km of the purified enzyme was 2.9 x 10(-5) M for fetuin, 5.4 x 10(-5) M for ovalbumin, 2.0 x 10(-5) M for IgG immlnoglobulin heavy chain, and 2.2 x 10(-5) M for UDP-galactose. About 20% of the total galactosyltransferase activity in lymph node homogenates was present in the cytosol fraction, and 80% was in the microsomal and Golgi fractions. The kinetic properties of the bound and soluble galactosyltransferases were similar,and both required Mn2+ for maximal activity. However, the bound enzyme required the addition of detergent, lysolecithin, GDP-mannose, and UDP-N-acetylglucosamine for maximum activity. These compounds did not influence the activity of the soluble transferase. The membrane preparations catalyzed the transfer of galactose from UDP-galactose and N-acetylglycosamine from UDP-N-acetylglucosamine to incomplete oligosaccharide chains of endogenous IgG immunoglobulin bound to these particles. The labeled products of these reactions were isolated, and the structures of their oligosaccharide chains were determined and compared with those isolated from the heavy chain of porcine IgG immunoglobulin. The glycopeptide prepared from the endogenous acceptor and the major glycopeptide prepared by proteolytic digestion of the heavy chain of porcine IgG immunoglobulin has identical structures. The following structure for the carbohydrate chains of porcine IgG immunoglobulin was determined by sequential enzymatic hydrolysis and methylation studies.
半乳糖基转移酶（UDP - 半乳糖：糖蛋白半乳糖基转移酶，EC 2.4.1.22）是从猪肠系膜中分离出来的。通过在含有共价结合的对氨基苯基 - β - D - N - 乙酰葡糖胺的琼脂糖4B柱上进行淋巴色谱法分离。该纯酶在圆盘凝胶电泳上显示出单一谱带，在37℃时的比活性为35 nmol·min⁻¹（mg蛋白质）⁻¹。通过排阻色谱法、蔗糖密度离心法和十二烷基硫酸钠 - 凝胶电泳法测得其分子量为57000。还原和烷基化后得到相同的分子量，这表明该酶仅由一条多肽链组成。该酶催化半乳糖与猪IgG免疫球蛋白重链、胎球蛋白、卵清蛋白和卵类粘蛋白可溶性制剂的游离末端N - 乙酰葡糖胺残基之间形成β1→4键。存在于颗粒状亚细胞制剂中的一种内源性糖蛋白也是该酶的良好底物，它被鉴定为不完整的IgG免疫球蛋白重链。纯化酶对胎球蛋白的Km为2.9×10⁻⁵M，对卵清蛋白为5.4×10⁻⁵M，对IgG免疫球蛋白重链为2.0×10⁻⁵M，对UDP - 半乳糖为2.2×10⁻⁵M。淋巴结匀浆中约20%的总半乳糖基转移酶活性存在于胞质溶胶部分，80%存在于微粒体和高尔基体部分。结合型和可溶性半乳糖基转移酶的动力学性质相似，两者都需要Mn²⁺才能达到最大活性。然而，结合型酶需要添加去污剂、溶血卵磷脂、GDP - 甘露糖和UDP - N - 乙酰葡糖胺才能达到最大活性。这些化合物不影响可溶性转移酶的活性。膜制剂催化UDP - 半乳糖中的半乳糖和UDP - N - 乙酰葡糖胺中的N - 乙酰葡糖胺转移到与这些颗粒结合的内源性IgG免疫球蛋白的不完整寡糖链上。分离出这些反应的标记产物，测定其寡糖链的结构，并与从猪IgG免疫球蛋白重链中分离出的结构进行比较。由内源性受体制备的糖肽和通过猪IgG免疫球蛋白重链的蛋白水解消化制备的主要糖肽具有相同的结构。通过顺序酶促水解和甲基化研究确定了猪IgG免疫球蛋白碳水化合物链的以下结构。
