Kaplan F, Hechtman P
Biochem J. 1984 Jan 15;217(2):353-64. doi: 10.1042/bj2170353.
Two enzymes that catalyse the transfer of galactose from UDP-galactose to GM2 ganglioside were partially purified from rat liver Golgi membranes. These preparations, designated enzyme I (basic) and enzyme II (acidic), utilized as acceptors GM2 ganglioside and asialo GM2 ganglioside as well as ovalbumin, desialodegalactofetuin, desialodegalacto-orosomucoid, desialo bovine submaxillary mucin and GM2 oligosaccharide. Enzyme II catalysed disaccharide synthesis in the presence of the monosaccharide acceptors N-acetylglucosamine and N-acetylgalactosamine. The affinity adsorbent alpha-lactalbumin-agarose, which did not retard GM2 ganglioside galactosyltransferase, was used to remove most or all of galactosyltransferase activity towards glycoprotein and monosaccharide acceptors from the extracted Golgi preparation. After treatment of the extracted Golgi preparation with alpha-lactalbumin-agarose, enzyme I and enzyme II GM2 ganglioside galactosyltransferase activities, prepared by using DEAE-Sepharose chromatography, were distinguishable from transferase activity towards GM2 oligosaccharide and glycoproteins by the criterion of thermolability. This residual galactosyltransferase activity towards glycoprotein substrates was also shown to be distinct from GM2 ganglioside galactosyltransferase in both enzyme preparations I and II by the absence of competition between the two acceptor substrates. The two types of transferase activities could be further distinguished by their response to the presence of the protein effector alpha-lactalbumin. GM2 ganglioside galactosyltransferase was stimulated in the presence of alpha-lactalbumin, whereas the transferase activity towards desialodegalactofetuin was inhibited in the presence of this protein. The results of purification studies, comparison of thermolability properties and competition analysis suggested the presence of a minimum of five galactosyltransferase species in the Golgi extract. Five peaks of galactosyltransferase activity were resolved by isoelectric focusing. Two of these peaks (pI 8.6 and 6.3) catalysed transfer of galactose to GM2 ganglioside, and three peaks (pI 8.1, 6.8 and 6.3) catalysed transfer to glycoprotein acceptors.
从大鼠肝脏高尔基体膜中部分纯化出两种催化半乳糖从尿苷二磷酸半乳糖转移至GM2神经节苷脂的酶。这些制剂，分别命名为酶I（碱性）和酶II（酸性），以GM2神经节苷脂、脱唾液酸GM2神经节苷脂以及卵清蛋白、去唾液酸去半乳糖胎球蛋白、去唾液酸去半乳糖血清类黏蛋白、去唾液酸牛下颌黏蛋白和GM2寡糖作为受体。在单糖受体N - 乙酰葡糖胺和N - 乙酰半乳糖胺存在的情况下，酶II催化二糖合成。不阻滞GM2神经节苷脂半乳糖基转移酶的亲和吸附剂α - 乳白蛋白 - 琼脂糖，用于从提取的高尔基体制剂中去除大部分或全部针对糖蛋白和单糖受体的半乳糖基转移酶活性。在用α - 乳白蛋白 - 琼脂糖处理提取的高尔基体制剂后，通过DEAE - 琼脂糖层析制备的酶I和酶II的GM2神经节苷脂半乳糖基转移酶活性，根据热稳定性标准可与针对GM2寡糖和糖蛋白的转移酶活性区分开来。在两种酶制剂I和II中，针对糖蛋白底物的这种残留半乳糖基转移酶活性也通过两种受体底物之间不存在竞争而显示与GM2神经节苷脂半乳糖基转移酶不同。这两种转移酶活性可通过它们对蛋白质效应物α - 乳白蛋白存在的反应进一步区分。在α - 乳白蛋白存在的情况下，GM2神经节苷脂半乳糖基转移酶受到刺激，而针对去唾液酸去半乳糖胎球蛋白的转移酶活性在这种蛋白质存在时受到抑制。纯化研究结果、热稳定性特性比较和竞争分析表明，高尔基体提取物中至少存在五种半乳糖基转移酶。通过等电聚焦解析出五个半乳糖基转移酶活性峰。其中两个峰（pI 8.6和6.3）催化半乳糖转移至GM2神经节苷脂，三个峰（pI 8.1、6.8和6.3）催化转移至糖蛋白受体。
