Myllylä R, Risteli L, Kivirikko K I
Eur J Biochem. 1976 Jan 2;61(1):59-67. doi: 10.1111/j.1432-1033.1976.tb09997.x.
A purification of over 2000-fold is reported for collagen glucosyltransferase from Triton X-100 extract of whole chick embryos and one of about 160-fold from similar extract of chick embryo cartilage. The addition of the detergent more than doubled the enzyme activity in the homogenates. The purified enzyme preparations from whole chick embryos showed one major band and two or three minor bands in polyacrylamide gel electrophoresis and were entirely free of collagen galactosyltransferase activity. The molecular weight of collagen glucosyltransferase from both sources was about 52000 -- 54000, as determined by gel filtration. In some enzyme preparations an additional form was observed, with an elution position corresponding to a molecular weight of about 130000. Manganese was the most effective metal co-factor for the purified enzyme, but partial replacement could be obtained with Co2+, Mg2+ and Ca2+, whereas no replacement was found with other metals. The activity of the purified enzyme was stimulated by the addition of dithiothreitol to the incubation system and inhibited by preincubation with p-mercuribenzoate. UDP-glucose or the collagen substrate partially protected the enzyme against p-mercuribenzoate inactivation in the presence of Mn2+ but not in its absence. Some protection was also noted with Mn2+ alone.
据报道，从全鸡胚的Triton X-100提取物中纯化胶原蛋白葡萄糖基转移酶可达到2000倍以上，从鸡胚软骨的类似提取物中纯化约160倍。去污剂的添加使匀浆中的酶活性增加了一倍多。从全鸡胚中纯化的酶制剂在聚丙烯酰胺凝胶电泳中显示出一条主要带和两三条次要带，并且完全没有胶原蛋白半乳糖基转移酶活性。通过凝胶过滤测定，来自两种来源的胶原蛋白葡萄糖基转移酶的分子量约为52000 - 54000。在一些酶制剂中观察到另一种形式，其洗脱位置对应于分子量约为130000。锰是纯化酶最有效的金属辅因子，但Co2+、Mg2+和Ca2+可部分替代，而其他金属则不能替代。向孵育系统中添加二硫苏糖醇可刺激纯化酶的活性，而与对巯基苯甲酸预孵育则可抑制其活性。在存在Mn2+的情况下，UDP-葡萄糖或胶原蛋白底物可部分保护酶免受对巯基苯甲酸的失活作用，但在不存在Mn2+的情况下则不能。单独使用Mn2+也有一定的保护作用。
