Shailubhai K, Saxena E S, Balapure A K, Vijay I K
Department of Animal Sciences, University of Maryland, College Park 20742.
J Biol Chem. 1990 Jun 15;265(17):9701-6.
Glucosidase I involved in the processing of N-linked glycoproteins was purified to homogeneity from the lactating rat mammary gland. The purified enzyme exhibited a single band at 85 kDa on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Polyclonal antibodies raised against the enzyme recognized a similar band on Western blots and also inhibited the enzyme activity. The enzyme levels gradually increased until the midlactation stage and thereafter declined sharply during the period of postlactation. A similar profile of the levels of immunoreactive glucosidase I was observed. These findings suggest that the accumulation of glucosidase I is modulated as a function of gland ontogeny. The results on hormonal regulation of glucosidase I indicate that the synthesis of the enzyme is stimulated by a combination of insulin, hydrocortisone, and prolactin; additionally, epidermal growth factor may play a role in this regulation. The above observation was substantiated by immunoprecipitation of [35S]methionine-labeled microsomal extracts with anti-glucosidase I antibodies. The immunoprecipitation of soluble extracts from [35S]methionine-labeled tissue with anti-rat alpha-lactalbumin antibodies indicates that these hormones not only stimulate the synthesis of alpha-lactalbumin but also play an important role in its glycosylation.
参与N-连接糖蛋白加工的葡糖苷酶I从泌乳大鼠乳腺中纯化至同质。在还原条件下，经10%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，纯化后的酶在85 kDa处呈现单一条带。针对该酶产生的多克隆抗体在蛋白质免疫印迹中识别出类似条带，并且还抑制了酶活性。酶水平在泌乳中期之前逐渐升高，此后在泌乳后期急剧下降。免疫反应性葡糖苷酶I水平呈现出类似的变化情况。这些发现表明，葡糖苷酶I的积累随着腺体个体发育而受到调节。关于葡糖苷酶I激素调节的结果表明，胰岛素、氢化可的松和催乳素共同刺激该酶的合成；此外，表皮生长因子可能在此调节过程中发挥作用。用抗葡糖苷酶I抗体对[35S]甲硫氨酸标记的微粒体提取物进行免疫沉淀，证实了上述观察结果。用抗大鼠α-乳白蛋白抗体对[35S]甲硫氨酸标记组织的可溶性提取物进行免疫沉淀表明，这些激素不仅刺激α-乳白蛋白的合成，而且在其糖基化过程中也发挥重要作用。
