Budzik G P, Swann D A, Hayashi A, Donahoe P K
Cell. 1980 Oct;21(3):909-15. doi: 10.1016/0092-8674(80)90454-7.
Mullerian inhibiting substance (MIS), a secreted testicular product responsible for regression of the Mullerian ducts in the male mammalian embryo, was purified 7000 fold, exploiting the glycoprotein nature of this important fetal regressor to achieve enhanced purification. The present procedure employs media incubation of newborn calf testis, passage through DEAE Bio-Gel A and CM Bio-Gel A and sequential lectin affinity chromatography on wheat germ lectin (WGL)-Sepharose 6MB and concanavalin A (Con A)-Sepharose 4B. Strongly bioactive MIS was released from both lectin columns in the bound glycoprotein fraction only after elution with lectin-specific sugar. Carbohydrate analysis of the highly purified glycoprotein fraction eluted from Con A indicated the presence of both N-acetyl glucosamine and mannose, as would be expected from its sequential lectin affinity, as well as of galactose, galactosamine and N-acetyl neuraminic acid. Electrophoresis of this fraction on polyacrylamide-SDS gels showed an identical band pattern after staining with either Coomassie blue or periodic acid-Schiff reagent, further indicating that MIS is a glycoprotein.
苗勒氏管抑制物质（MIS）是一种由睾丸分泌的产物，负责雄性哺乳动物胚胎中苗勒氏管的退化。利用这种重要的胎儿退化因子的糖蛋白性质，对其进行了7000倍的纯化，以实现更高程度的提纯。目前的方法包括对新生小牛睾丸进行培养基孵育，通过DEAE Bio-Gel A和CM Bio-Gel A进行分离，并依次在麦胚凝集素（WGL）-琼脂糖6MB和伴刀豆球蛋白A（Con A）-琼脂糖4B上进行凝集素亲和层析。只有在用凝集素特异性糖洗脱后，两种凝集素柱上结合的糖蛋白部分才释放出具有强生物活性的MIS。从Con A柱上洗脱的高度纯化的糖蛋白部分的碳水化合物分析表明，存在N-乙酰葡糖胺和甘露糖，这与其依次进行的凝集素亲和层析结果相符，同时还存在半乳糖、半乳糖胺和N-乙酰神经氨酸。该部分在聚丙烯酰胺-SDS凝胶上进行电泳，用考马斯亮蓝或过碘酸-希夫试剂染色后显示出相同的条带模式，进一步表明MIS是一种糖蛋白。
