Godine J E, Chin W W, Habener J F
J Biol Chem. 1981 Mar 10;256(5):2475-9.
Polyadenylated RNA prepared from pituitary glands of ovariectomized rats was translated in heterologous cell-free systems derived from wheat germ and rabbit reticulocytes in the absence and in the presence of pancreatic microsomal membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the major 14,000-dalton product immunoprecipitated by antisera to the alpha subunit of rat and bovine luteinizing hormone from membrane-free translations was processed to a major 21,000-dalton and a minor 17,000-dalton product in the presence of microsomal membranes. These membrane-dependent products were sensitive to cleavage by the glycosidase endo-beta-N-acetylglucosaminidase H, but the 14,000-dalton product was resistant. The 21,000-dalton product was sequestered within the microsomal vesicles as shown by its resistance to limited proteolysis capable of digesting the 14,000-dalton precursor. Glycosylated products with apparent Mr = 18,000 and 17,000 were identified from membrane-supplemented translations by immunoprecipitation using antisera to the beta subunit of rat luteinizing hormone. The immunological identity of the glycosylated subunits of luteinizing hormone was confirmed by showing competition of the immunoprecipitation reactions with unlabeled subunits.
从去卵巢大鼠垂体中制备的聚腺苷酸化RNA，在有无胰腺微粒体膜存在的情况下，于源自小麦胚芽和兔网织红细胞的异源无细胞体系中进行翻译。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳表明，在无膜翻译中，由抗大鼠和牛促黄体激素α亚基的抗血清免疫沉淀的主要14,000道尔顿产物，在微粒体膜存在的情况下被加工成主要的21,000道尔顿产物和次要的17,000道尔顿产物。这些依赖膜的产物对糖苷酶内切β-N-乙酰葡糖胺酶H的切割敏感，但14,000道尔顿产物具有抗性。21,000道尔顿产物被隔离在微粒体小泡内，这可通过其对能够消化14,000道尔顿前体的有限蛋白水解的抗性来证明。通过使用抗大鼠促黄体激素β亚基的抗血清进行免疫沉淀，从补充膜的翻译中鉴定出表观分子量为18,000和17,000的糖基化产物。通过显示免疫沉淀反应与未标记亚基的竞争，证实了促黄体激素糖基化亚基的免疫同一性。
