Glinoer D, Gershengorn M C, Robbins J
Biochim Biophys Acta. 1976 Jan 19;418(2):232-44. doi: 10.1016/0005-2787(76)90073-3.
Thyroxine-binding globulin biosynthesis was demonstrated in hepatocytes isolated from normal adult Rhesus monkeys. Dispersed cells were obtained by in situ liver perfusion with collagenase, hyaluronidase and EDTA. Conditions for optimum cell survival and incorporation of radioactive leucine into newly synthesized proteins were defined. Protein synthesis, and specifically thyroxine-binding globulin synthesis, were shown to continue throughout the incubation period, while cell survival remained high (75% excluded trypan blue after 6h). Incubation medium, cytosol and a particulate fraction (extracted with digitonin) were analyzed for thyroxine-binding globulin. After extensive dialysis and purification by affinity chromatography, newly synthesized thyroxine-binding globulin was identified by specific double-antibody immunoprecipitation and by immunodiffusion and immunoelectrophoresis with autoradiography. Newly synthesized thyroxine-binding globulin was present after 4 h of incubation. After 6 h, the total synthesized had increased to 150% of the 4 h value, while the fraction present in the medium and increased to 300%, indicating probable thyroxine-binding globulin secretion
在从正常成年恒河猴分离出的肝细胞中证实了甲状腺素结合球蛋白的生物合成。通过用胶原酶、透明质酸酶和乙二胺四乙酸原位肝脏灌注获得分散的细胞。确定了细胞最佳存活以及放射性亮氨酸掺入新合成蛋白质的条件。结果表明，在整个孵育期间蛋白质合成持续进行，特别是甲状腺素结合球蛋白的合成，而细胞存活率仍很高（6小时后75%的细胞排斥台盼蓝）。对孵育培养基、胞质溶胶和颗粒部分（用洋地黄皂苷提取）进行甲状腺素结合球蛋白分析。经过广泛透析和亲和层析纯化后，通过特异性双抗体免疫沉淀以及放射自显影的免疫扩散和免疫电泳鉴定新合成的甲状腺素结合球蛋白。孵育4小时后出现新合成的甲状腺素结合球蛋白。6小时后，总合成量增加到4小时值的150%，而培养基中存在的部分增加到300%，表明可能有甲状腺素结合球蛋白分泌
