Bachrach L K, Liu F R, Burrow G N, Eggo M C
Department of Pediatrics, Stanford University Medical Center, California 94305.
Endocrinology. 1989 Dec;125(6):2831-8. doi: 10.1210/endo-125-6-2831.
The insulin-like growth factors (IGFs) are bound by specific, high affinity binding proteins. Distinct classes of IGF-binding proteins have been described in human serum, amniotic fluid, cerebrospinal fluid, and conditioned medium from cultured cells. Sheep thyroid cells produce IGF-binding proteins under hormonal regulation. Cells grown without or with standard medium supplements (transferrin, glycyl-histidyl-lysine, hydrocortisone, somatostatin, insulin, and TSH) released binding proteins with apparent mol wt of 23, 29, and 32 kDa on Western ligand blot (nonreduced). Binding proteins from these cells appeared as 21, 26, 34, 36, and 41 kDa bands when cross-linked to [125I]IGF-I under reducing conditions. The addition of epidermal growth factor (EGF) or phorbol esters, thyroid cell mitogens stimulated the production of larger binding proteins with mol wt of 40-44 and 48-52 by ligand blot and cross-linking methods, respectively. Deglycosylation of conditioned medium cross-linked to [125I]IGF-I with endoglycosidase-F did not alter the size of the smaller binding proteins, but reduced EGF-stimulated binding proteins to 36-40 kDa. Similarly, tunicamycin treatment, which inhibits glycosylation, reduced only the size of this larger binding protein species. Polyclonal antisera directed against the human amniotic fluid binding protein (BP-28) immunoprecipitated the 32 kDa sheep thyroid binding protein seen on ligand blot and the cross-linked binding protein at 36-38 kDa. Antibody against the major human serum binding protein (BP-53) recognized only the larger EGF-stimulated binding proteins. In contrast to sheep thyroid cells, rat FRTL5 thyroid cells produced no detectable IGF-binding proteins. We conclude that the predominant binding proteins produced by sheep thyroid cells under standard culture conditions are non-glycosylated and immunoreact with antiserum directed against BP-28. EGF and phorbol esters stimulate production of larger glycosylated binding proteins antigenically related to BP-53.
胰岛素样生长因子（IGFs）与特异性高亲和力结合蛋白相结合。在人血清、羊水、脑脊液以及培养细胞的条件培养基中已发现不同种类的IGF结合蛋白。绵羊甲状腺细胞在激素调节下产生IGF结合蛋白。在无标准培养基补充物（转铁蛋白、甘氨酰 - 组氨酰 - 赖氨酸、氢化可的松、生长抑素、胰岛素和促甲状腺激素）或有标准培养基补充物的情况下培养的细胞，在Western配体印迹法（非还原）中释放出表观分子量为23、29和32 kDa的结合蛋白。在还原条件下与[125I]IGF - I交联时，这些细胞的结合蛋白表现为21、26、34、36和41 kDa的条带。添加表皮生长因子（EGF）或佛波酯（甲状腺细胞有丝分裂原）分别通过配体印迹法和交联法刺激产生分子量为40 - 44 kDa和48 - 52 kDa的更大结合蛋白。用内切糖苷酶 - F对与[125I]IGF - I交联的条件培养基进行去糖基化处理，不会改变较小结合蛋白的大小，但会将EGF刺激的结合蛋白减少到36 - 40 kDa。同样，抑制糖基化的衣霉素处理仅减小了这种较大结合蛋白种类的大小。针对人羊水结合蛋白（BP - 28）的多克隆抗血清免疫沉淀了在配体印迹中看到的32 kDa绵羊甲状腺结合蛋白以及交联后36 - 38 kDa的结合蛋白。针对主要人血清结合蛋白（BP - 53）的抗体仅识别较大的EGF刺激的结合蛋白。与绵羊甲状腺细胞不同，大鼠FRTL5甲状腺细胞未产生可检测到的IGF结合蛋白。我们得出结论，在标准培养条件下，绵羊甲状腺细胞产生的主要结合蛋白是非糖基化的，并且与针对BP - 28的抗血清发生免疫反应。EGF和佛波酯刺激产生与BP - 53抗原相关的更大糖基化结合蛋白。
