Wang J F, Becks G P, Buckingham K D, Hill D J
Lawson Research Institute, St Joseph's Health Centre, London, Ontario, Canada.
J Endocrinol. 1990 Jun;125(3):439-48. doi: 10.1677/joe.0.1250439.
We have characterized the insulin-like growth factor-binding proteins (IGF-BPs) released by isolated sheep thyroid epithelial cells. Thyroid follicles were isolated with collagenase and cultured in Coon's modified F-12 M (0H medium) supplemented with insulin, cortisol, transferrin, glycyl-histidyl-lysine and somatostatin (5H medium) and TSH (6H medium). Conditioned 0H medium specifically bound both 125I-labelled IGF-I and -II, although binding capacity was reduced following acid-gel filtration to separate endogenous IGF-BP complexes, suggesting some destruction of BPs. The binding of 125I-labelled IGF-I or -II to conditioned (0H) medium was progressively displaced by increasing amounts of unlabelled homologous peptides, while fractionation on concanavalin A-Sepharose showed that the IGF-BPs consisted of both glycoprotein and non-glycoprotein components. The molecular sizes of the IGF-BPs were resolved by separation of 0H medium on SDS-PAGE and ligand blot analysis with 125I-labelled IGF-I or -II. Conditioned medium contained four specific binding species for IGF-II of 19, 30, 38 and 46 kDa; all but the smallest also binding radiolabelled IGF-I. Prior fractionation on concanavalin A-Sepharose showed that the 46 kDa binding species was a glycoprotein. Competition studies with increasing concentrations of unlabelled IGF-I or -II during ligand blotting suggested that the 46 and 30 kDa binding species had a greater affinity for IGF-II than IGF-I, while the 38 kDa had a greater relative affinity for IGF-I. Incubation of cells in 5H medium reduced the abundance of the 46 kDa binding protein, while incubation in 6H medium decreased the release of all binding protein species. Results show that isolated thyroid follicles released several forms of IGF-BP with differing relative affinities for IGF-I and -II. Gross changes seen in the presence of BPs between 0H, 5H and 6H media suggest acute hormonal control of release.
我们已经对分离出的绵羊甲状腺上皮细胞释放的胰岛素样生长因子结合蛋白（IGF - BPs）进行了特性分析。用胶原酶分离甲状腺滤泡，并在补充了胰岛素、皮质醇、转铁蛋白、甘氨酰 - 组氨酰 - 赖氨酸和生长抑素的Coon改良F - 12 M（0H培养基）以及促甲状腺激素（TSH）（6H培养基）中进行培养。条件性0H培养基能特异性结合125I标记的IGF - I和 - II，不过在进行酸凝胶过滤以分离内源性IGF - BP复合物后，结合能力有所降低，这表明部分结合蛋白被破坏。随着未标记同源肽量的增加，125I标记的IGF - I或 - II与条件性（0H）培养基的结合逐渐被取代，而在伴刀豆球蛋白A - 琼脂糖上进行分级分离显示，IGF - BPs由糖蛋白和非糖蛋白成分组成。通过在SDS - PAGE上分离0H培养基并用125I标记的IGF - I或 - II进行配体印迹分析，解析了IGF - BPs的分子大小。条件性培养基含有四种对IGF - II具有特异性结合的蛋白，分子量分别为19、30、38和46 kDa；除了最小的那种外，其他几种也能结合放射性标记的IGF - I。先前在伴刀豆球蛋白A - 琼脂糖上进行的分级分离显示，46 kDa的结合蛋白是一种糖蛋白。在配体印迹过程中用浓度递增的未标记IGF - I或 - II进行竞争研究表明，46 kDa和30 kDa的结合蛋白对IGF - II的亲和力比对IGF - I的亲和力更高，而38 kDa的结合蛋白对IGF - I具有更高的相对亲和力。在5H培养基中培养细胞会降低46 kDa结合蛋白的丰度，而在6H培养基中培养则会减少所有结合蛋白种类的释放。结果表明，分离出的甲状腺滤泡释放出几种对IGF - I和 - II具有不同相对亲和力的IGF - BP形式。在0H、5H和6H培养基中存在结合蛋白时所观察到的总体变化表明存在对释放的急性激素控制。
