The E-domain peptide of rat pro-insulin-like growth factor II (proIGF-II): properties of the peptide in serum and production by rat cell lines.
The E-domain peptide of rat pro-insulin-like growth factor II (proIGF-II): properties of the peptide in serum and production by rat cell lines.
复制标题
大鼠胰岛素样生长因子 II (proIGF-II) 的 E 结构域肽：血清中肽的特性以及大鼠细胞系的产生。
DOI：
10.1016/0167-4889(90)90167-c
复制
发表时间：
1990
期刊：
影响因子：
--
通讯作者：
Straus,DS
中科院分区：
文献类型：
--
作者：
Hylka,VW;Straus,DS
我们之前在培养的 BRL-3A 大鼠肝细胞条件培养基中鉴定了一种天然存在的肽片段，该片段源自胰岛素原样生长因子 II (proIGF-II117–156) E 结构域的羧基末端区域。在本研究中，我们利用该肽的放射免疫测定（RIA）来测量培养基和血清中该肽的生理浓度。 E 结构域肽的血清水平在 5 天新生大鼠中非常高，此后下降到成年大鼠血清中的低水平。成年大鼠血清在 1 M 乙酸中的 Sephadex G-75 上进行色谱分析，产生 E 肽免疫反应性的单宽峰，与合成的 E 肽标准品共洗脱。然而，第 5 天新生大鼠血清在 Sephadex G-75 上的色谱分析产生了两个免疫反应性峰。其中一个峰与合成 E 肽标准品共洗脱，而另一个峰在通常洗脱较高分子量蛋白质的区域中洗脱。旨在确定成年大鼠血清是否含有 E 结构域肽的结合蛋白的实验表明：(1) 血清中几乎不含 E 结构域肽的结合蛋白（如果有的话），(2) 血清含有降解 E 结构域肽的蛋白酶活性，以及 (3) 可以通过乙酸/乙醇提取消除蛋白酶活性。在测试的几种大鼠细胞系（BRL-3A、大鼠胚胎成纤维细胞（REF）、肝癌细胞系（H4、HTC）、GH3垂体肿瘤细胞和正常大鼠肾成纤维细胞（NRK））中，只有 BRL-3A 和 REF 细胞将可测量的 E 结构域肽分泌到培养基中。此外，还发现血清的某些成分可以刺激BRL-3A和REF细胞分泌E结构域肽。在 1 M 乙酸中的 Sephadex G-75 上对 BRL-3A 和 REF 条件培养基的免疫反应性进行色谱分析，产生与合成 E 结构域肽标准品共洗脱的单峰。由于 E 结构域肽的分泌与 IGF-II 的表达平行，因此 proIGF-II E 结构域肽的 RIA 可用于研究不同生理条件下 IGF-II 的生物合成和分泌。 IGF-II E结构域肽的RIA与IGF-II的RIA相比具有两个技术优势，即缺乏IGF结合蛋白的干扰以及可以相对容易地合成大量纯抗原。
We previously identified a naturally occurring peptide fragment derived from the carboxyl terminal region of the E-domain of pro-insulin-like growth factor II (proIGF-II117–156) in medium conditioned by cultured BRL-3A rat liver cells. In the present study we utilized a radioimmunoassay (RIA) for this peptide to measure physiological concentrations of the peptide in media and serum. Serum levels of the E-domain peptide were very high in the 5-day neonatal rat and declined thereafter to reach low levels in adult rat serum. Chromatography of adult rat serum on Sephadex G-75 in 1 M acetic acid yielded a single broad peak of E-peptide immunoreactivity that coeluted with a synthetic E-peptide standard. However, chromatography of day 5 neonatal rat serum on Sephadex G-75 yielded two peaks of immunoreactivity. One of the peaks coeluted with a synthetic E-peptide standard, whereas the other peak eluted in a region where higher molecular weight proteins typically elute. Experiments aimed at determining whether adult rat serum contained a binding protein for the E-domain peptide revealed that: (1) serum contains little, if any, binding protein for the E-domain peptide, (2) serum contains a proteinase activity that degrades the E-domain peptide, and (3) the proteinase activity can be eliminated by acetic acid/ethanol extraction. Of several rat cell lines tested (BRL-3A, rat embryo fibroblasts (REF), hepatoma cell lines (H4, HTC), GH3pituitary tumor cells, and normal rat kidney fibroblasts (NRK)), only BRL-3A and REF cells secreted measurable E-domain peptide into the medium. In addition, it was found that some component(s) of serum could stimulate secretion of E-domain peptide from BRL-3A and REF cells. Chromatography of the immunoreactivity from BRL-3A and REF-conditioned media on Sephadex G-75 in 1 M acetic acid yielded a single peak that coeluted with a synthetic E-domain peptide standard. Since secretion of the E-domain peptide parallels the expression of IGF-II, the RIA for the proIGF-II E-domain peptide may be useful for studies of the biosynthesis and secretion of IGF-II under different physiological conditions. The RIA for the IGF-II E-domain peptide has two technical advantages over the RIA for IGF-II, namely, the lack of interference by IGF binding proteins and the relative ease with which large quantities of pure antigen can be synthesized.