Hylka V W, Straus D S
Division of Biomedical Sciences, University of California, Riverside.
Biochim Biophys Acta. 1990 Jan 23;1051(1):6-13. doi: 10.1016/0167-4889(90)90167-c.
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), GH3 pituitary 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.
我们之前在培养的BRL - 3A大鼠肝细胞条件培养基中鉴定出一种源自胰岛素样生长因子II（proIGF - II）E结构域羧基末端区域的天然存在的肽片段（proIGF - II117 - 156）。在本研究中，我们利用针对该肽的放射免疫测定法（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出生5天的新生大鼠中非常高，此后下降，在成年大鼠血清中降至低水平。成年大鼠血清在1 M乙酸中于Sephadex G - 75上进行色谱分析，产生了一个单一的宽峰E肽免疫反应性，它与合成E肽标准品共洗脱。然而，出生5天的新生大鼠血清在Sephadex G - 75上进行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，而另一个峰在通常较高分子量蛋白质洗脱的区域洗脱。旨在确定成年大鼠血清是否含有E结构域肽结合蛋白的实验表明：（1）血清中几乎没有（如果有的话）E结构域肽结合蛋白，（2）血清含有一种降解E结构域肽的蛋白酶活性，并且（3）该蛋白酶活性可通过乙酸/乙醇提取消除。在所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细胞向培养基中分泌可测量的E结构域肽。此外，发现血清中的某些成分可以刺激BRL - 3A和REF细胞分泌E结构域肽。BRL - 3A和REF条件培养基的免疫反应性在1 M乙酸中于Sephadex G - 75上进行色谱分析，产生了一个与合成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，proIGF - II E结构域肽的RIA可能有助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相对于IGF - II的RIA具有两个技术优势，即不受IGF结合蛋白的干扰以及相对容易大量合成纯抗原。
