Stromberg K, Hudgins W R
Cancer Res. 1986 Nov;46(11):6004-10.
Purified human epidermal growth factor (hEGF) from urine promotes anchorage-independent cell growth in soft agar medium. This growth is enhanced by transforming growth factor-beta (TGF-beta), and is specifically inhibited by hEGF antiserum. Transforming growth factors of the alpha type (TGF-alpha), potentially present in normal human urine or urine from tumor-bearing patients, also promote anchorage-independent cell growth and compete with EGF for membrane receptor binding. Consequently, TGF-alpha cannot be distinguished from urinary hEGF by these two functional assays. Therefore, a technique for separation of TGF-alpha and related peptides from urinary EGF based on biochemical characteristics would be useful. Radioiodination of characterized growth factors [mouse EGF (mEGF), hEGF, and rat TGF-alpha (rTGF-alpha)], which were then separately added to human urine, was used to evaluate a resolution scheme that separates TGF-alpha from the high level of background hEGF present in human urine. Commercially available methyl bonded microparticulate silica efficiently adsorbed the 125I-labeled mEGF, 125I-labeled hEGF, and 125I-labeled rTGF-alpha that were added to 24-h human urine samples. Fractional elution with acetonitrile (MeCN) of the adsorbed silica released approximately 70 to 80% of the 125I-labeled mEGF and 125I-labeled hEGF between 25 and 30% MeCN, and over 80% of the 125I-labeled rTGF-alpha between 15 and 25% MeCN, with retention after dialysis of less than 0.2 and 1.7% of the original urinary protein, respectively. Consequently, a single-step enrichment of about 400-fold for mEGF and hEGF, and 50-fold for rTGF-alpha were achieved rapidly. Subsequent Bio-Gel P-10 chromatography indicated that 125I-labeled mEGF and 125I-labeled hEGF eluted later than would be predicted on the basis of their reported molecular weight of approximately 6000, whereas 125I-labeled rTGF-alpha eluted from Bio-Gel P-10 at an approximate molecular weight of 8000 to 9000. 125I-labeled rTGF-alpha bound to carboxymethyl cellulose and eluted at a less acidic pH than did hEGF. On reverse phase high performance liquid chromatography using a linear gradient of 18 to 35% MeCN over 120 min, 125I-labeled rTGF-alpha was comparatively hydrophilic, eluting at 22% MeCN in contrast to the more hydrophobic 125I-labeled hEGF, which eluted at 27% MeCN. These observed biochemical differences between hEGF and TGF-alpha provide a rationale for resolution of these and perhaps other related putative transforming growth factors from bulk urine of tumor-bearing patients.
从尿液中提纯的人表皮生长因子（hEGF）可促进细胞在软琼脂培养基中进行非贴壁依赖性生长。转化生长因子-β（TGF-β）可增强这种生长，而hEGF抗血清则能特异性抑制该生长。α型转化生长因子（TGF-α）可能存在于正常人尿液或荷瘤患者尿液中，它也能促进非贴壁依赖性细胞生长，并与表皮生长因子（EGF）竞争膜受体结合。因此，通过这两种功能测定无法区分TGF-α和尿液中的hEGF。所以，基于生化特性从尿液EGF中分离TGF-α及相关肽的技术将很有用处。对具有特征性的生长因子[小鼠EGF（mEGF）、hEGF和大鼠TGF-α（rTGF-α）]进行放射性碘化，然后分别添加到人类尿液中，以此评估一种从人类尿液中高背景水平的hEGF中分离TGF-α的分离方案。市售的甲基键合微粒硅胶能有效吸附添加到24小时人类尿液样本中的125I标记的mEGF、125I标记的hEGF和125I标记的rTGF-α。用乙腈（MeCN）对吸附的硅胶进行分步洗脱，在25%至30% MeCN之间释放出约70%至80%的125I标记的mEGF和125I标记的hEGF，在15%至25% MeCN之间释放出超过80%的125I标记的rTGF-α，透析后保留的原始尿蛋白分别少于0.2%和1.7%。因此，能快速实现对mEGF和hEGF约400倍的单步富集以及对rTGF-α 50倍的单步富集。随后的Bio-Gel P-10柱色谱分析表明，125I标记的mEGF和125I标记的hEGF的洗脱时间比根据其报道的约6000分子量所预测的要晚，而125I标记的rTGF-α从Bio-Gel P-10柱上以约8000至9000的分子量洗脱。125I标记的rTGF-α与羧甲基纤维素结合，且在比hEGF更低的酸性pH值下洗脱。在使用120分钟内18%至35% MeCN线性梯度的反相高效液相色谱中，125I标记的rTGF-α相对亲水，在22% MeCN处洗脱，而疏水性更强的125I标记的hEGF在27% MeCN处洗脱。观察到的hEGF和TGF-α之间的这些生化差异为从荷瘤患者的大量尿液中分离这些以及可能的其他相关假定转化生长因子提供了理论依据。
