Stromberg K, Hudgins W R, Fryling C M, Hazarika P, Dedman J R, Pardue R L, Hargreaves W R, Orth D N
J Cell Biochem. 1986;32(4):247-59. doi: 10.1002/jcb.240320402.
Extracts of serum-free conditioned medium from human rhabdomyosarcoma A673 cells contain high molecular weight (HMW) transforming growth factors (TGFs) that can be partially purified by Bio-Gel P-100 and carboxymethyl (CM)-cellulose chromatography (Todaro et al: Proc Natl Acad Sci USA 77:5258, 1980). Reverse-phase high performance liquid chromatography (HPLC) revealed a principal peak of epidermal growth factor (EGF) radioreceptor assay (RRA) activity and anchorage-independent growth (AIG) activity that coeluted with 25-26% acetonitrile. If a trailing shoulder of EGF RRA activity from the CM-C chromatography was included in the material for HPLC analysis, additional active fractions were observed at 21-22% acetonitrile. Importantly, both active regions from HPLC failed to compete in radioimmunoassays under reduced and denatured conditions for human EGF (hEGF), human TGF-alpha (hTGF-alpha), or rat TGF-alpha (rTGF-alpha) and failed to give positive signals in Western blots under conditions in which TGF-alpha was readily detected when using an antisera raised against the 17 C-terminal amino acids of rTGF-alpha. Nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed EGF RRA and AIG activities in gel slices corresponding to Mr 15,000 and 22,000 in the 25-26% acetonitrile eluate and Mr 15,000, 20,000, 27,000, and 48,000 in the 21-22% acetonitrile eluate. The presence of multiple forms of EGF-receptor-binding peptides produced in vitro suggest size heterogeneity and possible immunologic diversity among high molecular weight members of the EGF/TGF-alpha family of growth-promoting polypeptides.
人横纹肌肉瘤A673细胞无血清条件培养基的提取物含有高分子量（HMW）转化生长因子（TGFs），这些因子可通过Bio-Gel P-100和羧甲基（CM）-纤维素色谱法进行部分纯化（托达罗等人：《美国国家科学院院刊》77:5258，1980年）。反相高效液相色谱（HPLC）显示，表皮生长因子（EGF）放射受体分析（RRA）活性和非锚定依赖性生长（AIG）活性的主峰与25 - 26%乙腈共同洗脱。如果将CM-C色谱中EGF RRA活性的拖尾峰包含在用于HPLC分析的材料中，则在21 - 22%乙腈处观察到额外的活性馏分。重要的是，HPLC的两个活性区域在还原和变性条件下的放射免疫分析中均不能与人EGF（hEGF）、人TGF-α（hTGF-α）或大鼠TGF-α（rTGF-α）竞争，并且在使用针对rTGF-α的17个C末端氨基酸产生的抗血清时，在易于检测到TGF-α的条件下，Western印迹中未能给出阳性信号。非还原十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）显示，在25 - 26%乙腈洗脱液中，对应于Mr 15,000和22,000的凝胶切片以及在21 - 22%乙腈洗脱液中对应于Mr 15,000、20,000、27,000和48,000的凝胶切片中存在EGF RRA和AIG活性。体外产生的多种形式的EGF受体结合肽的存在表明，促生长多肽EGF/TGF-α家族的高分子量成员之间存在大小异质性和可能的免疫多样性。
