Kobrin M S, Samsoondar J, Kudlow J E
J Biol Chem. 1986 Nov 5;261(31):14414-9.
Untransformed bovine anterior pituitary cells cultured in serum-free defined medium secrete an epidermal growth factor (EGF)-like peptide with an amino acid composition similar to rat or human alpha-transforming growth factor (alpha TGF). To further characterize the bovine pituitary alpha TGF, it was compared to a human alpha TGF partially purified from the conditioned medium of a human melanoma cell line. An anti-alpha TGF monoclonal antibody, MF9, was produced from hybridomas derived from mice immunized with a 17-residue synthetic peptide corresponding to the carboxyl-terminal sequence of rat alpha TGF. The hybridoma supernatants were initially screened for the ability to immunoprecipitate 125I-peptide and then tested for recognition of human alpha TGF. Only 2 of 36 antipeptide antibodies recognized the native alpha TGF. The binding of 125I-peptide to MF9 was displaced by human alpha TGF but not by EGF. Bovine pituitary alpha TGF also displaced the binding of 125I-peptide to MF9 in a similar manner to human alpha TGF. Both iodinated human and bovine pituitary alpha TGF were immunoprecipitated by MF9 whereas 125I-EGF was not. Recognition of alpha TGF by MF9 was strongly dependent on sulfhydryl reduction of the growth factors, suggesting that synthetic peptides representing sulfhydryl-rich protein are not ideal immunogens. Tryptic digests of both 125I-alpha TGFs chromatographed to give a single, indistinguishable peak of iodinated material on a reverse-phase C18 high performance liquid chromatography column when eluted with two different solvent systems, suggesting the generation of a single and identical tyrosine-containing tryptic peptide from both alpha TGFs. The comparisons of the bovine pituitary and human melanoma alpha TGF using a sequence-specific monoclonal antibody and peptide mapping suggest that these alpha TGFs are related and that alpha TGF production is not limited to transformed or fetal sources.
在无血清限定培养基中培养的未转化牛垂体前叶细胞分泌一种表皮生长因子（EGF）样肽，其氨基酸组成与大鼠或人α-转化生长因子（α-TGF）相似。为了进一步鉴定牛垂体α-TGF，将其与从人黑色素瘤细胞系条件培养基中部分纯化的人α-TGF进行比较。用对应于大鼠α-TGF羧基末端序列的17个残基合成肽免疫小鼠获得的杂交瘤产生了一种抗α-TGF单克隆抗体MF9。最初筛选杂交瘤上清液免疫沉淀125I-肽的能力，然后检测其对人α-TGF的识别。36种抗肽抗体中只有2种识别天然α-TGF。人α-TGF可取代125I-肽与MF9的结合，而EGF则不能。牛垂体α-TGF也以与人α-TGF相似的方式取代125I-肽与MF9的结合。碘化的人及牛垂体α-TGF均能被MF9免疫沉淀，而125I-EGF则不能。MF9对α-TGF的识别强烈依赖于生长因子的巯基还原，这表明代表富含巯基蛋白质的合成肽不是理想的免疫原。当用两种不同的溶剂系统洗脱时，两种125I-α-TGF的胰蛋白酶消化产物在反相C18高效液相色谱柱上进行色谱分析，得到一个单一的、无法区分的碘化物质峰，这表明两种α-TGF产生了单一且相同的含酪氨酸胰蛋白酶肽。使用序列特异性单克隆抗体和肽图谱对牛垂体和人黑色素瘤α-TGF进行的比较表明，这些α-TGF相关，且α-TGF的产生不限于转化细胞或胎儿来源。
