Luetteke N C, Michalopoulos G K, Teixidó J, Gilmore R, Massagué J, Lee D C
Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710.
Biochemistry. 1988 Aug 23;27(17):6487-94. doi: 10.1021/bi00417a043.
In addition to the mature 50 amino acid transforming growth factor alpha (TGF alpha), some transformed cells appear to produce multiple higher molecular weight forms. The structure and derivation of most of these larger soluble TGF alpha species remain to be established. We previously reported that a chemically induced rat hepatocellular carcinoma cell line, JM1, secreted acid-stable proteins which bind to epidermal growth factor receptors and stimulate DNA synthesis in primary cultures of normal adult rat hepatocytes. Purification and characterization of these hepatoma-derived growth factors have indicated their relationship to TGF alpha. Two EGF-competing activities of apparent Mr 30K and 10K were separated by gel filtration of concentrated JM1-conditioned medium and further purified by ion-exchange chromatography and reverse-phase HPLC. Both growth factors were detected by a radioimmunoassay specific for TGF alpha. Western blotting with antibodies to the 50 amino acid TGF alpha revealed that the lower molecular weight factor comigrated with the synthetic 6-kDa rat TGF alpha. The higher molecular weight TGF alpha appeared on immunoblots as a diffuse band of 18-21 kDa, which converted to the mature 6-kDa form upon digestion with elastase, confirming a precursor-product relationship. However, the 18-21-kDa proteins did not react with antibodies directed against the carboxy-terminal cytoplasmic segment of the transmembrane TGF alpha precursor. Enzymatic deglycosylation of the 18-21-kDa TGF alpha species by sequential removal of sialic acids and O- and N-linked carbohydrate reduced the molecular weight to 11K. The size and soluble nature of this polypeptide suggest that it represents the extracellular domain of the transmembrane TGF alpha precursor.(ABSTRACT TRUNCATED AT 250 WORDS)
除了成熟的50个氨基酸的转化生长因子α（TGFα）外，一些转化细胞似乎还产生多种更高分子量的形式。这些较大的可溶性TGFα物种中大多数的结构和来源仍有待确定。我们先前报道，一种化学诱导的大鼠肝细胞癌细胞系JM1分泌与表皮生长因子受体结合并刺激正常成年大鼠肝细胞原代培养物中DNA合成的酸稳定蛋白。这些肝癌衍生生长因子的纯化和表征表明了它们与TGFα的关系。通过对浓缩的JM1条件培养基进行凝胶过滤分离出两种表观分子量为30K和10K的表皮生长因子竞争活性，并通过离子交换色谱和反相高效液相色谱进一步纯化。两种生长因子均通过针对TGFα的放射免疫测定法检测。用针对50个氨基酸的TGFα的抗体进行蛋白质印迹分析表明，较低分子量的因子与合成的6 kDa大鼠TGFα迁移一致。较高分子量的TGFα在免疫印迹上表现为18 - 21 kDa的弥散条带，经弹性蛋白酶消化后转变为成熟的6 kDa形式，证实了前体 - 产物关系。然而，18 - 21 kDa的蛋白质不与针对跨膜TGFα前体羧基末端细胞质片段的抗体反应。通过依次去除唾液酸以及O - 连接和N - 连接的碳水化合物对18 - 21 kDa的TGFα物种进行酶促去糖基化，使分子量降至11K。这种多肽的大小和可溶性性质表明它代表跨膜TGFα前体的细胞外结构域。（摘要截断于250字）
