Cappelluti E, Strom S C, Harris R B
Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Richmond 23298.
Biochemistry. 1993 Jan 19;32(2):551-60. doi: 10.1021/bi00053a021.
Transforming growth factor-alpha (TGF-alpha) is a mitogenic peptide produced by tumor cells and by virally and chemically transformed cells in culture. TGF-alpha is almost certainly derived from its precursor protein (pro-TGF-alpha) by limited proteolysis, but the physiologically relevant processing enzyme(s) is(are) unknown. We now report that oncogenically transformed rat liver epithelial cells (known to secrete TGF-alpha) and Schwann cells in culture transfected with SV40 T-antigen (which are now reported to express mRNA encoding pro-TGF-alpha) contain membrane associated, neutral pH, serine proteinases which are elastase-like in their substrate specificity, but elastase is not known to be associated with these cell types. In both cell types, the enzyme is associated with a subcellular fraction enriched for microsomes and plasma membranes. Furthermore, the enzyme appears to be specifically induced 4-fold in the transformed epithelial cells as compared with the level of enzyme present in the nontransformed parental cells. The enzymes have been purified approximately 20,000-fold to near homogeneity (50-60 units/mg) and are virtually identical with regard to their molecular weights (38,000) and other physiochemical properties. Results obtained with numerous synthetic peptide substrates show the enzymes prefer nonpolar residues such as Ala and Val in the P1 and P2 positions, but promiscuity of cleavage specificity observed with long-chain peptide substrates is attributed to the absence of structure in these peptides. Thus, although these enzymes may be involved in processing pro-TGF-alpha at the plasma membrane of the cell, it is just as likely that these enzymes play other physiological roles in the parental and/or transformed cells and that there is no specific endoproteolytic processing enzyme of pro-TGF-alpha.
转化生长因子α（TGF-α）是一种有丝分裂原性肽，由肿瘤细胞以及培养中的病毒转化细胞和化学转化细胞产生。TGF-α几乎肯定是通过有限的蛋白水解作用从其前体蛋白（前TGF-α）衍生而来，但生理相关的加工酶尚不清楚。我们现在报告，致癌转化的大鼠肝上皮细胞（已知分泌TGF-α）和用SV40 T抗原转染的培养中的雪旺细胞（现在据报道表达编码前TGF-α的mRNA）含有与膜相关的、中性pH的丝氨酸蛋白酶，其底物特异性类似于弹性蛋白酶，但弹性蛋白酶与这些细胞类型无关。在这两种细胞类型中，该酶都与富含微粒体和质膜的亚细胞部分相关。此外，与未转化的亲代细胞中存在的酶水平相比，该酶在转化的上皮细胞中似乎被特异性诱导了4倍。这些酶已被纯化了约20000倍，达到近乎均一的程度（50 - 60单位/毫克），并且在分子量（38000）和其他物理化学性质方面几乎相同。用多种合成肽底物获得的结果表明，这些酶在P1和P2位置更喜欢非极性残基，如Ala和Val，但在长链肽底物中观察到的切割特异性的混杂性归因于这些肽中缺乏结构。因此，尽管这些酶可能参与细胞质膜上的前TGF-α加工，但同样有可能这些酶在亲代和/或转化细胞中发挥其他生理作用，并且不存在前TGF-α的特异性内切蛋白水解加工酶。
