Chan J C, Scanlon M, Zhang H Z, Murray J L
Department of Tumor Biology, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Biochem Biophys Res Commun. 1991 Jul 31;178(2):453-9. doi: 10.1016/0006-291x(91)90128-t.
Previously, we reported that transformation associated protein (TAP) was over-expressed in the 6m2 line, but not in their normal counterparts (1,2). 6m2 is a culture of NRK cells transformed by the ts-110 mutant of MSV-M. The synthesis of TAP and the expression of transformation properties in the 6m2 cells are all temperature-sensitive (2; 3; 4). TAP is secreted as two polypeptides of 64 kD and 68 kD (P64 and P68) (2). Experiments were carried out to determine whether any metalloproteinase (MP) activity was associated with TAP. Results of zymograms indicated that the two forms of purified TAP (P64 and P68) had MP activity, using gelatin or collagen type IV as substrates. Serum-free medium (SFM) of 6m2 cells incubated at 33 degrees C also showed two bands of MP activity, while the corresponding SFM from 6m2 cells at 39 degrees C lacked such MP activity, indicating that the synthesis of MP was temperature-sensitive. The association of MP activity with the P64 and P68 bands of TAP (purified or in SFM) was confirmed by simultaneous Western blot analysis, which showed the reactivity of the two MP bands with monoclonal or polyclonal antibodies to TAP. Accordingly, what we previously designated as TAP is apparently one form of MP, which are known to be involved in tumor cell metastasis.
此前，我们报道过转化相关蛋白（TAP）在6m2细胞系中过表达，而在其正常对应细胞中则不然（1,2）。6m2是由MSV-M的ts-110突变体转化的NRK细胞培养物。6m2细胞中TAP的合成及转化特性的表达均对温度敏感（2；3；4）。TAP以64kD和68kD的两种多肽形式（P64和P68）分泌（2）。开展了实验以确定是否有任何金属蛋白酶（MP）活性与TAP相关。酶谱分析结果表明，以明胶或IV型胶原为底物时，两种纯化形式的TAP（P64和P68）具有MP活性。在33℃孵育的6m2细胞的无血清培养基（SFM）也显示出两条MP活性条带，而来自39℃的6m2细胞的相应SFM缺乏这种MP活性，这表明MP的合成对温度敏感。通过同时进行的蛋白质免疫印迹分析证实了MP活性与TAP的P64和P68条带（纯化的或在SFM中）的关联，该分析显示两条MP条带与抗TAP的单克隆或多克隆抗体发生反应。因此，我们之前称为TAP的显然是MP的一种形式，已知MP参与肿瘤细胞转移。
