Overproduction of stable ornithine decarboxylase and antizyme in the difluoromethylornithine-resistant cell line DH23b.
Overproduction of stable ornithine decarboxylase and antizyme in the difluoromethylornithine-resistant cell line DH23b.
复制标题
二氟甲基鸟氨酸抗性细胞系 DH23b 中稳定鸟氨酸脱羧酶和抗酶的过量产生。
DOI：
10.1042/bj3170811
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Leyser,A
中科院分区：
文献类型：
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作者：
Mitchell,JL;Choe,CY;Judd,GG;Daghfal,DJ;Kurzeja,RJ;Leyser,A
DH23b 细胞是因对二氟甲基鸟氨酸具有抗性而选择的 HTC 细胞系的一种变体，其鸟氨酸脱羧酶 (ODC) 稳定性和多胺转运的反馈调节存在缺陷，并且 ODC 蛋白累积量超过正常浓度的 1000 倍。人们对多胺反馈调节系统的组成部分进行了检查，试图理解这些不寻常的反应。 Southern 印迹分析显示 ODC DNA 序列扩增（约 10 倍），但抗酶没有任何伴随增加。此外，扩增的 ODC 序列包含一个单碱基取代，导致 Cys-441 转化为 Trp。先前已证明这种修饰可导致 HMOA 细胞中 ODC 的稳定性。尽管在 DH23b 细胞中尚未发现抗酶活性，但蛋白质印迹分析显示，抗酶蛋白的积累量是亲本 HTC 细胞中诱导的积累量的 50 倍以上。这种增加与这些细胞中抗酶半衰期增加 6-9 倍一致，这是由于突变型 ODC-抗酶复合物无法被 26 S 蛋白酶体降解的结果。与 DH23b 和 HMOA 细胞中抗酶的稳定性相关的是另外两种形式的抗酶蛋白的出现，其表观分子质量分别为 22 和 18.5 kDa。这表明这些是由于从抗酶的 N 末端蛋白水解去除离散片段的结果，这可能表明抗酶快速周转的初始步骤。
DH23b cells, a variant of the HTC line selected for their resistance to difluoromethylornithine, exhibit defective feedback regulation of ornithine decarboxylase (ODC) stability and polyamine transport, and accumulate ODC protein to > 1000 times normal concentrations. The components of the polyamine feedback regulation system have been examined in an attempt to understand these unusual responses. Southern-blot analysis revealed an amplification (approx. 10-fold) in ODC DNA sequence without any concomitant increase in antizyme. Moreover, the amplified ODC sequence contains a single base substitution that results in the conversion of Cys-441 into Trp. This modification has previously been shown to cause ODC stability in HMOA cells. Although antizyme activity has not been noted in DH23b cells, Western-blot analysis revealed the accumulation of antizyme protein to > 50 times that induced in parental HTC cells. This increase is consistent with a 6–9-fold increase in the half-life of antizyme in these cells, a consequence of the inability of the mutant ODC–antizyme complex to be degraded by 26 S proteasome. Associated with the stabilization of antizyme in both DH23b and HMOA cells is the appearance of two additional forms of antizyme protein with apparent molecular masses of 22 and 18.5 kDa. It is suggested that these result from proteolytic removal of discrete fragments from the N-terminal end of antizyme, perhaps an indication of an initial step in rapid antizyme turnover.