Mitchell J L, Hoff J A, Bareyal-Leyser A
Department of Biological Sciences, Northern Illinois University, DeKalb 60115.
Arch Biochem Biophys. 1991 Oct;290(1):143-52. doi: 10.1016/0003-9861(91)90600-n.
Ornithine decarboxylase (ODC) is extremely unstable in mammalian cells. This unusual characteristic facilitates rapid fluctuations in the activity of this enzyme in response to variations in its biosynthesis. Unfortunately, very little is known about the mechanism or regulation of this ODC-specific proteolytic pathway. This study describes the production and characterization of a variant of the rat hepatoma HTC cell line that is strikingly deficient in this pathway. This cell variant was induced by selection for growth in stepwise increasing concentrations (up to 10 mM) of the irreversible ODC inhibitor, alpha-difluoromethylornithine (DFMO). Resistance to this inhibitor appears to result from a combination of elevated (10X) ODC biosynthesis and inhibited degradation, producing greater than a 2000-fold increase in the level of ODC protein. In these variant cells (DH23b) inhibition of protein synthesis by cycloheximide did not result in rapid loss of enzyme activity or ODC protein determined by radioimmunoassay. Pulse-chase studies with [35S]methionine confirmed that this enzyme was not preferentially degraded, even when spermidine was added to the media. ODC purified from the variant cells was found to be identical to the control cell enzyme in size, isoelectric point, substrate binding kinetics, and sensitivity to the inhibitor DFMO. Also, as in the control cells, a major fraction of the ODC molecules extracted from DH23b cells was shown to be phosphorylated on a serine residue. The inability to detect physical or kinetic differences between the parent and the variant cell ODC suggests that the unusual stability of ODC in this cell is associated with a defect in a cellular mechanism for ODC-specific degradation.
鸟氨酸脱羧酶（ODC）在哺乳动物细胞中极不稳定。这一不同寻常的特性使得该酶的活性能够随着其生物合成的变化而迅速波动。遗憾的是，对于这条ODC特异性蛋白水解途径的机制或调控，人们了解甚少。本研究描述了大鼠肝癌HTC细胞系的一个变体的产生及特性，该变体在这条途径中明显存在缺陷。这个细胞变体是通过在逐步增加浓度（高达10 mM）的不可逆ODC抑制剂α-二氟甲基鸟氨酸（DFMO）中进行生长筛选诱导产生的。对这种抑制剂的抗性似乎源于ODC生物合成升高（10倍）和降解受到抑制的共同作用，导致ODC蛋白水平增加超过2000倍。在这些变体细胞（DH23b）中，用环己酰亚胺抑制蛋白质合成并不会导致通过放射免疫测定法测定的酶活性或ODC蛋白迅速丧失。用[35S]甲硫氨酸进行的脉冲追踪研究证实，即使向培养基中添加亚精胺，这种酶也不会被优先降解。从变体细胞中纯化的ODC在大小、等电点、底物结合动力学以及对抑制剂DFMO的敏感性方面与对照细胞的酶相同。此外，与对照细胞一样，从DH23b细胞中提取的大部分ODC分子在一个丝氨酸残基上被磷酸化。无法检测到亲本细胞和变体细胞的ODC在物理或动力学上的差异，这表明ODC在这种细胞中的异常稳定性与ODC特异性降解的细胞机制缺陷有关。
