Institute of Animal Physiology and Genetics CAS, v.v.i., Brno, Czech Republic.
In Vitro Cell Dev Biol Anim. 2012 Oct;48(9):545-9. doi: 10.1007/s11626-012-9542-8. Epub 2012 Sep 14.
Caspases are key enzymatic components of the intracellular apoptotic machinery, and their role in mammalian systems is often studied using fluoromethylketone (FMK) inhibitors. Despite many advantages of such approach, efficiency of the inhibitor and membrane permeability speed are often questioned. This work therefore focuses on an exact evaluation of caspase-3 FMK inhibition dynamics in camptothecin-induced mesenchymal micromasses. Two parameters of caspase-3 FMK inhibitor were investigated: first, the stability of the inhibitory potential in the time course of cultivation and, simultaneously, the dynamics of caspase-3 FMK inhibition after camptothecin-induced apoptosis peak. A photon-counting chemiluminescence approach was applied for quantification of active caspase-3. The sensitivity of the photon-counting method allowed for evaluation of active caspase-3 concentration in femtogram amounts per cell. The inhibitor penetrated the cells within the first minute after its application, and the peak of caspase-3 started to decline to the blank level after 30 min. The inhibitory effect of the FMK inhibitor was unchanged during the entire 48 h of cultivation.
半胱天冬酶是细胞内凋亡机制的关键酶学组成部分，其在哺乳动物系统中的作用通常使用氟甲基酮 (FMK) 抑制剂进行研究。尽管这种方法有许多优点，但抑制剂的效率和膜通透性速度经常受到质疑。因此，这项工作重点研究了喜树碱诱导的间充质微团中 caspase-3 FMK 抑制的动态变化。研究了 caspase-3 FMK 抑制剂的两个参数：首先，在培养过程中的时间进程中抑制潜力的稳定性，以及同时，喜树碱诱导细胞凋亡高峰后 caspase-3 FMK 抑制的动力学。光子计数化学发光法用于定量活性 caspase-3。光子计数法的灵敏度允许评估每个细胞中活性 caspase-3 的皮克数量级浓度。抑制剂在应用后的第一分钟内穿透细胞，并且 caspase-3 的峰值在 30 分钟后开始下降到空白水平。在整个 48 小时的培养过程中，FMK 抑制剂的抑制作用保持不变。
