A novel method for fluorescence microscopic demonstration of caspase activity

我们已经开发了一种基于4-甲氧基-2-萘酰胺（MNA）的底物，Z-Asp（OME）-Glu（OME）-Val-Asp（OME）-MNA（DEVD-MNA），其对触发多种凋亡细胞变性的主要半胱天冬酶之一的半胱天冬酶3具有相对特异性。caspase-3释放的MNA在340 nm激发波长和425 nm发射波长处产生荧光。使用这种底物，我们首先确定了一氧化氮（NO）-半胱氨酸蛋白酶-3-样酶活性的时间过程中处理的小鼠破骨细胞样细胞的细胞裂解物，NOC 18。Caspase-3样酶活性在NOC 18处理后8 h出现短暂的峰值，此后下降。MNA与5-亚硝基水杨醛（NSA）在弱酸性条件下（pH = 6.0）形成席夫碱配合物，配合物呈针状荧光晶体生长。当DEVD-MNA和NSA应用于NOC 18处理的鼠破骨细胞样细胞（8小时）时，在细胞中形成小针状荧光晶体。半胱天冬酶抑制剂DEVD-苯甲酮（FMK）可完全抑制晶体的形成.因此，用这种荧光底物观察到胱天蛋白酶的细胞内活化。经NOC 18处理16 h后，细胞内晶体形成明显减少，出现典型的细胞核浓缩、细胞皱缩等凋亡形态。晶体形成的效率与通过DEVD-MNA在体外测量的半胱天冬酶-3样酶活性的变化密切相关。可见的caspase活性先于凋亡形态学变化。我们开发的方法的优点是（1）用这种荧光底物染色的细胞是可固定的，（2）在混合细胞群中，如破骨细胞培养物，可以特异性地检测经历凋亡变性的细胞。

We have developed a 4-methoxy-2-naphthylamide (MNA)-based substrate, Z-Asp(OME)-Glu(OME) -Val-Asp(OME) -MNA (DEVD-MNA), which is relatively specific for caspase-3, one of the major caspases that triggers multiple apoptotic cellular degeneration. MNA, released by caspase-3 showed fluorescence with an exitation wave length of 340nm and emission wave length of 425nm. Using this substrate, we first determined the time course of caspase-3-like enzyme activity in cell lysates of murine osteoclast-like cells treated with nitric oxide (NO)-releaser, NOC18. Caspase-3-like enzyme activity showed a transient peak at 8 h after NOC18 treatment and decreased thereafter. MNA forms a Schiff-base complex with 5-nitrosalycylaldehyde (NSA) under mild acidic condition (〜pH 6.0), and the complex grows as needle-like fluorescence crystals. When DEVD-MNA and NSA were applied to NOC18-treated murine osteoclast-like cells (8 h), small needle-like fluorescence crystals were formed in the cells. The crystal formation was completely inhibited with caspase inhibitor DEVD- fenylmethylketone (FMK). Thus, intracellular activation of caspase(s) was visualized with this fluorescence substrate. After 16 h of NOC18-treatment, the crystal formation in cells was markedly reduced, and typical apoptotic morphologies of nuclear condensation and cell shrinkage, were observed. The efficiency of the crystal formation correlated well with changes in caspase-3-like enzyme activity measured by DEVD-MNA in vitro. The visualized caspase activity preceded the apoptotic morphological changes. The advantages of the method we have developed are (1) stained cells with this fluorescence substrate were fixable and (2) among mixed cell populations, such as osteoclast culture, cells undergoing apoptotic degeneration could be specifically detected.