Synthesis and characterization of a new two photon excitable acid sphingomyelinase FRET probe.
Synthesis and characterization of a new two photon excitable acid sphingomyelinase FRET probe.
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DOI：
10.1016/j.bmc.2021.116303
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发表时间：
2021-07
影响因子：
3.5
通讯作者：
Zainelabdeen H. Mohamed;C. Rhein;B. Schmid;P. Tripal;J. Kornhuber;C. Arenz
中科院分区：
文献类型：
--
作者：
Zainelabdeen H. Mohamed;C. Rhein;B. Schmid;P. Tripal;J. Kornhuber;C. Arenz
最近，FRET探针的酸性鞘磷脂酶（ASM）已经使观察酶的活性在完整的细胞中的第一次。在这里，我们提出了一个ASM FRET探针专门优化的双光子激发。为了便于探针表征和与先前探针的比较，我们将两个完整探针与限定量的探针的神经酰胺切割产物混合，并将它们固定在脂质珠上。直接激发的NBD FRET受体荧光被证明是一种有用的参考手段，并表明新探针比以前的探针更亮，尽管只是中等程度。然后，新的探针被用于检测抑制各种ASM抑制剂显微镜下的第一次。此外，在细胞中，直接激发受体荧光被证明是一个有用的参数，除了FRET可视化抑制ASM。
Recently, FRET probes for acid sphingomyelinase (ASM) have enabled the observation of enzyme activity in intact cells for the first time. Here we present an ASM FRET probe specifically optimized for 2-photon excitation. To facilitate probe characterization and comparison to the previous probe, we mixed the two intact probes with defined amounts of the probes' ceramide cleavage products and mounted them on lipid beads. Directly excited NBD FRET acceptor fluorescene proved to be a useful means of reference and showed that the new probe is brighter, albeit only moderately, than the previous one. The new probe was then used to detect inhibition by various ASM inhibitors microscopically for the first time. Also in cells, directly excited acceptor fluorescence proved to be a useful parameter in addition to FRET to visualize inhibition of ASM.