Amplification of a FRET Probe by Lipid–Water Partition for the Detection of Acid Sphingomyelinase in Live Cells. Issue 10 (3rd February 2017)

• Record Type: Journal Article
• Authors: Pinkert, Thomas; Furkert, David; Korte, Thomas; Herrmann, Andreas; Arenz, Christoph
• Is Part Of: Angewandte Chemie international edition. Volume 56:Issue 10(2017)
• Page Start: 2790
• Page End: 2794
• Publication Date: 2017-02-03
• Subjects: enzymes -- fluorescence spectroscopy -- FRET probes -- membranes -- sphingolipids; Chemistry -- Periodicals; 540
• DOI: 10.1002/anie.201611706
• Languages: English
• ISSNs: 1433-7851

Abstract

Abstract: Real‐time monitoring of acid sphingomyelinase (ASM) activity is crucial for investigating its role in lipid‐mediated signaling processes. In this study, we synthesized fluorescent phosphosphingolipids capable of FRET by phosphorodichloridate chemistry. These sphingomyelin analogues are substrates for recombinant human ASM and can be used to monitor ASM activity by fluorescence spectroscopy. Incubation with cell lysates from wild‐type and knock‐out mice further confirmed probe cleavage to be exclusive to ASM. We also systematically exploited the environmental sensitivity of the fluorophores to achieve significant increases in responsiveness. This concept may be transferred to other lipid probes in the future. The ASM activity in live cells was imaged by two‐photon‐excitation microscopy. Abstract : A FRET probe for acid sphingomyelinase was designed. Whereas a first probe suffered from quenching by the environment (top), a second‐generation probe made use of the same phenomenon to suppress background fluorescence (bottom). The probe was used to detect the activity of acid sphingomyelinase in vitro as well as in live cells by two‐photon‐excitation microscopy.