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SNAREs pp 163-173 | Cite as

SNAP-25 S-Guanylation and SNARE Complex Formation

  • Yusuke Kishimoto
  • Takaaki Akaike
  • Hideshi Ihara
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1860)

Abstract

8-Nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP), which is the second messenger in nitric oxide/reactive oxygen species redox signaling, covalently binds to protein thiol groups (called S-guanylation) and exerts various biological functions. Synaptosomal associated protein 25 (SNAP-25), a member of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, plays an important role in the process of membrane fusion. We previously showed that SNAP-25 is S-guanylated at cysteine 90. In addition, we revealed that S-guanylation of SNAP-25 increases SNARE complex formation, but decreases the affinity of SNARE complex for complexin. Since SNAP-25 plays a critical role in regulating exocytosis, it is important to elucidate the physiological or pathophysiological meanings of S-guanylation of this protein. Here we describe a protocol for detecting 8-nitro-cGMP and S-guanylated proteins in cells by immunocytochemistry, and methods to detect SNARE complex in 8-nitro-cGMP-treated cells.

Key words

8-Nitro-cGMP SNAP-25 SNARE complex Nitric oxide Redox signal 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Yusuke Kishimoto
    • 1
  • Takaaki Akaike
    • 2
  • Hideshi Ihara
    • 1
  1. 1.Department of Biological Science, Graduate School of ScienceOsaka Prefecture UniversitySakaiJapan
  2. 2.Department of Environmental Medicine and Molecular ToxicologyTohoku University Graduate School of MedicineSendaiJapan

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