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Updated: Oct 8, 2025

Forward Genetic Screen Using Transgenic Calcium Reporter Aequorin to Identify Novel Targets in Calcium Signaling
Published on: August 1, 2020
Formation of Coelenteramine from 2-Peroxycoelenterazine in the Ca2+ -Binding Photoprotein Aequorin
Satoshi Inouye1, Mitsuhiro Nakamura2, Takamitsu Hosoya3
1Yokohama Research Center, JNC Co. 5-1, Okawa, Yokohama, 236-8605, Japan.
Abstract:
Aequorin consists of apoprotein (apoAequorin) and (S)-2-peroxycoelenterazine (CTZ-OOH) and is considered to be a transient-state complex of an enzyme (apoAequorin) and a substrate (coelenterazine and molecular oxygen) in the enzymatic reaction. The degradation process of CTZ-OOH in aequorin was characterized under various conditions of protein denaturation. By acid treatment, the major product from CTZ-OOH was coelenteramine (CTM), but not coelenteramide (CTMD), and no significant luminescence was observed. The counterparts of CTM from CTZ-OOH were identified as 4-hydroxyphenylpyruvic acid (4HPPA) and 4-hydroxyphenylacetic acid (4HPAA) by liquid chromatography/electrospray ionization-time-of-flight mass spectrometry (LC/ESI-TOF-MS). In the luminescence reaction of aequorin with Ca2+ , similar amounts of 4HPPA and 4HPAA were detected, indicating that CTM is formed by two pathways from CTZ-OOH through dioxetanone anion and not by hydrolysis from CTMD.
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