Application of new semisynthetic aequorins with long half-decay time of luminescence to G-protein-coupled receptor assay

Aequorin is a Ca 2+-binding photoprotein and consists of an apoprotein (apoaequorin) and a 2-peroxide of coelenterazine. Eight new coelenterazine analogues modified at the C2-position were synthesized and incorporated into recombinant apoaequorin with O 2 to yield different semisynthetic aequorins....

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Bibliographic Details
Published in:Analytical biochemistry 2010-12, Vol.407 (2), p.247-252
Main Authors: Inouye, Satoshi, Iimori, Rie, Sahara, Yuiko, Hisada, Sunao, Hosoya, Takamitsu
Format: Article
Language:English
Subjects:
Aequorin
Aequorin - chemistry
Aequorin - genetics
Aequorin - metabolism
Animals
Apoproteins - chemistry
Apoproteins - genetics
Apoproteins - metabolism
Calcium - chemistry
Cell-based GPCR assay
CHO Cells
Coelenterazine synthesis
Cricetinae
Cricetulus
Fluorescent protein
Half-Life
High-throughput screening
Imidazoles - chemical synthesis
Imidazoles - chemistry
Kinetics
Luminescent Agents - chemistry
Oxygen - chemistry
Pyrazines - chemical synthesis
Pyrazines - chemistry
Receptors, G-Protein-Coupled - metabolism
Receptors, Muscarinic - metabolism
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Spectrometry, Fluorescence - methods
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Summary:Aequorin is a Ca 2+-binding photoprotein and consists of an apoprotein (apoaequorin) and a 2-peroxide of coelenterazine. Eight new coelenterazine analogues modified at the C2-position were synthesized and incorporated into recombinant apoaequorin with O 2 to yield different semisynthetic aequorins. The luminescence properties and the sensitivity to Ca 2+ of these semisynthetic aequorins were characterized. Two semisynthetic aequorins, namely me- and cf3-aequorin, showed a slow decay of the luminescence pattern with less sensitivity to Ca 2+ and were useful for the cell-based G-protein-coupled receptor (GPCR) reporter assays.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2010.08.025