How much H2O2 is produced by recombinant D-Amino acid oxidase in mammalian cells?

Matlashov, M. E., Belousov, V. V., Enikolopov, G. (2014) How much H2O2 is produced by recombinant D-Amino acid oxidase in mammalian cells? Antioxidants and Redox Signaling, 20 (7). pp. 1039-1044. ISSN 15230864 (ISSN)

URL: http://www.ncbi.nlm.nih.gov/pubmed/24020354
DOI: 10.1089/ars.2013.5618

Abstract

Yeast D-amino acid oxidase (DAO) can serve as a genetically encoded producer of reactive oxygen species (ROS) in redox signaling studies. However, dynamics of hydrogen peroxide production and its sensitivity to externally added D-alanine (D-Ala) in cells have not been determined. Here we show that DAO, fused to a genetically encoded H2O2 indicator HyPer, can be used for controlled production of ROS in living eukaryotic cells. We found a clear heterogeneity in ROS production dynamics between individual cells. Moreover, different cell lines demonstrated distinct sensitivity to added D-Ala. Finally, by comparing signals generated by the HyPer-DAO fusion protein versus coexpressed HyPer and DAO proteins, we show that the fusion system is more sensitive to hydrogen peroxide production. Our results show the utility of the HyPer-DAO genetically encoded system for redox signaling studies and suggest that H2O2 produced by DAO in the cytoplasm acts locally in close proximity to the enzyme.

Item Type: Paper
Subjects: organs, tissues, organelles, cell types and functions > cell types and functions > cell functions
organs, tissues, organelles, cell types and functions > cell types and functions
bioinformatics > genomics and proteomics > small molecules > hydrogen peroxide
organism description > animal > mammal
CSHL Authors:
Communities: CSHL Cancer Center Program > Signal Transduction
CSHL labs > Enikopolov lab
CSHL Cancer Center Shared Resources > Microscopy Service
Depositing User: Matt Covey
Date: 2014
Date Deposited: 14 Mar 2014 15:02
Last Modified: 29 Oct 2015 14:52
PMCID: PMC3928830
Related URLs:
URI: http://repository.cshl.edu/id/eprint/29673

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