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)
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: | https://repository.cshl.edu/id/eprint/29673 |
Actions (login required)
 |
Administrator's edit/view item |