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Analysis of Plasmodium falciparum Mitochondrial Electron Transport Chain Activity Using Seahorse XFe96 Extracellular Flux Assays.
- Source :
-
Bio-protocol [Bio Protoc] 2023 Nov 05; Vol. 13 (21), pp. e4863. Date of Electronic Publication: 2023 Nov 05 (Print Publication: 2023). - Publication Year :
- 2023
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Abstract
- The mitochondrial electron transport chain (ETC) is a multi-component pathway that mediates the transfer of electrons from metabolic reactions that occur in the mitochondrion to molecular oxygen (O <subscript>2</subscript> ). The ETC contributes to numerous cellular processes, including the generation of cellular ATP through oxidative phosphorylation, serving as an electron sink for metabolic pathways such as de novo pyrimidine biosynthesis and for maintaining mitochondrial membrane potential. Proper functioning of the mitochondrial ETC is necessary for the growth and survival of apicomplexan parasites including Plasmodium falciparum , a causative agent of malaria. The mitochondrial ETC of P. falciparum is an attractive target for antimalarial drugs, due to its essentiality and its differences from the mammalian ETC. To identify novel P. falciparum ETC inhibitors, we have established a real-time assay to assess ETC function, which we describe here. This approach measures the O <subscript>2</subscript> consumption rate (OCR) of permeabilized P. falciparum parasites using a Seahorse XFe96 flux analyzer and can be used to screen compound libraries for the identification of ETC inhibitors and, in part, to determine the targets of those inhibitors. Key features • With this protocol, the effects of candidate inhibitors on mitochondrial O <subscript>2</subscript> consumption in permeabilized asexual P. falciparum parasites can be tested in real time. • Through the sequential injection of inhibitors and substrates into the assay, the molecular targets of candidate inhibitors in the ETC can, in part, be determined. • The assay is applicable for both drug discovery approaches and enquiries into a fundamental aspect of parasite mitochondrial biology.<br />Competing Interests: Competing interestsThe authors declare that they have no competing interests.<br /> (©Copyright : © 2023 The Authors; This is an open access article under the CC BY-NC license.)
Details
- Language :
- English
- ISSN :
- 2331-8325
- Volume :
- 13
- Issue :
- 21
- Database :
- MEDLINE
- Journal :
- Bio-protocol
- Publication Type :
- Academic Journal
- Accession number :
- 37969754
- Full Text :
- https://doi.org/10.21769/BioProtoc.4863