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BMC Biochemistry (2)
Rustin, Pierre (2)
Bébéar, Cécile M (1)
Bénit, Paule (1)
Chretien, Dominique (1)
Darin, Niklas (1)
Dassa, Emmanuel P (1)
Favier, Judith (1)
Gimenez-Roqueplo, Anne-Paule (1)
Goncalves, Sergio (1)
Kadhom, Norman (1)
Munnich, Arnold (1)
Paupe, Vincent (1)
Rötig, Agnès (1)
Year of Publication
Rapid determination of tricarboxylic acid cycle enzyme activities in biological samples
Dassa, Emmanuel P
In the last ten years, deficiencies in tricarboxylic acid cycle (TCAC) enzymes have been shown to cause a wide spectrum of human diseases, including malignancies and neurological and cardiac diseases. A prerequisite to the identification of disease-causing TCAC enzyme deficiencies is the availability of effective enzyme assays.
We developed three assays that measure the full set of TCAC enzymes. One assay relies on the sequential addition of reagents to measure succinyl-CoA ligase activity, followed by succinate dehydrogenase, fumarase and, finally, malate dehydrogenase. Another assay measures the activity of α-ketoglutarate dehydrogenase followed by aconitase and isocitrate dehydrogenase. The remaining assay measures citrate synthase activity using a standard procedure. We used these assays successfully on extracts of small numbers of human cells displaying various severe or partial TCAC deficiencies and on frozen heart homogenates from heterozygous mice harboring an SDHB gene deletion.
This set of assays is rapid and simple to use and can immediately detect even partial defects, as the activity of each enzyme can be readily compared with one or more other activities measured in the same sample.
Mitochondrial activities in human cultured skin fibroblasts contaminated by Mycoplasma hyorhinis
Bébéar, Cécile M
Mycoplasma contaminations are a recurrent problem in the use of cultured cells, including human cells, especially as it has been shown to impede cell cycle, triggering cell death under various conditions. More specific consequences on cell metabolism are poorly known.
Here we report the lack of significant consequence of a heavy contamination by the frequently encountered mycoplasma strain, M. hyorhinis, on the determination of respiratory chain activities, but the potential interference when assaying citrate synthase. Contamination by M. hyorhinis was detected by fluorescent imaging and further quantified by the determination of the mycoplasma-specific phosphate acetyltransferase activity. Noticeably, this latter activity was not found equally distributed in various mycoplasma types, being exceptionally high in M. hyorhinis.
While we observed a trend for respiration reduction in heavily contaminated cells, no significant and specific targeting of any respiratory chain components could be identified. This suggested a potential interference with cell metabolism rather than direct interaction with respiratory chain components.
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