PT  - JOURNAL ARTICLE
AU  - Lucia Fernandez-del-Rio
AU  - Cristiane Benincá
AU  - Frankie Villalobos
AU  - Cynthia Shu
AU  - Linsey Stiles
AU  - Marc Liesa
AU  - Ajit S Divakaruni
AU  - Rebeca Acin-Perez
AU  - Orian S Shirihai
TI  - A novel approach to measure complex V ATP hydrolysis in frozen cell lysates and tissue homogenates
AID  - 10.26508/lsa.202201628
DP  - 2023 Apr 01
TA  - Life Science Alliance
PG  - e202201628
VI  - 6
IP  - 4
4099  - https://www.life-science-alliance.org/content/6/4/e202201628.short
4100  - https://www.life-science-alliance.org/content/6/4/e202201628.full
SO  - Life Sci. Alliance2023 Apr 01; 6
AB  - Mitochondrial depolarization can initiate reversal activity of ATP synthase, depleting ATP by its hydrolysis. We have recently shown that increased ATP hydrolysis contributes to ATP depletion leading to a maladaptation in mitochondrial disorders, where maximal hydrolytic capacity per CV content is increasing. However, despite its importance, ATP hydrolysis is not a commonly studied parameter because of the limitations of the currently available methods. Methods that measure CV hydrolytic activity indirectly require the isolation of mitochondria and involve the introduction of detergents, preventing their utilization in clinical studies or any high-throughput analyses. Here, we describe a novel approach to assess maximal ATP hydrolytic capacity and maximal respiratory capacity in a single assay in cell lysates, PBMCs, and tissue homogenates that were previously frozen. The methodology described here has the potential to be used in clinical samples to determine adaptive and maladaptive adjustments of CV function in diseases, with the added benefit of being able to use frozen samples in a high-throughput manner and to explore ATP hydrolysis as a drug target for disease treatment.