RT Journal Article
SR Electronic
T1 A novel approach to measure complex V ATP hydrolysis in frozen cell lysates and tissue homogenates
JF Life Science Alliance
JO Life Sci. Alliance
FD Life Science Alliance LLC
SP e202201628
DO 10.26508/lsa.202201628
VO 6
IS 4
A1 Lucia Fernandez-del-Rio
A1 Cristiane Benincá
A1 Frankie Villalobos
A1 Cynthia Shu
A1 Linsey Stiles
A1 Marc Liesa
A1 Ajit S Divakaruni
A1 Rebeca Acin-Perez
A1 Orian S Shirihai
YR 2023
UL https://www.life-science-alliance.org/content/6/4/e202201628.abstract
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.