Oxidative phosphorylation K0.5ADP in vitro depends on substrate oxidative capacity: Insights from a luciferase-based assay to evaluate ADP kinetic parameters
AffiliationDepartment of Medicine, Division of Endocrinology, University of Arizona
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CitationWillis, W., Willis, E., Kuzmiak-Glancy, S., Kras, K., Hudgens, J., Barakati, N., ... & Mandarino, L. (2021). Oxidative Phosphorylation K0. 5ADP In Vitro Depends on Substrate Oxidative Capacity: Insights from a Luciferase-Based Assay to Evaluate ADP Kinetic Parameters. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 148430.
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AbstractThe K0.5ADP of oxidative phosphorylation (OxPhos) identifies the cytosolic ADP concentration which elicits one-half the maximum OxPhos rate. This kinetic parameter is commonly measured to assess mitochondrial metabolic control sensitivity. Here we describe a luciferase-based assay to evaluate the ADP kinetic parameters of mitochondrial ATP production from OxPhos, adenylate kinase (AK), and creatine kinase (CK). The high sensitivity, reproducibility, and throughput of the microplate-based assay enabled a comprehensive kinetic assessment of all three pathways in mitochondria isolated from mouse liver, kidney, heart, and skeletal muscle. Carboxyatractyloside titrations were also performed with the assay to estimate the flux control strength of the adenine nucleotide translocase (ANT) over OxPhos in human skeletal muscle mitochondria. ANT flux control coefficients were 0.91 ± 0.07, 0.83 ± 0.06, and 0.51 ± 0.07 at ADP concentrations of 6.25, 12.5, and 25 μM, respectively, an [ADP] range which spanned the K0.5ADP. The oxidative capacity of substrate combinations added to drive OxPhos was found to dramatically influence ADP kinetics in mitochondria from several tissues. In mouse skeletal muscle ten different substrate combinations elicited a 7-fold range of OxPhos Vmax, which correlated positively (R2 = 0.963) with K0.5ADP values ranging from 2.3 ± 0.2 μM to 11.9 ± 0.6 μM. We propose that substrate-enhanced capacity to generate the protonmotive force increases the OxPhos K0.5ADP because flux control at ANT increases, thus K0.5ADP rises toward the dissociation constant, KdADP, of ADP-ANT binding. The findings are discussed in the context of top-down metabolic control analysis. © 2021 Elsevier B.V.
Note12 month embargo; available online 20 April 2021
VersionFinal accepted manuscript
SponsorsNational Institutes of Health