TY - JOUR
T1 - Oxidative phosphorylation K0.5ADP in vitro depends on substrate oxidative capacity
T2 - Insights from a luciferase-based assay to evaluate ADP kinetic parameters
AU - Willis, Wayne
AU - Willis, Elizabeth
AU - Kuzmiak-Glancy, Sarah
AU - Kras, Katon
AU - Hudgens, Jamie
AU - Barakati, Neusha
AU - Stern, Jennifer
AU - Mandarino, Lawrence
N1 - Funding Information:
WW would like to thank Dr. GJ Kemp for helpful discussion and advice. The authors have no conflicts or competing interests of any type. This work was supported by National Institutes of Health Grants R01 DK047936 (L.J.M.) and DK066483 (L.J.M.). This work was performed at the University of Arizona Department of Medicine, Tucson, AZ, USA.
Publisher Copyright:
© 2021 Elsevier B.V.
PY - 2021/8/1
Y1 - 2021/8/1
N2 - The 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.
AB - The 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.
KW - Adenine nucleotide translocase
KW - Metabolic control
KW - Michaelis-Menten kinetics
KW - Respiration
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U2 - 10.1016/j.bbabio.2021.148430
DO - 10.1016/j.bbabio.2021.148430
M3 - Article
C2 - 33887230
AN - SCOPUS:85104926823
VL - 1862
JO - Biochimica et Biophysica Acta - Bioenergetics
JF - Biochimica et Biophysica Acta - Bioenergetics
SN - 0005-2728
IS - 8
M1 - 148430
ER -