Oxidative phosphorylation K0.5ADP in vitro depends on substrate oxidative capacity: Insights from a luciferase-based assay to evaluate ADP kinetic parameters

The K(0.5)ADP 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 mu M, respectively, an [ADP] range which spanned the K(0.5)ADP. 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 V-max, which correlated positively (R-2 = 0.963) with K(0.5)ADP values ranging from 2.3 +/- 0.2 mu M to 11.9 +/- 0.6 mu M. We propose that substrate-enhanced capacity to generate the protonmotive force increases the OxPhos K(0.5)ADP because flux control at ANT increases, thus K(0.5)ADP rises toward the dissociation constant, KdADP, of ADP-ANT binding. The findings are discussed in the context of top-down metabolic control analysis.

Automated summary

The study presents a luciferase-based assay for evaluating ADP kinetic parameters in mitochondria, showing that oxidative capacity and substrate combinations can dramatically influence ADP kinetics. The research also suggests that substrate-enhanced capacity to generate protonmotive force increases the OxPhos K(0.5)ADP, leading to flux control at ANT and subsequently affecting ADP-ANT binding dissociation constant.