PMID- 33887230
OWN - NLM
STAT- MEDLINE
DCOM- 20210924
LR  - 20220802
IS  - 1879-2650 (Electronic)
IS  - 0005-2728 (Print)
IS  - 0005-2728 (Linking)
VI  - 1862
IP  - 8
DP  - 2021 Aug 1
TI  - Oxidative phosphorylation K(0.5)ADP in vitro depends on substrate oxidative 
      capacity: Insights from a luciferase-based assay to evaluate ADP kinetic 
      parameters.
PG  - 148430
LID - S0005-2728(21)00063-3 [pii]
LID - 10.1016/j.bbabio.2021.148430 [doi]
AB  - 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 muM, 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 muM to 
      11.9 +/- 0.6 muM. 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, K(d)ADP, of 
      ADP-ANT binding. The findings are discussed in the context of top-down metabolic 
      control analysis.
CI  - Copyright (c) 2021 Elsevier B.V. All rights reserved.
FAU - Willis, Wayne
AU  - Willis W
AD  - Department of Medicine, Division of Endocrinology, University of Arizona, Tucson, 
      AZ, United States; Center for Disparities in Diabetes, Obesity, and Metabolism, 
      University of Arizona, Tucson, AZ, United States. Electronic address: 
      waynewillis@deptofmed.arizona.edu.
FAU - Willis, Elizabeth
AU  - Willis E
AD  - College of Medicine, University of Kentucky, Lexington, KY, United States.
FAU - Kuzmiak-Glancy, Sarah
AU  - Kuzmiak-Glancy S
AD  - Department of Kinesiology, University of Maryland, College Park, MD, United 
      States.
FAU - Kras, Katon
AU  - Kras K
AD  - Department of Medicine, Division of Endocrinology, University of Arizona, Tucson, 
      AZ, United States.
FAU - Hudgens, Jamie
AU  - Hudgens J
AD  - College of Pharmacy, Midwestern University, Glendale, AZ, United States.
FAU - Barakati, Neusha
AU  - Barakati N
AD  - Department of Medicine, Division of Endocrinology, University of Arizona, Tucson, 
      AZ, United States.
FAU - Stern, Jennifer
AU  - Stern J
AD  - Department of Medicine, Division of Endocrinology, University of Arizona, Tucson, 
      AZ, United States; Center for Disparities in Diabetes, Obesity, and Metabolism, 
      University of Arizona, Tucson, AZ, United States.
FAU - Mandarino, Lawrence
AU  - Mandarino L
AD  - Department of Medicine, Division of Endocrinology, University of Arizona, Tucson, 
      AZ, United States; Center for Disparities in Diabetes, Obesity, and Metabolism, 
      University of Arizona, Tucson, AZ, United States.
LA  - eng
GR  - R01 DK047936/DK/NIDDK NIH HHS/United States
GR  - R01 DK066483/DK/NIDDK NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20210420
PL  - Netherlands
TA  - Biochim Biophys Acta Bioenerg
JT  - Biochimica et biophysica acta. Bioenergetics
JID - 101731706
RN  - 61D2G4IYVH (Adenosine Diphosphate)
RN  - EC 1.13.12.- (Luciferases)
SB  - IM
MH  - Adenosine Diphosphate/*metabolism
MH  - Animals
MH  - Humans
MH  - Kidney/*metabolism
MH  - Kinetics
MH  - Luciferases/*metabolism
MH  - Mice
MH  - Mitochondria/*metabolism
MH  - Mitochondria, Liver/*metabolism
MH  - Muscle, Skeletal/*metabolism
MH  - *Oxidative Phosphorylation
MH  - Oxidative Stress
MH  - Oxygen Consumption
PMC - PMC8725320
MID - NIHMS1701235
OTO - NOTNLM
OT  - Adenine nucleotide translocase
OT  - Metabolic control
OT  - Michaelis-Menten kinetics
OT  - Respiration
COIS- Declaration of interests The authors declare that they have no known competing 
      financial interests or personal relationships that could have appeared to 
      influence the work reported in this paper.
EDAT- 2021/04/23 06:00
MHDA- 2021/09/25 06:00
PMCR- 2022/08/01
CRDT- 2021/04/22 20:11
PHST- 2020/12/21 00:00 [received]
PHST- 2021/03/30 00:00 [revised]
PHST- 2021/04/07 00:00 [accepted]
PHST- 2021/04/23 06:00 [pubmed]
PHST- 2021/09/25 06:00 [medline]
PHST- 2021/04/22 20:11 [entrez]
PHST- 2022/08/01 00:00 [pmc-release]
AID - S0005-2728(21)00063-3 [pii]
AID - 10.1016/j.bbabio.2021.148430 [doi]
PST - ppublish
SO  - Biochim Biophys Acta Bioenerg. 2021 Aug 1;1862(8):148430. doi: 
      10.1016/j.bbabio.2021.148430. Epub 2021 Apr 20.