scholarly journals A Fluorescence-Based Method to Measure ADP/ATP Exchange of Recombinant Adenine Nucleotide Translocase in Liposomes

Biomolecules ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 685
Author(s):  
Jürgen Kreiter ◽  
Eric Beitz ◽  
Elena E. Pohl
Keyword(s):  
Exchange Rates ◽  
Adenine Nucleotide ◽  
Membrane Conductance ◽  
Adenine Nucleotide Translocase ◽  
Mitochondrial Proteins ◽  
Binding Affinities ◽  
Substrate Transport ◽  
Bongkrekic Acid ◽  
Aspartate Glutamate Carrier ◽  
Specific Inhibitors

Several mitochondrial proteins, such as adenine nucleotide translocase (ANT), aspartate/glutamate carrier, dicarboxylate carrier, and uncoupling proteins 2 and 3, are suggested to have dual transport functions. While the transport of charge (protons and anions) is characterized by an alteration in membrane conductance, investigating substrate transport is challenging. Currently, mainly radioactively labeled substrates are used, which are very expensive and require stringent precautions during their preparation and use. We present and evaluate a fluorescence-based method using Magnesium Green (MgGrTM), a Mg2+-sensitive dye suitable for measurement in liposomes. Given the different binding affinities of Mg2+ for ATP and ADP, changes in their concentrations can be detected. We obtained an ADP/ATP exchange rate of 3.49 ± 0.41 mmol/min/g of recombinant ANT1 reconstituted into unilamellar liposomes, which is comparable to values measured in mitochondria and proteoliposomes using a radioactivity assay. ADP/ATP exchange calculated from MgGrTM fluorescence solely depends on the ANT1 content in liposomes and is inhibited by the ANT-specific inhibitors, bongkrekic acid and carboxyatractyloside. The application of MgGrTM to investigate ADP/ATP exchange rates contributes to our understanding of ANT function in mitochondria and paves the way for the design of other substrate transport assays.

scholarly journals Mitochondrial Uncoupling Proteins (UCP1-UCP3) and Adenine Nucleotide Translocase (ANT1) Enhance the Protonophoric Action of 2,4-Dinitrophenol in Mitochondria and Planar Bilayer Membranes

Biomolecules ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1178
Author(s):  
Kristina Žuna ◽  
Olga Jovanović ◽  
Ljudmila Khailova ◽  
Sanja Škulj ◽  
Zlatko Brkljača ◽  
...  
Keyword(s):  
Lipid Membranes ◽  
Adenine Nucleotide ◽  
Specific Inhibitor ◽  
Membrane Conductance ◽  
Uncoupling Proteins ◽  
Site Directed Mutagenesis ◽  
Adenine Nucleotide Translocase ◽  
Planar Bilayer ◽  
Mitochondrial Uncoupling ◽  
Dynamics Simulations

2,4-Dinitrophenol (DNP) is a classic uncoupler of oxidative phosphorylation in mitochondria which is still used in “diet pills”, despite its high toxicity and lack of antidotes. DNP increases the proton current through pure lipid membranes, similar to other chemical uncouplers. However, the molecular mechanism of its action in the mitochondria is far from being understood. The sensitivity of DNP’s uncoupling action in mitochondria to carboxyatractyloside, a specific inhibitor of adenine nucleotide translocase (ANT), suggests the involvement of ANT and probably other mitochondrial proton-transporting proteins in the DNP’s protonophoric activity. To test this hypothesis, we investigated the contribution of recombinant ANT1 and the uncoupling proteins UCP1-UCP3 to DNP-mediated proton leakage using the well-defined model of planar bilayer lipid membranes. All four proteins significantly enhanced the protonophoric effect of DNP. Notably, only long-chain free fatty acids were previously shown to be co-factors of UCPs and ANT1. Using site-directed mutagenesis and molecular dynamics simulations, we showed that arginine 79 of ANT1 is crucial for the DNP-mediated increase of membrane conductance, implying that this amino acid participates in DNP binding to ANT1.

scholarly journals Inhibition of Ca2+-induced large-amplitude swelling of liver and heart mitochondria by cyclosporin is probably caused by the inhibitor binding to mitochondrial-matrix peptidyl-prolyl cis-trans isomerase and preventing it interacting with the adenine nucleotide translocase

1990 ◽  
Vol 268 (1) ◽  
pp. 153-160 ◽  
Author(s):  
A P Halestrap ◽  
A M Davidson
Keyword(s):  
Adenine Nucleotide ◽  
Mitochondrial Protein ◽  
Preceding Paper ◽  
Adenine Nucleotide Translocase ◽  
Heart Mitochondria ◽  
Mitochondrial Matrix ◽  
Isomerase Activity ◽  
Mitochondrial Inner Membrane ◽  
Pore Opening ◽  
Bongkrekic Acid

1. Isolated rat liver and heart mitochondria incubated in 150 mM-KSCN or sucrose medium in the presence of respiratory-chain inhibitors showed a large increase in swelling when exposed to 250 microM-Ca2+. Swelling was inhibited by bongkrekic acid and cyclosporin A in both media and by ADP in KSCN medium; the effect of ADP was reversed by carboxyatractyloside. These results demonstrate that this is a suitable technique with which to study the opening of the Ca2(+)-induced non-specific pore of the mitochondrial inner membrane and implicate the adenine nucleotide carrier in this process. 2. Titration of the rate of swelling with increasing concentrations of cyclosporin showed the number of cyclosporin-binding sites (+/- S.E.M.) in liver and heart mitochondria to be respectively 113.7 +/- 5.0 (n = 9) and 124.3 +/- 11.2 (n = 10) pmol/mg of protein, with a Ki of about 5 nM. 3. Liver and heart mitochondrial-matrix fractions were prepared free of membrane and cytosolic contamination and shown to contain cyclosporin-sensitive peptidyl-prolyl cis-trans isomerase (cyclophilin) activity. Titration of isomerase activity with cyclosporin gave values (+/- S.E.M.) of 110.6 +/- 10.1 (n = 5) and 165.4 +/- 15.0 (n = 3) pmol of enzyme/mg of liver and heart mitochondrial protein respectively, with a Ki of 2.5 nM. The similarity of these results to those from the swelling experiments suggest that the isomerase may be involved in the Ca2(+)-induced swelling. 4. The rapid light-scattering change induced in energized heart mitochondria exposed to submicromolar Ca2+ [Halestrap (1987) Biochem. J. 244, 159-164] was inhibited by ADP and bongkrekic acid, the former effect being reversed by carboxyatractyloside. These results suggest an interaction of Ca2+ with the adenine nucleotide carrier when the ‘c’ conformation. 5. A model is proposed in which mitochondrial peptidyl-prolyl cis-trans isomerase interacts with the adenine nucleotide carrier in the presence of Ca2+ to cause non-specific pore opening. The model also explains the involvement of the adenine nucleotide translocase in the PPi-mediated cyclosporin-insensitive increase in K+ permeability described in the preceding paper [Davidson & Halestrap (1990) Biochem. J. 268, 147-152]. 6. The physiological and pathological implications of the model are discussed in relation to reperfusion injury and cyclosporin toxicity.

scholarly journals Recruitment of NF-κB into Mitochondria Is Involved in Adenine Nucleotide Translocase 1 (ANT1)-induced Apoptosis

2004 ◽  
Vol 279 (37) ◽  
pp. 38415-38423 ◽  
Author(s):  
Monica Zamora ◽  
Claudia Meroño ◽  
Octavi Viñas ◽  
Teresa Mampel
Keyword(s):  
Adenine Nucleotide ◽  
Adenine Nucleotide Translocase ◽  
Induced Apoptosis
Sign in / Sign up

Export Citation Format

Share Document