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. 1981 Dec;78(12):7473–7477. doi: 10.1073/pnas.78.12.7473

Synthesis of adenosine triphosphate in respiration-inhibited submitochondrial particles induced by microsecond electric pulses

Justin Teissie 1, Barry E Knox 1, Tian Yow Tsong 1,*, Janna Wehrle 1
PMCID: PMC349290  PMID: 6950390

Abstract

Phosphorylation of ADP to ATP was induced in nonrespiring submitochondrial particles (SMP) from rat liver by the application of electric pulses with field strengths of 10-35 kV/cm and a decay time of 60 μs. In all cases respiration was inhibited completely by using cyanide or rotenone. Newly formed ATP was measured by two independent methods, (i) the luciferase/luciferin bioluminescence assay and (ii) synthesis of [32P]ATP from ADP and 32Pi. Both methods gave consistent and essentially identical results. Above 10 kV/cm the amount of ATP synthesized increased with increasing field strength, and at 30 kV/cm, approximately 40 pmol of ATP was synthesized per mg of SMP protein per pulse. ATP synthesis was shown to be related to the field-induced transmembrane potential, not to Joule heating of the suspension. Synthesis was abolished by the uncouplers carbonyl cyanide p-trifluoromethoxyphenylhydrazone, carbonyl cyanide m-chlorophenylhydrazone, and 2,4-dinitrophenol. The ionophores valinomycin and A23187 reduced the level of synthesis by 75% and 50%, respectively. ATP synthesis was also blocked by inhibitors of the F0F1 ATPase complex, oligomycin, N,N′-dicyclohexyl carbodiimide, venturicidin, and aurovertin. The activities of the adenine nucleotide translocator and adenylate kinase, as well as release of bound nucleotides, could be excluded as sources of the new ATP. The data indicate that the minimal applied field at which ATP synthesis could be detected is approximately 8 kV/cm, corresponding to a maximal induced membrane potential of 60 mV in SMP. The maximal synthesis occurred at around 30 kV/cm, or an induced transmembrane potential of 200 mV. The duration of the applied pulse was also found to be critical, with 8 μs being the minimal triggering time for the synthesis. The induction of ATP synthesis in nonrespiring SMP by an externally applied electrical field is a direct demonstration of the transformation, by the mitochondrial inner membrane, of electrical energy into the chemical bond energy of ATP.

Keywords: ATPase, bioenergetics, transmembrane potential

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Azzone G. F., Pozzan T., Massari S. Proton electrochemical gradient and phosphate potential in mitochondria. Biochim Biophys Acta. 1978 Feb 9;501(2):307–316. doi: 10.1016/0005-2728(78)90036-1. [DOI] [PubMed] [Google Scholar]
  2. BRADLEY L. B., JACOB M., JACOBS E. E., SANADI D. R. Uncoupling of oxidative phosphorylation by cadmium ion. J Biol Chem. 1956 Nov;223(1):147–156. [PubMed] [Google Scholar]
  3. COOPER C., LEHNINGER A. L. Oxidative phosphorylation by an enzyme complex from extracts of mitochondria. V. The adenosine triphosphate-phosphate exchange reaction. J Biol Chem. 1957 Jan;224(1):561–578. [PubMed] [Google Scholar]
  4. Gräber P., Witt H. T. Relations between the electrical potential, pH gradient, proton flux and phosphorylation in the photosynthetic membrane. Biochim Biophys Acta. 1976 Feb 16;423(2):141–163. doi: 10.1016/0005-2728(76)90174-2. [DOI] [PubMed] [Google Scholar]
  5. Harris D. A., Crofts A. R. The initial stages of photophosphorylation. Studies using excitation by saturating, short flashes of light. Biochim Biophys Acta. 1978 Apr 11;502(1):87–102. doi: 10.1016/0005-2728(78)90134-2. [DOI] [PubMed] [Google Scholar]
  6. Jagendorf A. T., Uribe E. ATP formation caused by acid-base transition of spinach chloroplasts. Proc Natl Acad Sci U S A. 1966 Jan;55(1):170–177. doi: 10.1073/pnas.55.1.170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kinosita K., Jr, Tsong T. T. Hemolysis of human erythrocytes by transient electric field. Proc Natl Acad Sci U S A. 1977 May;74(5):1923–1927. doi: 10.1073/pnas.74.5.1923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kozlov I. A., Skulachev V. P. H+-Adenosine triphosphatase and membrane energy coupling. Biochim Biophys Acta. 1977 Jun 21;463(1):29–89. doi: 10.1016/0304-4173(77)90003-9. [DOI] [PubMed] [Google Scholar]
  9. Lemasters J. J., Hackenbrock C. R. Continuous measurement and rapid kinetics of ATP synthesis in rat liver mitochondria, mitoplasts and inner membrane vesicles determined by firefly-luciferase luminescence. Eur J Biochem. 1976 Aug 1;67(1):1–10. doi: 10.1111/j.1432-1033.1976.tb10625.x. [DOI] [PubMed] [Google Scholar]
  10. Lemasters J. J., Hackenbrock C. R. Continuous measurement of adenosine triphosphate with firefly luciferase luminescence. Methods Enzymol. 1979;56:530–544. doi: 10.1016/0076-6879(79)56051-0. [DOI] [PubMed] [Google Scholar]
  11. Lienhard G. E., Secemski I. I. P 1 ,P 5 -Di(adenosine-5')pentaphosphate, a potent multisubstrate inhibitor of adenylate kinase. J Biol Chem. 1973 Feb 10;248(3):1121–1123. [PubMed] [Google Scholar]
  12. NIELSEN S. O., LEHNINGER A. L. Phosphorylation coupled to the oxidation of ferrocytochrome c. J Biol Chem. 1955 Aug;215(2):555–570. [PubMed] [Google Scholar]
  13. Pedersen P. L. ATP-dependent reactions catalyzed by inner membrane vesicles of rat liver mitochondria. Kinetics, substrate specificity, and bicarbonate sensitivity. J Biol Chem. 1976 Feb 25;251(4):934–940. [PubMed] [Google Scholar]
  14. Pedersen P. L., Hullihen J. Adenosine triphosphatase of rat liver mitochondria. Capacity of the homogeneous F1 component of the enzyme to restore ATP synthesis in urea-treated membranes. J Biol Chem. 1978 Apr 10;253(7):2176–2183. [PubMed] [Google Scholar]
  15. Schlodder E., Witt H. T. Relation between the initial kinetics of ATP synthesis and of conformational changes in the chloroplast ATPase studied by external field pulses. Biochim Biophys Acta. 1981 May 13;635(3):571–584. doi: 10.1016/0005-2728(81)90115-8. [DOI] [PubMed] [Google Scholar]
  16. Schwarz G., Bauer P. J. Structural flexibility and fast proton transfer reflected by the dielectric properties of poly-L-proline in aqueous solution. Biophys Chem. 1974 Apr;1(4):257–265. doi: 10.1016/0301-4622(74)80012-8. [DOI] [PubMed] [Google Scholar]
  17. Slater E. C. Mechanism of oxidative phosphorylation. Annu Rev Biochem. 1977;46:1015–1026. doi: 10.1146/annurev.bi.46.070177.005055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Teissie J., Tsong T. Y. Electric field induced transient pores in phospholipid bilayer vesicles. Biochemistry. 1981 Mar 17;20(6):1548–1554. doi: 10.1021/bi00509a022. [DOI] [PubMed] [Google Scholar]
  19. Thayer W. S., Hinkle P. C. Synthesis of adenosine triphosphate by an artificially imposed electrochemical proton gradient in bovine heart submitochondrial particles. J Biol Chem. 1975 Jul 25;250(14):5330–5335. [PubMed] [Google Scholar]
  20. Wehrle J. P., Cintrón N. M., Pedersen P. L. Phosphate transport in rat liver mitochondria. Energy-dependent accumulation of phosphate by inverted inner membrane vesicles. J Biol Chem. 1978 Dec 10;253(23):8598–8603. [PubMed] [Google Scholar]
  21. Witt H. T. Energy conversion in the functional membrane of photosynthesis. Analysis by light pulse and electric pulse methods. The central role of the electric field. Biochim Biophys Acta. 1979 Mar 14;505(3-4):355–427. doi: 10.1016/0304-4173(79)90008-9. [DOI] [PubMed] [Google Scholar]

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