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. 1975 Mar;146(3):601–608. doi: 10.1042/bj1460601

Evidence of a calcium-ion-transport system in mitochondria isolated from flight muscle of the developing sheep blowfly Lucilia cuprina.

F L Bygrave, A A Daday, F A Doy
PMCID: PMC1165349  PMID: 807204

Abstract

The EGTA (ethanedioxybis(ethylamine)tetra-acetic acid)-Ruthenium Red-quench technique (Reed & Bygrave, 1974a) was used to measure initial rates of Ca-2+ transport in mitochondria from flight muscle of the blowfly Lucilia cuprina. Evidence is provided for the existence in these mitochondria of a Ca-2+-transport system that has many features in common with that known to exist in rat liver mitochondria. These include requirement for energy, saturation at high concentrations of Ca-2+, a sigmoidal relation between initial rates of Ca-2+ transport and Ca-2+ concentration, a high affinity for free Ca-2+ (Km approx. 5 muM) and high affinity for the Ca-2+-transport inhibitoy, Ruthenium Red (approx. 0.03 nmol of carrier-specific binding-sites/mg of protein; Ki approx. 1.6 x 10- minus 8 M). Controlled respiration can be stimulated by Ca-2+ after a short lag-period provided the incubation medium contains KCl and not sucrose. The ability of Lucilia mitochondria to transport Ca-2+ critically depends on the stage of mitochondrial development; Ca-2+ transport is minimal in mitochondria from pharate adults, is maximal between 0 and 2h post-emergence and thereafter rapidly declines to reach less than 20% of the maximum value by about 2-3 days post-emergence. Respiration in mitochondria from newly emerged flies does not respond to added Ca-2+; that from 3-5-day-old flies is stimulated approx. 50%. Whereas very low concentrations of Ca-2+ inhibit ADP-stimulated respiration and oxidative phosphorylation in mitochondria from newly emerged flies (Ki approx. 60 ng-ions of Ca-2+/mg of protein); much higher concentrations (approx. 200 ng-ion/mg of protein) are needed to inhibit these processes in those from older flies. The potential of this system for studying the function and development of metabolite transport systems in mitochondria is discussed.

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Selected References

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  1. Akerman K. E., Saris N. E., Järvisalo J. O. Mitochondrial "high-affinity" binding sites for Ca2+ - fact or artefact? Biochem Biophys Res Commun. 1974 Jun 4;58(3):801–807. doi: 10.1016/s0006-291x(74)80488-2. [DOI] [PubMed] [Google Scholar]
  2. Bygrave F. L., Reed K. C. On the role of the adenosine diphosphate-adenosine triphosphate exchange reaction in oxidative phosphorylation: Effect of calcium. FEBS Lett. 1970 May 1;7(4):339–342. doi: 10.1016/0014-5793(70)80200-9. [DOI] [PubMed] [Google Scholar]
  3. Bygrave F. L., Reed K. C., Spencer T. Cooperative interactions in energy-dependent accumulation of Ca2+ by isolated rat liver mitochondria. Nat New Biol. 1971 Mar 17;230(11):89–89. doi: 10.1038/newbio230089a0. [DOI] [PubMed] [Google Scholar]
  4. Carafoli E., Hansford R. G., Sackton B., Lehninger A. L. Interaction of Ca2+ with blowfly flight muscle mitochondria. J Biol Chem. 1971 Feb 25;246(4):964–972. [PubMed] [Google Scholar]
  5. Carafoli E., Lehninger A. L. A survey of the interaction of calcium ions with mitochondria from different tissues and species. Biochem J. 1971 May;122(5):681–690. doi: 10.1042/bj1220681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. DRAHOTA Z., CARAFOLI E., ROSSI C. S., GAMBLE R. L., LEHNINGER A. L. THE STEADY STATE MAINTENANCE OF ACCUMULATED CA++ IN RAT LIVER MITOCHONDRIA. J Biol Chem. 1965 Jun;240:2712–2720. [PubMed] [Google Scholar]
  7. Hansford R. G., Chappell J. B. The effect of Ca2+ on the oxidation of glycerol phosphate by blowfly flight-muscle mitochondria. Biochem Biophys Res Commun. 1967 Jun 23;27(6):686–692. doi: 10.1016/s0006-291x(67)80090-1. [DOI] [PubMed] [Google Scholar]
  8. Henderson P. J. A linear equation that describes the steady-state kinetics of enzymes and subcellular particles interacting with tightly bound inhibitors. Biochem J. 1972 Apr;127(2):321–333. doi: 10.1042/bj1270321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Luft J. H. Ruthenium red and violet. I. Chemistry, purification, methods of use for electron microscopy and mechanism of action. Anat Rec. 1971 Nov;171(3):347–368. doi: 10.1002/ar.1091710302. [DOI] [PubMed] [Google Scholar]
  10. Moore C. L. Specific inhibition of mitochondrial Ca++ transport by ruthenium red. Biochem Biophys Res Commun. 1971 Jan 22;42(2):298–305. doi: 10.1016/0006-291x(71)90102-1. [DOI] [PubMed] [Google Scholar]
  11. Morrison J. F. Kinetics of the reversible inhibition of enzyme-catalysed reactions by tight-binding inhibitors. Biochim Biophys Acta. 1969;185(2):269–286. doi: 10.1016/0005-2744(69)90420-3. [DOI] [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. Reed K. C. An oxygen polarograph designed for undergraduate use. Anal Biochem. 1972 Nov;50(1):206–212. doi: 10.1016/0003-2697(72)90500-3. [DOI] [PubMed] [Google Scholar]
  14. Reed K. C., Bygrave F. L. A re-evaluation of energy-independent calcium-ion binding by rat liver mitochondria. Biochem J. 1974 Sep;142(3):555–566. doi: 10.1042/bj1420555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Reed K. C., Bygrave F. L. The inhibition of mitochondrial calcium transport by lanthanides and ruthenium red. Biochem J. 1974 May;140(2):143–155. doi: 10.1042/bj1400143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. SZARKOWSKA L., KLINGENBERG M. ON THE ROLE OF UBIQUINONE IN MITOCHONDRIA. SPECTROPHOTOMETRIC AND CHEMICAL MEASUREMENTS OF ITS REDOX REACTIONS. Biochem Z. 1963;338:674–697. [PubMed] [Google Scholar]
  17. Southard J. H., Green D. E. High affinity binding of Ca++ in mitochondria: a reappraisal. Biochem Biophys Res Commun. 1974 Jul 10;59(1):30–37. doi: 10.1016/s0006-291x(74)80169-5. [DOI] [PubMed] [Google Scholar]
  18. Spencer T., Bygrave F. L. The role of mitochondria in modifying the cellular ionic environment: studies of the kinetic accumulation of calcium by rat liver mitochondria. J Bioenerg. 1973 Apr;4(3):347–362. doi: 10.1007/BF01648977. [DOI] [PubMed] [Google Scholar]
  19. Thorne R. F., Bygrave F. L. Calcium does not uncouple oxidative phosphorylation in tightly-coupled mitochondria from Ehrlich ascites tumour cells. Nature. 1974 Mar 22;248(446):348–351. doi: 10.1038/248348a0. [DOI] [PubMed] [Google Scholar]
  20. Vasington F. D., Gazzotti P., Tiozzo R., Carafoli E. The effect of ruthenium red on Ca 2+ transport and respiration in rat liver mitochondria. Biochim Biophys Acta. 1972 Jan 21;256(1):43–54. doi: 10.1016/0005-2728(72)90161-2. [DOI] [PubMed] [Google Scholar]
  21. Vinogradov A., Scarpa A. The initial velocities of calcium uptake by rat liver mitochondria. J Biol Chem. 1973 Aug 10;248(15):5527–5531. [PubMed] [Google Scholar]
  22. Walker A. C., Birt L. M. Development of respiratory activity and oxidative phosphorylation in flight muscle mitochondria of the blowfly, Lucilia cuprina. J Insect Physiol. 1969 Feb;15(2):305–317. doi: 10.1016/0022-1910(69)90277-7. [DOI] [PubMed] [Google Scholar]
  23. Wohlrab H. Respiration-linked calcium ion uptake by flight muscle mitochondria from the blowfly Sarcophaga bullata. Biochemistry. 1974 Sep 10;13(19):4014–4018. doi: 10.1021/bi00716a030. [DOI] [PubMed] [Google Scholar]

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