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. 1982 Jun 15;204(3):731–735. doi: 10.1042/bj2040731

A procedure for the rapid preparation of mitochondria from rat liver.

P H Reinhart, W M Taylor, F L Bygrave
PMCID: PMC1158413  PMID: 7126163

Abstract

A technique for the rapid preparation of mitochondria from rat liver is described. Tissue fractionation is performed by a single centrifugation step with a discontinuous Percoll density gradient. Total preparation times of 5--6 min are achieved by using this method. The mitochondrial fraction obtained is relatively free of contaminating organelles, as judged by marker-enzyme activity determinations. Mitochondria isolated by Percoll-density-gradient centrifugation differ from mitochondria obtained by differential centrifugation [Taylor, Prpić, Exton & Bygrave (1980) Biochem. J. 188, 443--450] in that the former exhibit a higher acceptor control ratio and a higher calcium content. Values obtained for the protonmotive force are not significantly different between the two preparations. The technique described may be widely applicable for studies requiring the rapid preparation of functionally intact and relatively uncontaminated mitochondria.

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Selected References

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  1. APPELMANS F., WATTIAUX R., DE DUVE C. Tissue fractionation studies. 5. The association of acid phosphatase with a special class of cytoplasmic granules in rat liver. Biochem J. 1955 Mar;59(3):438–445. doi: 10.1042/bj0590438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. BAUDHUIN P., BEAUFAY H. [An electron microscopic examination of purified fractions of cytoplasmic organites of rat liver]. Arch Int Physiol Biochim. 1963 Jan;71:119–120. [PubMed] [Google Scholar]
  3. Belsham G. J., Denton R. M., Tanner M. J. Use of a novel rapid preparation of fat-cell plasma membranes employing Percoll to investigate the effects of insulin and adrenaline on membrane protein phosphorylation within intact fat-cells. Biochem J. 1980 Nov 15;192(2):457–467. doi: 10.1042/bj1920457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blume H. Die Isolierung hochgereinigter Mitochondrienpräparationen für Untersuchungen zur Biotransformation von Arzneistoffen. Arch Pharm (Weinheim) 1979 Jul;312(7):561–572. doi: 10.1002/ardp.19793120702. [DOI] [PubMed] [Google Scholar]
  5. Booth R. F., Clark J. B. A method for the rapid separation of soluble and particulate components of rat brain synaptosomes. FEBS Lett. 1979 Nov 15;107(2):387–392. doi: 10.1016/0014-5793(79)80414-7. [DOI] [PubMed] [Google Scholar]
  6. Bustamante E., Soper J. W., Pedersen P. L. A high-yield preparative method for isolation of rat liver mitochondria. Anal Biochem. 1977 Jun;80(2):401–408. doi: 10.1016/0003-2697(77)90661-3. [DOI] [PubMed] [Google Scholar]
  7. Bygrave F. L., Heaney T. P., Ramachandran C. Submitochondrial location of ruthenium red-sensitive calcium-ion transport and evidence for its enrichment in a specific population of rat liver mitochondria. Biochem J. 1978 Sep 15;174(3):1011–1019. doi: 10.1042/bj1741011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bygrave F. L., Tranter C. J. The subcellular location, maturation and response to increased plasma glucagon of ruthenium red-insensitive calcium-ion transport in rat liver. Biochem J. 1978 Sep 15;174(3):1021–1030. doi: 10.1042/bj1741021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. CHANCE B., WILLIAMS G. R. The respiratory chain and oxidative phosphorylation. Adv Enzymol Relat Subj Biochem. 1956;17:65–134. doi: 10.1002/9780470122624.ch2. [DOI] [PubMed] [Google Scholar]
  10. Dawson A. P., Fulton D. V. The action of Nupercaine on calcium efflux from rat liver mitochondria. Biochem J. 1980 Jun 15;188(3):749–755. doi: 10.1042/bj1880749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dulley J. R. Determination of inorganic phosphate in the presence of detergents or protein. Anal Biochem. 1975 Jul;67(1):91–96. doi: 10.1016/0003-2697(75)90275-4. [DOI] [PubMed] [Google Scholar]
  12. Gellerfors P., Nelson B. D. A rapid method for the isolation of intact mitochondria from isolated rat liver cells. Anal Biochem. 1979 Feb;93(1):200–203. [PubMed] [Google Scholar]
  13. Harris E. J., Al-Shaikhaly M., Baum H. Stimulation of mitochondrial calcium ion efflux by thiol-specific reagents and by thyroxine. The relationship to adenosine diphosphate retention and to mitochondrial permeability. Biochem J. 1979 Aug 15;182(2):455–464. doi: 10.1042/bj1820455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  15. 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]
  16. Murphy E., Coll K., Rich T. L., Williamson J. R. Hormonal effects on calcium homeostasis in isolated hepatocytes. J Biol Chem. 1980 Jul 25;255(14):6600–6608. [PubMed] [Google Scholar]
  17. Nicholls D. G. The influence of respiration and ATP hydrolysis on the proton-electrochemical gradient across the inner membrane of rat-liver mitochondria as determined by ion distribution. Eur J Biochem. 1974 Dec 16;50(1):305–315. doi: 10.1111/j.1432-1033.1974.tb03899.x. [DOI] [PubMed] [Google Scholar]
  18. Reinhart P. H., Taylor W. M., Bygrave F. L. Studies on alpha-adrenergic-induced respiration and glycogenolysis in perfused rat liver. J Biol Chem. 1982 Feb 25;257(4):1906–1912. [PubMed] [Google Scholar]
  19. Schnaitman C., Greenawalt J. W. Enzymatic properties of the inner and outer membranes of rat liver mitochondria. J Cell Biol. 1968 Jul;38(1):158–175. doi: 10.1083/jcb.38.1.158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Scott I. D., Akerman K. E., Nicholls D. G. Calcium-ion transport by intact synaptosomes. Intrasynaptosomal compartmentation and the role of the mitochondrial membrane potential. Biochem J. 1980 Dec 15;192(3):873–880. doi: 10.1042/bj1920873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Taylor W. M., Prpić V., Exton J. H., Bygrave F. L. Stable changes to calcium fluxes in mitochondria isolated from rat livers perfused with alpha-adrenergic agonists and with glucagon. Biochem J. 1980 May 15;188(2):443–450. doi: 10.1042/bj1880443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Tischler M. E., Hecht P., Williamson J. R. Determination of mitochondrial/cytosolic metabolite gradients in isolated rat liver cells by cell disruption. Arch Biochem Biophys. 1977 May;181(1):278–293. doi: 10.1016/0003-9861(77)90506-9. [DOI] [PubMed] [Google Scholar]
  23. Wattiaux R., Wattiaux-De Coninck S., Ronveaux-Dupal M. F. Deterioration of rat-liver mitochondria during centrifugation in a sucrose gradient. Eur J Biochem. 1971 Sep 13;22(1):31–39. doi: 10.1111/j.1432-1033.1971.tb01511.x. [DOI] [PubMed] [Google Scholar]

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