Skip to main content
NCBI home page
The Journal of Biophysical and Biochemical Cytology logoLink to The Journal of Biophysical and Biochemical Cytology
. 1955 Mar 25;1(2):139–153. doi: 10.1083/jcb.1.2.139

A METHOD FOR ISOLATING INTACT MITOCHONDRIA AND NUCLEI FROM THE SAME HOMOGENATE, AND THE INFLUENCE OF MITOCHONDRIAL DESTRUCTION ON THE PROPERTIES OF CELL NUCLEI

Alexander L Dounce 1, Robert F Witter 1, Kenneth J Monty 1, Sidney Pate 1, Mary A Cottone 1
PMCID: PMC2223770  PMID: 14381436

Abstract

1. An improved type of ground glass homogenizer for soft tissues has been described which brings about a high degree of cell disruption and liberation of nuclei without causing appreciable damage to mitochondria. The gentleness and effectiveness of the new homogenizer in respect to isolation of mitochondria have been ascertained by comparing the ATP-ase activities of mitochondria isolated in 0.25 M sucrose solution without pH adjustment using a previous type of homogenizer with those of mitochondria isolated under the same conditions with the aid of the new homogenizer. In these experiments sucrose of 0.25 molarity without pH adjustment has been used in order to maintain the mitochondria in a rather sensitive state so as to make slightly deleterious effects of homogenization readily apparent. 2. A new method is described for the isolation of morphologically intact mitochondria and cell nuclei from the same homogenate. In this procedure the pH of the homogenate in 0.44 M sucrose is maintained at 6.0–6.2 with citric acid during the homogenization. An alternative method employing 0.44 M sucrose plus 0.005 M CaCl2 is given for the isolation of nuclei from tumor cells. However, the latter method does not produce unaltered mitochondria. 3. The α-ketoglutarate, malate, succinate, and hexanoate oxidases of the "intact" mitochondria isolated in 0.44 M sucrose adjusted to pH 6.0–6.2 with very dilute citric acid as described in this paper have been investigated, and it has been shown that the mitochondria compare favorably to those isolated in 0.25 M sucrose by a previously described method. 4. Mitochondria have been found to contain an enzyme which causes nuclei to lose their ability to form gels in dilute alkali. This enzyme is released from the mitochondria when the latter are disrupted. 5. Some properties of nuclei isolated by the new method have been briefly discussed.

Full Text

The Full Text of this article is available as a PDF (1,001.6 KB).

Selected References

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

  1. ALLFREY V., MIRSKY A. E. Some aspects of the desoxyribonuclease activities of animal tissues. J Gen Physiol. 1952 Nov;36(2):227–241. doi: 10.1085/jgp.36.2.227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. COPENHAVER J. H., Jr, LARDY H. A. Oxidative phosphorylations; pathways and yield in mitochondrial preparations. J Biol Chem. 1952 Mar;195(1):225–238. [PubMed] [Google Scholar]
  3. DOUNCE A. L., MONTY K. J. Factors influencing the ability of isolated cell nuclei to from gels in dilute alkali. J Biophys Biochem Cytol. 1955 Mar;1(2):155–160. doi: 10.1083/jcb.1.2.155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. HOGEBOOM G. H., SCHNEIDER W. C. Cytochemical studies. VI. The synthesis of diphosphopyridine nucleotide by liver cell nuclei. J Biol Chem. 1952 May;197(2):611–620. [PubMed] [Google Scholar]
  5. HOGEBOOM G. H., SCHNEIDER W. C., STRIEBICH M. J. Cytochemical studies. V. On the isolation and biochemical properties of liver cell nuclei. J Biol Chem. 1952 May;196(1):111–120. [PubMed] [Google Scholar]
  6. KIELLEY W. W., KIELLEY R. K. Myokinase and adenosinetriphosphatase in oxidative phosphorylation. J Biol Chem. 1951 Aug;191(2):485–500. [PubMed] [Google Scholar]
  7. SCHNEIDER W. C. Biochemical constitution of mammalian mitochondria. J Histochem Cytochem. 1953 Jul;1(4):212–233. doi: 10.1177/1.4.212. [DOI] [PubMed] [Google Scholar]
  8. SCHNEIDER W. C., HOGEBOOM G. H. Intracellular distribution of enzymes. X. Desoxyribonuclease and ribonuclease. J Biol Chem. 1952 Sep;198(1):155–163. [PubMed] [Google Scholar]
  9. SIEKEVITZ P., POTTER V. R. Intramitochondrial regulation of oxidative rate. J Biol Chem. 1953 Mar;201(1):1–13. [PubMed] [Google Scholar]
  10. WEBB M. The intracellular distribution of the deoxypentosenucleases of mammalian tissues. Exp Cell Res. 1953 Sep;5(1):16–26. doi: 10.1016/0014-4827(53)90090-1. [DOI] [PubMed] [Google Scholar]
  11. WITTER R. F., NEWCOMB E. H., STOTZ E. Studies of the mechanism of action of dinitrophenol. J Biol Chem. 1953 May;202(1):291–303. [PubMed] [Google Scholar]
  12. WITTER R. F., WATSON M. L., COTTONE M. A. Morphology and ATP-ase of isolated mitochondria. J Biophys Biochem Cytol. 1955 Mar;1(2):127–138. doi: 10.1083/jcb.1.2.127. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Biophysical and Biochemical Cytology are provided here courtesy of The Rockefeller University Press

RESOURCES