Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1978 Apr 1;77(1):134–147. doi: 10.1083/jcb.77.1.134

A study of rapid mitochondrial structural changes in vitro by spray- freeze-etching

PMCID: PMC2110030  PMID: 566274

Abstract

The spray-freeze-etching technique has been used to study energy-linked mitochondrial structural changes in rat liver mitochondria incubated in vitro. The technique involves spraying the suspension of mitochondria into liquid propane at -190 degrees C, and does not require the use of cryoprotectants or chemical fixatives. The results confirmed that freshly isolated mitochondria have a condensed matrix and that this expands at the expense of the outer compartment to give the orthodox configuration when the mitochondria are incubated in a K+ medium in the presence of substrate and phosphate. Addition of adenosine diphosphate (ADP) caused a rapid shrinkage of the matrix compartment, and the time- course and extent of this shrinkage has been measured quantitatively by coupling a rapid sampling device to the spray-freezing apparatus. These data show that for orthodox mitochondria the onset of phosphorylation is accompanied by a reduction of 30% in the matrix volume in 20's, and there is no evidence that the decrease in matrical volume affects the phosphorylation efficiency. These results suggest that natural ionophores in the mitochondrial inner membrane make it permeable enough to permit a rapid readjustment of matrix volume after the addition of ADP, and that the associated ion movement does not cause uncoupling of oxidative phosphorylation.

Full Text

The Full Text of this article is available as a PDF (3.0 MB).

Selected References

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

  1. Bachmann L., Schmitt W. W. Improved cryofixation applicable to freeze etching. Proc Natl Acad Sci U S A. 1971 Sep;68(9):2149–2152. doi: 10.1073/pnas.68.9.2149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Butler W. H., Judah J. D. Preparation of isolated rat liver mitochondria for electron microscopy. J Cell Biol. 1970 Feb;44(2):278–289. doi: 10.1083/jcb.44.2.278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. CHANCE B., WILLIAMS G. R. Respiratory enzymes in oxidative phosphorylation. III. The steady state. J Biol Chem. 1955 Nov;217(1):409–427. [PubMed] [Google Scholar]
  4. CLARK L. C., Jr, WOLF R., GRANGER D., TAYLOR Z. Continuous recording of blood oxygen tensions by polarography. J Appl Physiol. 1953 Sep;6(3):189–193. doi: 10.1152/jappl.1953.6.3.189. [DOI] [PubMed] [Google Scholar]
  5. Green D. E., Asai J., Harris R. A., Penniston J. T. Conformational basis of energy transformations in membrane systems. 3. Configurational changes in the mitochondrial inner membrane induced by changes in functional states. Arch Biochem Biophys. 1968 May;125(2):684–705. doi: 10.1016/0003-9861(68)90626-7. [DOI] [PubMed] [Google Scholar]
  6. Hackenbrock C. R. Energy-linked ultrastructural transformations in isolated liver mitochondria and mitoplasts. Preservation of configurations by freeze-cleaving compared to chemical fixation. J Cell Biol. 1972 May;53(2):450–465. doi: 10.1083/jcb.53.2.450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hackenbrock C. R. States of activity and structure in mitochondrial membranes. Ann N Y Acad Sci. 1972 Jun 20;195:492–505. [PubMed] [Google Scholar]
  8. Hackenbrock C. R. Ultrastructural bases for metabolically linked mechanical activity in mitochondria. I. Reversible ultrastructural changes with change in metabolic steady state in isolated liver mitochondria. J Cell Biol. 1966 Aug;30(2):269–297. doi: 10.1083/jcb.30.2.269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hackenbrock C. R. Ultrastructural bases for metabolically linked mechanical activity in mitochondria. II. Electron transport-linked ultrastructural transformations in mitochondria. J Cell Biol. 1968 May;37(2):345–369. doi: 10.1083/jcb.37.2.345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Höchli M., Hackenbrock C. R. Fluidity in mitochondrial membranes: thermotropic lateral translational motion of intramembrane particles. Proc Natl Acad Sci U S A. 1976 May;73(5):1636–1640. doi: 10.1073/pnas.73.5.1636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. KIELLEY W. W., BRONK J. R. Oxidative phosphorylation in mitochondrial fragments obtained by sonic vibration. J Biol Chem. 1958 Jan;230(1):521–533. [PubMed] [Google Scholar]
  12. KIELLEY W. W., KIELLEY R. K. Myokinase and adenosinetriphosphatase in oxidative phosphorylation. J Biol Chem. 1951 Aug;191(2):485–500. [PubMed] [Google Scholar]
  13. Lang R. D., Crosby P., Robards A. W. An inexpensive spray-freezing unit for preparing specimens for freeze-etching. J Microsc. 1976 Sep;108(1):101–104. doi: 10.1111/j.1365-2818.1976.tb01084.x. [DOI] [PubMed] [Google Scholar]
  14. Muscatello U., Bobyleva V. G., Pasquali-Ronchetti I., Ballotti-Ricci A. M. Configurational changes in isolated rat liver mitochondria as revealed by negative staining. III. Modifications caused by uncoupling agents. J Ultrastruct Res. 1975 Jul;52(1):1–12. doi: 10.1016/s0022-5320(75)80017-7. [DOI] [PubMed] [Google Scholar]
  15. Muscatello U., Guarriera-Bobyleva V., Buffa P. Configurational changes in isolated rat liver mitochondria as revealed by negative staining. II. Modifications caused by changes in respiratory states. J Ultrastruct Res. 1972 Aug;40(3):235–260. doi: 10.1016/s0022-5320(72)90098-6. [DOI] [PubMed] [Google Scholar]
  16. Muscatello U., Guarriero-Bobyleva V. Effect of negative stains used in electron microscopy on some biochemical parameters of the mitochondrial activity. J Ultrastruct Res. 1970 May;31(3):337–348. doi: 10.1016/s0022-5320(70)90136-x. [DOI] [PubMed] [Google Scholar]
  17. Muscatello U., Horne R. W. Effect of the tonicity of some negative-staining solutions on the elementary structure of membrane-bounded systems. J Ultrastruct Res. 1968 Oct;25(1):73–83. doi: 10.1016/s0022-5320(68)80061-9. [DOI] [PubMed] [Google Scholar]
  18. Packer L., Wrigglesworth J. M., Fortes P. A., Pressman B. C. Expansion of the inner membrane compartment and its relation to mitochondrial volume and ion transport. J Cell Biol. 1968 Nov;39(2):382–391. doi: 10.1083/jcb.39.2.382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Robards A. W., Parish G. R. Freeze-etching. Lab Pract. 1972 Apr;21(4):254–260. [PubMed] [Google Scholar]
  20. Stoner C. D., Sirak H. D. Osmotically-induced alterations in volume and ultrastructure of mitochondria isolated from rat liver and bovine heart. J Cell Biol. 1969 Dec;43(3):521–538. doi: 10.1083/jcb.43.3.521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Stoner C. D., Sirak H. D. Passive induction of the "energized-twisted" conformational state in bovine heart mitochondria. Biochem Biophys Res Commun. 1969 Apr 10;35(1):59–66. doi: 10.1016/0006-291x(69)90482-3. [DOI] [PubMed] [Google Scholar]
  22. Vail W. J., Riley K. R. Ultrastructure of isolated heavy beef heart mitochondria revealed by the freeze-etching technique. Nature. 1971 Jun 25;231(5304):525–527. doi: 10.1038/231525a0. [DOI] [PubMed] [Google Scholar]
  23. Vail W. J., Riley R. K., Williams C. H. The morphology and configurational states of isolated heavy beef heart mitochondria by the freeze fracture technique. J Bioenerg. 1972 Dec;3(6):467–479. doi: 10.1007/BF01539056. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES