Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1985 Feb 1;100(2):435–441. doi: 10.1083/jcb.100.2.435

Mobility of fluorescent derivatives of cytochrome c in mitochondria

PMCID: PMC2113438  PMID: 2981887

Abstract

Motion of cytochrome c bound to giant (2-10-micron diam) mitochondria isolated from the waterbug Lethocerus indicus was examined using the technique of fluorescence recovery after photobleaching. Fluorescent cytochrome c was exchanged for native cytochrome c through partly damaged outer membrane. Recovery profiles were not statistically different when the fluorescence from iron-free cytochrome c or fluorescein-labeled cytochrome c was used and were essentially the same in the presence or absence of an uncoupler. In the presence of excess porphyrin cytochrome c, the apparent diffusion coefficient was 6 X 10(- 11) cm2/s in 0.3 M sucrose-mannitol-EDTA and 3 X 10(-10) cm2/s in 0.10 M KCl/0.10 M sucrose. At concentrations of porphyrin cytochrome c that are stoichiometric with cytochrome c oxidase and for mitochondria in which excess cytochrome c was washed away, two components were observed in the recovery profile. The diffusion coefficient of the fast component was 1 X 10(-10) cm2/s. The second component showed no recovery during the time scale of measurement (D less than 10(-12) cm2/s). We speculate on the origin of the immobile fraction.

Full Text

The Full Text of this article is available as a PDF (750.0 KB).

Selected References

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

  1. Axelrod D., Koppel D. E., Schlessinger J., Elson E., Webb W. W. Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys J. 1976 Sep;16(9):1055–1069. doi: 10.1016/S0006-3495(76)85755-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barisas B. G., Leuther M. D. Fluorescence photobleaching recovery measurement of protein absolute diffusion constants. Biophys Chem. 1979 Sep;10(2):221–229. doi: 10.1016/0301-4622(79)85044-9. [DOI] [PubMed] [Google Scholar]
  3. Bosshard H. R., Zürrer M., Schägger H., von Jagow G. Binding of cytochrome c to the cytochrome bc1 complex (complex III) and its subunits cytochrome c1 and b1. Biochem Biophys Res Commun. 1979 Jul 12;89(1):250–258. doi: 10.1016/0006-291x(79)90971-9. [DOI] [PubMed] [Google Scholar]
  4. Branton D., Cohen C. M., Tyler J. Interaction of cytoskeletal proteins on the human erythrocyte membrane. Cell. 1981 Apr;24(1):24–32. doi: 10.1016/0092-8674(81)90497-9. [DOI] [PubMed] [Google Scholar]
  5. Erecińska M., Davis J. S., Wilson D. F. Interactions of cytochrome c with mitochondrial membranes. Binding to succinate-cytochrome c reductase. J Biol Chem. 1980 Oct 25;255(20):9653–9658. [PubMed] [Google Scholar]
  6. FERNANDEZ MORAN H., ODA T., BLAIR P. V., GREEN D. E. A MACROMOLECULAR REPEATING UNIT OF MITOCHONDRIAL STRUCTURE AND FUNCTION. CORRELATED ELECTRON MICROSCOPIC AND BIOCHEMICAL STUDIES OF ISOLATED MITOCHONDRIA AND SUBMITOCHONDRIAL PARTICLES OF BEEF HEART MUSCLE. J Cell Biol. 1964 Jul;22:63–100. doi: 10.1083/jcb.22.1.63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ferguson-Miller S., Brautigan D. L., Margoliash E. Correlation of the kinetics of electron transfer activity of various eukaryotic cytochromes c with binding to mitochondrial cytochrome c oxidase. J Biol Chem. 1976 Feb 25;251(4):1104–1115. [PubMed] [Google Scholar]
  8. GORNALL A. G., BARDAWILL C. J., DAVID M. M. Determination of serum proteins by means of the biuret reaction. J Biol Chem. 1949 Feb;177(2):751–766. [PubMed] [Google Scholar]
  9. Gupte S., Wu E. S., Hoechli L., Hoechli M., Jacobson K., Sowers A. E., Hackenbrock C. R. Relationship between lateral diffusion, collision frequency, and electron transfer of mitochondrial inner membrane oxidation-reduction components. Proc Natl Acad Sci U S A. 1984 May;81(9):2606–2610. doi: 10.1073/pnas.81.9.2606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hochman J. H., Schindler M., Lee J. G., Ferguson-Miller S. Lateral mobility of cytochrome c on intact mitochondrial membranes as determined by fluorescence redistribution after photobleaching. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6866–6870. doi: 10.1073/pnas.79.22.6866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Höchli M., Hackenbrock C. R. Lateral translational diffusion of cytochrome c oxidase in the mitochondrial energy-transducing membrane. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1236–1240. doi: 10.1073/pnas.76.3.1236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jacobson K., Hou Y., Wojcieszyn J. Evidence for lack of damage during photobleaching measurements of the lateral mobility of cell surface components. Exp Cell Res. 1978 Oct 1;116(1):179–189. doi: 10.1016/0014-4827(78)90074-5. [DOI] [PubMed] [Google Scholar]
  13. Kawato S., Sigel E., Carafoli E., Cherry R. J. Cytochrome oxidase rotates in the inner membrane of intact mitochondria and submitochondrial particles. J Biol Chem. 1980 Jun 25;255(12):5508–5510. [PubMed] [Google Scholar]
  14. Kawato S., Sigel E., Carafoli E., Cherry R. J. Rotation of cytochrome oxidase in phospholipid vesicles. Investigations of interactions between cytochrome oxidases and between cytochrome oxidase and cytochrome bc1 complex. J Biol Chem. 1981 Jul 25;256(14):7518–7527. [PubMed] [Google Scholar]
  15. Koppel D. E. Fluorescence redistribution after photobleaching. A new multipoint analysis of membrane translational dynamics. Biophys J. 1979 Nov;28(2):281–291. doi: 10.1016/S0006-3495(79)85176-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Koppel D. E., Sheetz M. P., Schindler M. Matrix control of protein diffusion in biological membranes. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3576–3580. doi: 10.1073/pnas.78.6.3576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. LEHNINGER A. L., UL HASSAN M., SUDDUTH H. C. Phosphorylation coupled to the oxidation of ascorbic acid by isolated mitochondria. J Biol Chem. 1954 Oct;210(2):911–922. [PubMed] [Google Scholar]
  18. Sowers A. E., Hackenbrock C. R. Rate of lateral diffusion of intramembrane particles: measurement by electrophoretic displacement and rerandomization. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6246–6250. doi: 10.1073/pnas.78.10.6246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Thompson N. L., Burghardt T. P., Axelrod D. Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy. Biophys J. 1981 Mar;33(3):435–454. doi: 10.1016/S0006-3495(81)84905-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Utsumi K., Packer L. Glutaraldehyde-fixed mitochondria. I. Enzyme activity, ion translocation, and conformational changes. Arch Biochem Biophys. 1967 Sep;121(3):633–640. doi: 10.1016/0003-9861(67)90048-3. [DOI] [PubMed] [Google Scholar]
  21. Vanderkooi J. M., Erecińska M. Cytochrome c interaction with membranes. Absorption and emission spectra and binding characteristics of iron-free cytochrome c. Eur J Biochem. 1975 Dec 1;60(1):199–207. doi: 10.1111/j.1432-1033.1975.tb20992.x. [DOI] [PubMed] [Google Scholar]
  22. Vanderkooi J. M., Glatz P., Casadei J., Woodrow G. V., 3rd Cytochrome c interaction with yeast cytochrome b2. Heme distances determined by energy transfer in fluorescence resonance. Eur J Biochem. 1980 Sep;110(1):189–196. doi: 10.1111/j.1432-1033.1980.tb04854.x. [DOI] [PubMed] [Google Scholar]
  23. Vanderkooi J. M., Landesberg R., Hayden G. W., Owen C. S. Metal-free and metal-substituted cytochromes c. Use in characterization of the cytochrome c binding site. Eur J Biochem. 1977 Dec 1;81(2):339–347. doi: 10.1111/j.1432-1033.1977.tb11957.x. [DOI] [PubMed] [Google Scholar]
  24. Vanderkooi J., Erecińska M., Chance B. Cytochrome c interaction with membranes. I. Use of a fluorescent chromophore in the study of cytochrome c interaction with artificial and mitochondrial membranes. Arch Biochem Biophys. 1973 Jan;154(1):219–229. doi: 10.1016/0003-9861(73)90052-0. [DOI] [PubMed] [Google Scholar]
  25. Wolf D. E., Henkart P., Webb W. W. Diffusion, patching, and capping of stearoylated dextrans on 3T3 cell plasma membranes. Biochemistry. 1980 Aug 19;19(17):3893–3904. doi: 10.1021/bi00558a002. [DOI] [PubMed] [Google Scholar]
  26. Yguerabide J., Schmidt J. A., Yguerabide E. E. Lateral mobility in membranes as detected by fluorescence recovery after photobleaching. Biophys J. 1982 Oct;40(1):69–75. doi: 10.1016/S0006-3495(82)84459-7. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES