Logo of jcellbiolHomeThe Rockefeller University PressEditorsContactInstructions for AuthorsThis issue
J Cell Biol. Jul 1, 1968; 38(1): 1–14.
PMCID: PMC2107464

NONDROPLET ULTRASTRUCTURAL DEMONSTRATION OF CYTOCHROME OXIDASE ACTIVITY WITH A POLYMERIZING OSMIOPHILIC REAGENT, DIAMINOBENZIDINE (DAB)

Abstract

A new method for demonstrating cytochrome oxidase activity, based upon the oxidative polymerization of 3,3'-diaminobenzidine (DAB) to an osmiophilic reaction product, has improved the localization of this enzyme over methods based upon the Nadi reaction, in both the light and electron microscopes. The reaction product occurs in nondroplet form, which more accurately delineates the localization of cytochrome oxidase in mitochondria of heart, liver, and kidney. In electron microscopic preparations the excess reaction product is found to overflow into the intracristate spaces and into the outer compartment between inner and outer limiting mitochondrial membranes. This finding suggests that the enzymatic activity of cytochrome c is located on the inner surface of the intracristate space which is the outer surface of the inner mitochondrial membrane. Succinic dehydrogenase activity has also been located at this site by using an osmiophilic ditetrazolium salt, TC-NBT. Considered together, the sites of reactivity of both parts of the respiratory chain have implications for the chemiosomotic hypothesis of Mitchell who suggests a mechanism of energy conservation during electron transport in the respiratory chain of the mitochondrion.

Full Text

The Full Text of this article is available as a PDF (1.2M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • BURSTONE MS. New histochemical techniques for the demonstration of tissue oxidase (cytochrome oxidase). J Histochem Cytochem. 1959 Mar;7(2):112–122. [PubMed]
  • Chance B, Lee CP, Mela L. Control and conservation of energy in the cytochrome chain. Fed Proc. 1967 Sep;26(5):1341–1354. [PubMed]
  • Graham RC, Jr, Karnovsky MJ. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J Histochem Cytochem. 1966 Apr;14(4):291–302. [PubMed]
  • Hackenbrock CR. 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. [PMC free article] [PubMed]
  • HANKER JS, SEAMAN AR, WEISS LP, UENO H, BERGMAN RA, SELIGMAN AM. OSMIOPHILIC REAGENTS: NEW CYTOCHEMICAL PRINCIPLE FOR LIGHT AND ELECTRON MICROSCOPY. Science. 1964 Nov 20;146(3647):1039–1043. [PubMed]
  • Haydon GB, Smith SO. Observations on mitochondrial staining for succinic dehydrogenase. J Histochem Cytochem. 1967 Dec;15(12):752–754. [PubMed]
  • Kerpel-Fronius S, Hajós F. A method for the electron microscopic demonstration of cytochrome oxidase in fresh and formalin-prefixed tissues. Histochemie. 1967;10(3):216–223. [PubMed]
  • Lehninger AL. Energy coupling in electron transport. Fed Proc. 1967 Sep;26(5):1333–1334. [PubMed]
  • MITCHELL P. Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism. Nature. 1961 Jul 8;191:144–148. [PubMed]
  • Mitchell P. Proton-translocation phosphorylation in mitochondria, chloroplasts and bacteria: natural fuel cells and solar cells. Fed Proc. 1967 Sep;26(5):1370–1379. [PubMed]
  • NACHLAS MM, CRAWFORD DT, GOLDSTEIN TP, SELIGMAN AM. The histochemical demonstration of cytochrome oxidase with a new reagent for the Nadi reaction. J Histochem Cytochem. 1958 Nov;6(6):445–456. [PubMed]
  • NACHLAS MM, MARGULIES SI, SELIGMAN AM. Sites of electron transfer to tetrazolium salts in the succinoxidase system. J Biol Chem. 1960 Sep;235:2739–2743. [PubMed]
  • Plapinger RE, Linus SL, Kawashima LT, Deb C, Seligman AM. Preparation and structure-activity relationship of reagents for cytochrome oxidase activity: potential for light and electron microscopy. Histochemie. 1968;14(1):1–16. [PubMed]
  • RICHARDSON KC, JARETT L, FINKE EH. Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technol. 1960 Nov;35:313–323. [PubMed]
  • Schnaitman C, Erwin VG, Greenawalt JW. The submitochondrial localization of monoamine oxidase. An enzymatic marker for the outer membrane of rat liver mitochondria. J Cell Biol. 1967 Mar;32(3):719–735. [PMC free article] [PubMed]
  • Seligman AM, Plapinger RE, Wasserkrug HL, Deb C, Hanker JS. Ultrastructural demonstration of cytochrome oxidase activity by the Nadi reaction with osmiophilic reagents. J Cell Biol. 1967 Sep;34(3):787–800. [PMC free article] [PubMed]
  • Seligman AM, Ueno H, Morizono Y, Wasserkrug HL, Katzoff L, Hanker JS. Electron microscopic demonstration of dehydrogenase activity with a new osmiophilic ditetrazolium salt (TC-NBT). J Histochem Cytochem. 1967 Jan;15(1):1–13. [PubMed]
  • Seligman AM, Wasserkrug HL, Hanker JS. A new staining method (OTO) for enhancing contrast of lipid--containing membranes and droplets in osmium tetroxide--fixed tissue with osmiophilic thiocarbohydrazide(TCH). J Cell Biol. 1966 Aug;30(2):424–432. [PMC free article] [PubMed]
  • Sottocasa GL, Kuylenstierna B, Ernster L, Bergstrand A. An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study. J Cell Biol. 1967 Feb;32(2):415–438. [PMC free article] [PubMed]
  • STRAUS W. FACTORS AFFECTING THE CYTOCHEMICAL REACTION OF PEROXIDASE WITH BENZIDINE AND THE STABILITY OF THE BLUE REACTION PRODUCT. J Histochem Cytochem. 1964 Jun;12:462–469. [PubMed]

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

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...