Skip to main content

Pitfalls in the light microscopical detection of NADH oxidase

The Histochemical Journal volume 22, pages 155–161 (1990)Cite this article

Summary

NADH oxidase activity has been detected at the ultrastructural level using cerium ions to trap H2O2 generated by the enzyme (via intermediate reactive oxygen species). In an attempt to localize NADH oxidase activity at the light microscope level using the cerium-diaminobenzidine (DAB)-nickel-H2O2, the cerium-DAB-cobalt-H2O2 or the cerium-alkaline lead procedures, the distribution patterns of the revealed enzyme were found to be identical to those for non-specific alkaline phosphatase and especially 5′-nucleotidase activity. With the cerium-DAB-cobalt-H2O2 visualization procedure, the distribution pattern of the inial reaction product was similar to that obtained with the other two techniques but much less final reaction product was formed. Incubations for NADH oxidase activity performed in the presence of exogenous catalase or in the absence of catalase or peroxidase inhibitors did not affect the staining intensity, whereas inhibitors of 5′-nucleotidase (EDTA) and non-specific alkaline phosphatase (levamisole) always did. Therefore, phosphatases contribute to the formation of the final reaction product. Since NADH initially cannot be hydrolysed by either of these two phosphatases, then presumably nucleotide prophosphatase (E.C.3.6.1.9) cleaves NADH into 5′-AMP and nicotinamide mononucleotide in a first step. Both nucleotides can be hydrolysed further by the two monophosphatases. These then generate cerium phosphate which is detected by the DAB-nickel-H2O2, DAB-cobalt-H2O2 or lead visualization methods.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

References

  • Angermüller, S. &Fahimi, H. D. (1988a) Light microscopic visualization of the reaction product of cerium used for localization of peroxisomal oxidases.J. Histochem. Cytochem. 36, 23–8.

    Google Scholar 

  • Angermüller, S. &Fahimi, H. D. (1988b) Heterogenous staining ofd-amino acid oxidase in peroxisomes of rat liver and kidney. A light and electron microscopical study.Histochemistry 88, 277–85.

    Google Scholar 

  • Björkmann, U., Ekholm, R. &Denef, J.-F. (1981) Cytochemical localization of hydrogen peroxide in isolated thyroid follicles.J. Ultrastruct. Res. 74, 105–15.

    Google Scholar 

  • Borgers, M. (1973) The cytochemical application of new potent inhibitors of alkaline phosphatases.J. Histochem. Cytochem. 21, 812–24.

    Google Scholar 

  • Borgers, M. &Thone, F. (1975) The inhibition of alkaline phosphatase byl-p-bromotetramisole.Histochemistry 44, 277–80.

    Google Scholar 

  • Briggs, R. T., Drath, D. B., Karnovsky, M. L. &Karnovsky, M. J. (1975) Localization of NADH oxidase on the surface of human polymorphonuclear leucocytes by a new cytochemical method.J. Cell. Biol. 67, 566–85.

    Google Scholar 

  • Burstone, M. S. (1962)Enzyme Histochemistry and its Application in the Study of Neoplasms. New York, London: Academic Press.

    Google Scholar 

  • Christie, K. N. &Stoward, P. J. (1982) The cytochemical reactivity of cerium ions with cardiac muscle.Acta Histochem. Cytochem. 15, 656–72.

    Google Scholar 

  • Dawson, T. P., Gandhi, R., Lehir, M. &Kaissling, B. (1989) Ecto-5′-nucleotidase: localization in rat kidney by light microscopic, histochemical and immunohistochemical methods.J. Histochem. Cytochem. 37, 39–47.

    Google Scholar 

  • De Pierre, J. W. &Karnovsky, M. L. (1974) Ecto-enzymes of the guinea-pig polymorphonuclear leukocyte II. Properties and suitability as markers for the plasma membrane.J. Biol. Chem. 249, 7121–9.

    Google Scholar 

  • Dixon, M. &Webb, E. C. (1979)Enzymes. 3rd edn. New York, San Francisco: Academic Press.

    Google Scholar 

  • Evans, W. H. (1974) Nucleotide pyrophosphatase, a sialoglycoprotein located on the hepatocyte surface.Nature 250, 391–4.

    Google Scholar 

  • Evans, W. H., Hood, D. O. &Gurd, J. W. (1973) Purification and properties of a mouse liver plasmamembrane glycoprotein hydrolysing nucleotide pyrophosphatase and phosphodiester bonds.Biochem. J. 135, 819–26.

    Google Scholar 

  • Frederks, W. M. &Marx, F. (1988) A quantitative histochemical study of 5′-nucleotidase activity in rat liver using the lead salt method and polyvinyl alcohol.Histochem. J. 20, 207–14.

    Google Scholar 

  • Gossrau, R. (1978) Tetrazoliummethoden zum histochemischen Hydrolasennachweis.Histochemistry 58, 203–18.

    Google Scholar 

  • Gossrau, R. (1989) Plasma membrane oxidases revealed by the Ce-DAB-H2O2−Ni-procedure. Fact or artefact?Histochem. J. In press.

  • Gossrau, R., Van Noorden, C. J. F. &Frederiks, W. M. (1989) Enhanced light microscopic visualization of oxidase activity with the cerium capture method.Histochemistry. 92, 349–53.

    Google Scholar 

  • Nalbhuber, K. J., Gossrau, R., Möller, U., Hulstaert, C. E., Zimmermann, N. &Feuerstein, H. (1988) The cerium perhydroxide-diaminobenzidine (Ce−H2O2-DAB) procedure. New methods for light microscopic phosphatase histochemistry and immunohistochemistry.Histochemistry 90, 289–98.

    Google Scholar 

  • Hsu, S.-M. &Soban, E. (1982) Color modification of diaminobenzidine (DAB) precipitation by metallic ions and its application for double immunohistochemistry.J. Histochem. Cytochem. 30, 1079–82.

    Google Scholar 

  • Ishikawa, Y., Hirai, K.-J. &Ogawa, K. (1981) Electron cytochemical studies on an NAD(P)H-dependent H2O2-generating enzyme activity in the rat small intestine.Acta Histochem. Cytochem. 14, 727–33.

    Google Scholar 

  • Karnovsky, M. J. &Robinson, J. M. (1981) Contribution of oxidative cytochemistry to our understanding of the phagocytic process. InHistochemistry. The Widening Horizons (edited byStoward, P. J. &Polak, J. M.), pp. 47–66. Chichester, New York, Brisbane, Toronto, Singapore: J. Wiley & Sons Ltd.

    Google Scholar 

  • Lehir, M., Dubach, U. C. &Angielski, S. (1985) Localization of nucleotide pyrophosphatase in the rat kidney.Histochemistry 86, 207–10.

    Google Scholar 

  • Lojda, Z., Gossrau, R. &Schiebler, T. H. (1979)Enzyme Histochemistry. A Laboratory Manual. Berlin, Heidelberg, New York: Springer.

    Google Scholar 

  • McGadey, J. (1970) A tetrazolium method for non-specific alkaline phosphatase.Histochemie 23, 180–84.

    Google Scholar 

  • Mizukami, Y., Matsubara, F., Matsukawa, S. &Izumi, R. (1983) Cytochemical localization of glutaraldehyderesistant NAD(P)H-oxidase in rat hepatocytes.Histochemistry 78, 259–67.

    Google Scholar 

  • Mizukami, Y., Matsubara, F. &Matsukawa, S. (1986) Cytochemical localization of peroxidase and hydrogenperoxide-producing NAD(P)H-oxidase in thyroid follicular cells of propylthiouracil-treated rats.Histochemistry 82, 263–8.

    Google Scholar 

  • Parson, J. D. (1987) Nucleotide metabolism. InMammalian Ectoenzymes (edited byKenny, A. J. &Turner, A. J.), pp. 139–67. Amsterdam, New York, Oxford: Elsevier.

    Google Scholar 

  • Racker, E. (1955) Mechanism of action and properties of pyridine nucleotide linked enzymes.Physiol. Rev. 35, 1–56.

    Google Scholar 

  • Robinson, J. M., Karnovsky, M. J., Stoward, P. J. &Lewis, P. R. (1990) Oxidases. InHistochemistry: Theoretical and Applied (edited byStoward, P. J. &Pearse, A. G. E.), Vol. III. Edinburgh, London, New York: Churchill Livingstone.

    Google Scholar 

  • Schmidt, H. (1988) Phenol oxidase (EC 1.14. 18.1). A marker enzyme for defense cells.Progr. Histochem. Cytochem. 17, 1–194.

    Google Scholar 

  • Van Noorden, C. J. F. &Jonges, G. N. (1987) Quantification of the histochemical reaction for alkaline phosphatase activity using the indoxyl-tetranitro BT method.Histochem. J. 19, 94–102.

    Google Scholar 

  • Wachstein, M. &Meisel, E. (1957) Histochemistry of hepatic phosphatases at a physiologic pH with special reference to the demonstration of bile canaliculi.Am. J. Clin. Pathol. 27, 13–23.

    Google Scholar 

  • Zimmermann, H. &Pearse, A. G. E. (1959) Limitations in the histochemical demonstrations of pyridine nucleotide-linked dehydrogenases (‘nothing-dehydrogenase’).J. Histochem. Cytochem. 7, 270–75.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Anatomy, Free University of Berlin, Königin-Luise-Strasse 15, D-1000, Berlin 33, Germany

    R. Gossrau

  2. Laboratory of Cell Biology and Histology, University of Amsterdam, Academic Medical Centre, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands

    C. J. F. Van Noorden & W. M. Frederiks

Authors
  1. R. Gossrau
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. C. J. F. Van Noorden
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. W. M. Frederiks
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Gossrau, R., Van Noorden, C.J.F. & Frederiks, W.M. Pitfalls in the light microscopical detection of NADH oxidase. Histochem J 22, 155–161 (1990). https://doi.org/10.1007/BF01003535

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01003535

Keywords

  • NADH
  • Catalase
  • Cerium
  • Levamisole
  • Intermediate Reactive Oxygen