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Histochemie

, Volume 11, Issue 2, pp 112–128 | Cite as

Histochemical demonstration of carbonic anhydrase activity

  • Holger P. J. Hansson
Article

Summary

Freeze-dried frozen sections are floated on the surface of the freshly prepared incubation mixture (CoSO4 1.75 × 10−3M, H2SO4 5.3 × 10−2M, NaHCO3 1.57 × 10−2M and KH2PO4 1.17 to 11.7 × 10−3M; demonstration of weak activity requires high phosphate). A compound containing cobalt and phosphorous precipitates at carbonic anhydrase sites and is converted to CoS. Adequate staining requires only 2–10 minutes of incubation. Actazolamide inhibits the staining reaction in specific concentrations. Actazolamidein vivo, 20 mg/kgi.v. to mice 30 minutes before sacrifice also inhibited the staining. The proportion phosphorous in the specific precipitate increases with KH2PO4 of the medium (shown by the addition of60Co and32P). An explanation of the reaction mechanism is given, based on the catalyzed loss of CO2 in the surface layer. The inclusion of phosphate in the medium makes this modification ofHäusler's method so sensitive that it shows carbonic anhydrase activity in for instance stratum spinosum of the skin.

Keywords

Cobalt Surface Layer H2SO4 Reaction Mechanism KH2PO4 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. Bleyl, U.: Zur Spezifität des histoohemischen Carboanhydratasenachweises im Inselorgan der Bauchspeicheldrüse. Histochemie4, 286–311 (1964).Google Scholar
  2. Datta, P. K., andT. H. Shepard: Intracellular localization of carbonic anhydrase in rat liver and kidney tissues. Arch. Biochem.81, 124–129 (1959).Google Scholar
  3. Davenport, H. W.: Gastric carbonic anhydrase. J. Physiol. (Lond.)97, 32–43 (1939).Google Scholar
  4. Fand, S. B., H. J. Levine, andH. L. Erwin: A reappraisal of the histochemical method for carbonic anhydrase. J. Histochem. Cytochem.7, 27–33 (1959).Google Scholar
  5. Giacobini, E.: A cytochemical study of the localization of carbonic anhydrase in the nervous system. J. Neurochem.9, 169–177 (1962).Google Scholar
  6. Hansson, H. P. J.: Demonstration of carbonic anhydrase by means of fluorescent antibodies in human erythrocytes. Life Sci.4, 965–968 (1965).Google Scholar
  7. Häusler, G.: Zur Technik und Spezifität des histochemischen Carboanhydrasenachweises im Modellversuch und in Gewebsschnitten von Rattennieren. Histochemie1, 29–47 (1958).Google Scholar
  8. Karler, R., andD. M. Woodbury: Intracellular distribution of carbonic anhydrase. Biochem. J.75, 538–543 (1959).Google Scholar
  9. Kern, D. M.: The hydration of carbon dioxide. J. Chem. Educat.37, 14–23 (1960).Google Scholar
  10. Kernohan, J. C.: The pH-activity curve of bovine carbonic anhydrase and its relationship to the inhibition of the enzyme by anions. Biochim. biophys. Acta (Amst.)96, 304–317 (1965).Google Scholar
  11. —,W. W. Forrest, andF. J. W. Roughton: The activity of concentrated solutions of carbonic anhydrase. Biochim. biophys. Acta (Amst.)67, 31–41 (1963).Google Scholar
  12. Korhonen, E., andL. K. Korhonen: Histochemical demonstration of carbonic anhydrase activity in the eyes of rat and mouse. Acta ophthal. (Kbh.)43, 475–481 (1965).Google Scholar
  13. ——: Histochemical demonstration of carbonic anhydrase activity in mast cells. Experientia (Basel)21, 628 (1965a).Google Scholar
  14. ——: Electrophoretic and histochemical studies of carbonic anhydrase activity. Histochemie5, 279–288 (1965b).Google Scholar
  15. Korhonen, L. K., E. Näätänen, andM. Hyyppä: A histochemical study of carbonic anhydrase in some parts of the mouse brain. Acta histochem. (Jena)18, 336–347 (1964).Google Scholar
  16. Maren, T. H.: Carbonic anhydrase inhibition. V. N5-substituted 2-acetylamino-1,3,4-thiadiazole-5-sulfonamides: Metabolic conversion and use as control substances. J. Pharmacol. exp. Ther.117, 385–401 (1956).Google Scholar
  17. —,V. I. Ash, andE. M. Baily jr.: Carbonic anhydrase inhibition. II. A method for determination of carbonic anhydrase inhibitors, particularly of Diamox®. Bull. Hopkins Hosp.95, 244–255 (1954).Google Scholar
  18. Mustakallio, K. K., J. Raekallio, andE. Raekallio: The histochemical demonstration of carbonic anhydrase. An attempt to localize its inhibition by acetazolamide (Diamox®) in rat kidney. Ann. Med. exp. Fenn.38, 247–251 (1960).Google Scholar
  19. Muther, T. F.: On the non-specificity of histochemical methods for carbonic anhydrase. Fed. Proc.25, 320 (1966).Google Scholar
  20. Nachlas, M. M., W. Prinn, andA. M. Seligman: Quantitative estimation of lyoand desmoenzymes in tissue sections with and without fixation. J. biophys. biochem. Cytol.2, 487–502 (1956).Google Scholar
  21. —,A. C. Young, andA. M. Seligman: Problems of enzymatic localization by chemical reactions applied to tissue sections. J. Histochem. Cytochem.5, 565–583 (1957).Google Scholar
  22. Waldeyer, A., andG. Häusler: Histochemische Studien über die Carboanhydraseaktivität der Samenwege und ihrer Anhangdrüsen von Mus rattus. Acta biol. med. germ.2, 568–589 (1959).Google Scholar
  23. Wistrand, P. J.: Comparison by direct measurement of sulfonamide carbonic anhydrase inhibition in whole cells and in enzyme solutions. In: Drugs and enzymes. Proc. of the 2nd Internat. Pharmacological Meeting, Prague 1963, vol. 2, p. 176–186. Oxford: Pergamon Press 1965.Google Scholar
  24. — andS. N. Rao: Immunological and kinetic properties of carbonic anhydrases from various tissues. Biochem, biophys. Acta (Amst.) (in press) (1967).Google Scholar

Copyright information

© Springer-Verlag 1967

Authors and Affiliations

  • Holger P. J. Hansson
    • 1
  1. 1.Departments of Pharmacology and DermatologyUniversity of UppsalaUppsalaSweden

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