Summary
Freeze-dried frozen sections are floated on the surface of the freshly prepared incubation mixture (CoSO4 1.75 × 10−3 M, H2SO4 5.3 × 10−2 M, NaHCO3 1.57 × 10−2 M and KH2PO4 1.17 to 11.7 × 10−3 M; 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.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Bleyl, U.: Zur Spezifität des histoohemischen Carboanhydratasenachweises im Inselorgan der Bauchspeicheldrüse. Histochemie4, 286–311 (1964).
Datta, P. K., andT. H. Shepard: Intracellular localization of carbonic anhydrase in rat liver and kidney tissues. Arch. Biochem.81, 124–129 (1959).
Davenport, H. W.: Gastric carbonic anhydrase. J. Physiol. (Lond.)97, 32–43 (1939).
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).
Giacobini, E.: A cytochemical study of the localization of carbonic anhydrase in the nervous system. J. Neurochem.9, 169–177 (1962).
Hansson, H. P. J.: Demonstration of carbonic anhydrase by means of fluorescent antibodies in human erythrocytes. Life Sci.4, 965–968 (1965).
Häusler, G.: Zur Technik und Spezifität des histochemischen Carboanhydrasenachweises im Modellversuch und in Gewebsschnitten von Rattennieren. Histochemie1, 29–47 (1958).
Karler, R., andD. M. Woodbury: Intracellular distribution of carbonic anhydrase. Biochem. J.75, 538–543 (1959).
Kern, D. M.: The hydration of carbon dioxide. J. Chem. Educat.37, 14–23 (1960).
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).
—,W. W. Forrest, andF. J. W. Roughton: The activity of concentrated solutions of carbonic anhydrase. Biochim. biophys. Acta (Amst.)67, 31–41 (1963).
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).
——: Histochemical demonstration of carbonic anhydrase activity in mast cells. Experientia (Basel)21, 628 (1965a).
——: Electrophoretic and histochemical studies of carbonic anhydrase activity. Histochemie5, 279–288 (1965b).
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).
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).
—,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).
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).
Muther, T. F.: On the non-specificity of histochemical methods for carbonic anhydrase. Fed. Proc.25, 320 (1966).
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).
—,A. C. Young, andA. M. Seligman: Problems of enzymatic localization by chemical reactions applied to tissue sections. J. Histochem. Cytochem.5, 565–583 (1957).
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).
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.
— andS. N. Rao: Immunological and kinetic properties of carbonic anhydrases from various tissues. Biochem, biophys. Acta (Amst.) (in press) (1967).
Author information
Authors and Affiliations
Additional information
This investigation was supported by grants from the Medical Faculty, University of Uppsala and from the U.S. National Institutes of Health (Grant NB 3060 to E.Bárány).
Rights and permissions
About this article
Cite this article
Hansson, H.P.J. Histochemical demonstration of carbonic anhydrase activity. Histochemie 11, 112–128 (1967). https://doi.org/10.1007/BF00571716
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00571716