Skip to main content
SpringerLink
Log in

Über den Histochemischen Nachweis der β-Glucuronidase, α-Mannosidase und α-Galactosidase mit 1-Naphthylglykosiden

On the histochemical demonstration of β-glucuronidase, α-mannosidase and α-galactosidase using 1-naphthyl glycosides

  • Published:
Histochemie Aims and scope Submit manuscript

Summary

By means of simultaneous azo coupling using 1-naphthyl glycosides as substrates the distribution and activity of β-glucuronidase, α-mannosidase and α-galactosidase have been investigated in rats, mice and guinea-pigs.

For β-glucuronidase the incubation medium consists of 5–10 mg 1-naphthyl-β-glucuronide (dissolved in 0.4 ml NN-dimethyl formamide) and 0.6 ml 2% hexazonium-p-rosaniline in 9 ml 0.2 M acetate buffer, pH 5; for α-mannosidase and α-galactosidase of the same quantities of 1-naphthyl-α-mannoside and α-galactoside respectively and p-rosaniline in 9 ml 0.1 M citrate, citric acid-phosphate or acetate buffer, pH 5. Qualitative and quantitative inhibition tests using various 1–4 lactones and galactose prove the reaction specifity of the methods presented here.

β-Glucuronidase can be detected especially in lysosomes of rat organs, e.g. kidney, epididymis, uterus, vesicular gland, intestine and respiratory tract; tissues from mice and guineapigs exhibit a slower splitting rate for 1-naphthyl glucuronide. As to α-mannosidase its lysosomal localization becomes apparent in many organs also by means of histochemistry. The urogenital system, intestine and the salivary glands belong to the structures with the highest amount of α-mannosidase, and in the mouse kidney sex differences occur. For the first time α-galactosidase can be demonstrated unequivocally in the lysosomes of rat, mouse and guineapig tissues in which this enzyme displays a high overall activity. 6-Br-2-Naphthyl-α-galactoside and Fast Blue B for postcoupling are not able to detect the lysosomal localization of α-galactosidase.

Fluorometric measurements of these 3 glycosidases by means of the corresponding 1-naphthyl glycoside reveal inhibition rates between 90 and 98% following fixation in formol or glutaraldehyde. Washing in sugar solution raises enzyme activity two or three times.

Zusammenfassung

Mit simultanen Azokupplungsverfahren und 1-Naphthylglykosiden als Substraten werden Verteilung und Aktivität von β-Glucuronidase, α-Mannosidase und α-Galactosidase bei Ratte, Maus und Meerschweinchen untersucht.

Für die β-Glucuronidase besteht das Inkubationsmedium aus 5–10 mg 1-Naphthylβ-glucuronid (gelöst in 0.4 ml NN-Dimethylformamid) und 0.6 ml 2% Hexazonium-p-rosanilin in 9 ml 0.2 M Acetat-Puffer, pH 5; für die α-Mannosidase und α-Galactosidase aus der gleichen Menge 1-Naphthyl-α-mannosid bzw. -α-galactosid und p-Rosanilin in 9 ml 0.1 M CitratCitronensäure-Phosphatoder Acetat-Puffer, pH 5 bzw. 5.2. Die Spezifität der Nachweisreaktionen sichern qualitative und quantitative Hemmversuche mit verschiedenen 1–4-Lactonen und Galactose ab.

Die β-Glucuronidase kann bei der Ratte vor allem intralysosomal nachgewiesen werden, z.B. in Niere, Nebenhoden, Uterus, Samenblase, Darm und Respirationstrakt; Mäuseund Meerschweinchengewebe setzen 1-Naphthyl-β-glucuronid langsamer um. Für die α-Mannosidase läßt sich in zahlreichen Organen auch histochemisch die lysosomale Lokalisation des Enzyms beweisen, wobei die Aktivität in Urogenitalsystem, Darm und Speicheldrüsen besonders hoch ist, und in der Mäuseniere geschlechtsspezifische Unterschiede vorkommen. Erstmalig wird die intralysosomale Lokalisation der α-Galactosidase gezeigt, die ubiquitär in teilweise hoher Aktivität anzutreffen ist. 6-Br-2-Naphthyl-α-galactosid eignet sich in Verbindung mit Fast Blue B nicht zur intralysosomalen Lokalisation der α-Galaotosidase.

Fluorometrische Messungen aller 3 Glykosidasen mit dem entsprechenden 1-Naphthylglykosid ergeben nach Fixation in Formol oder Glutaraldehyd Hemmraten zwischen 90 und 98 %; anschließendes Waschen in Zuckerlösung verdoppelt oder verdreifacht die Restaktivität.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

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

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Literatur

  • Ballantyne, B., Bright, J. E.: The histochemical localization of β-D-glucuronidases in the rat kidney: a comparison of methods. Ann. Histoohim. 16, 313–322 (1971)

    Google Scholar 

  • Barman, Th. E.: Enzyme handbook, vol. II. Berlin-Heidelberg-New York: Springer 1969

    Google Scholar 

  • Barrett, A. J.: Properties of lysosomal enzymes. In: Lysosomes (Dingle, J. T., Fell, H. B., eds.), vol. I, p. 245–312. Amsterdam-London: North-Holland Publishing Co. 1969

    Google Scholar 

  • Beaufay, H.: Methods for the isolation of lysosomes. In: Lysosomes (Dingle, J. T., Fell, H. B., eds.), vol. II, p. 516–546. Amsterdam-New York: North-Holland Publishing Co. 1969

    Google Scholar 

  • Bergmeyer, H. U., Bernt, E., Gutman, I.: Raffinose. In: Methoden der enzymatischen Analyse (H. U. Bergmeyer, Hrsg.), Bd. II, S. 1139–1142. Weinheim: Chemie 1970

    Google Scholar 

  • Bowers, W. E., de Duve, C.: Lysosomes in lymphoid tissue. II. Intracellular distribution of acid hydrolases. J. Cell Biol. 32, 339–348 (1967)

    Google Scholar 

  • Bulmer, D. (1967), zit. nach Pearse (1972)

  • Burton, J. F., Pearse, A. G. E.: A critical study of the methods for histochemical localization of β-glucuronidases. Brit. J. exp. Path. 33, 87–97 (1952)

    Google Scholar 

  • Coleman, R. L.: Seperation of an α-galactosidase from rat uterus. Biochim. biophys. Acta (Amst.) 159, 192–193 (1968)

    Google Scholar 

  • Conchie, J., Findlay, J., Levvy, G. A.: Mammalian glycosidases. Distribution in the body. Biochem. J. 71, 318–325 (1959)

    Google Scholar 

  • Conchie, J., Hay, A. J.: Mammalian glycosidases. 2. Properties of α-mannosidase and β-galactosidase from rat epididymis. Biochem. J. 73, 327–334 (1959)

    Google Scholar 

  • Conchie, J., Hay, A. J.: Mammalian glycosidases. 4. Intracellular localization of β-galactosidase, α-mannosidase, β-N-acetylglucosaminidase and α-L-fucosidase in mammalian tissues. Biochem. J. 87, 354–361 (1963)

    Google Scholar 

  • Conchie, J., Hay, A. J., Levvy, G. A.: 3. The intracellular localization of β-glucuronidase in different mammalian tissues. Biochem. J. 79, 324–330 (1961)

    Google Scholar 

  • Courtois, J. E., Petek, F.: α-Galactosidase from coffee beans. In: Methods in enzymology (Colowick, S. P., Kaplan, N. O., eds.), vol. VIII. Complex carbohydrates, p. 565–570. New York-London: Academic Press 1966

    Google Scholar 

  • Dott, H. M.: Lysosomes and lysosomal enzymes in the reproductive tract. In: Lysosomes (Dingle, J. T., Fell, H. B., eds.), vol. II, p. 330–360. Amsterdam-New York: NorthHolland Publishing Co. 1969

    Google Scholar 

  • Fishman, W. H., Baker, J. R.: Cellular localization of β-glucuronidase in rat tissues. J. Histochem. Cytochem. 4, 570–587 (1956)

    Google Scholar 

  • Fishman, W. H., Goldman, S. S., De Lellis, R.: Dual localization of β-glucuronidase in endoplasmic reticulum and in lysosomes. Nature (Lond.) 213, 457–460 (1967)

    Google Scholar 

  • Fishman, W. H., Ide, H., Rufo, T.: Dual localization of acid hydrolases in endoplasmic reticulum and in lysosomes. I. β-glucuronidase staining reactions and cytochemical studies in androgen-stimulated mice. Histochemie 20, 287–299 (1969)

    Google Scholar 

  • Ginsburg, V., Neufeld, E. F.: Complex heterosaccharides of animals. Ann. Rev. Biochem. 38, 371–388 (1969)

    Google Scholar 

  • Gossrau, R.: Über den histochemischen Nachweis der β-Glucosidase mit 1-NaphthyI-β-glucopyranosid. Histochemie 34, 163–176 (1973a)

    Google Scholar 

  • Gossrau, R.: Über die β-Glucosidase und Lactase im Darm von Vertebraten. Histochemie 35, 143–151 (1973b)

    Google Scholar 

  • Gossrau, R.: Über den histochemischen und mikrochemischen Nachweis der β-Galactosidase mit 1-Naphthyl-β-galactopyranosid. Histochemie 35, 199–218 (1973c)

    Google Scholar 

  • Gossrau, R.: Untersuchung der N-Acetyl-β-glucosaminidase mit 1-Naphthyl-N-acetylβ-glucosaminid. Histochemie (1973d, im Druck)

  • Gossrau, R.: Zur Spaltung von Naphthol-AS-BI-β-galactopyranosid durch die saureβ-Galaotosidase. Histochemie (1973e, im Druck)

  • Hayashi, M.: Distribution of β-glucuronidase activity in rat tissues employing the naphthol AS-BI glucuronide hexazonium pararosanilin method. J. Histochem. Cytochem. 12, 659–669 (1964)

    Google Scholar 

  • Hayashi, M.: Comparative histochemical localization of lysosomal enzymes in rat tissues. J. Histochem. Cytochem. 15, 83–92 (1967)

    Google Scholar 

  • Hayashi, M., Nakajima, Y., Fishman, W. H.: The cytologic demonstration of β-glucuronidase employing naphthol AS-BI and hexazonium-p-rosanilin; a preliminary report. J. Histochem. 12, 293–297 (1964)

    Google Scholar 

  • Hayashi, M., Shirahama, T., Cohen, A. S.: Combined cytochemical and electron microscopic demonstration of β-glucuronidase activity in rat liver with the use of a simultaneous coupling azo dye technique. J. Cell Biol. 36, 289–297 (1968)

    Google Scholar 

  • Hopwood, D.: Some aspects of fixation with glutaraldehyde. J. Anat. (Lond.) 101, 83–92 (1967)

    Google Scholar 

  • Janigan, D. T.: Tissue enzyme fixation studies. I. The effects of aldehyde fixation on β-glucuronidase, β-galactosidase, N-acetyl-β-glucosaminidase and β-glucosidase in tissue blocks. Lab. Invest. 13, 1038–1050 (1964)

    Google Scholar 

  • Levvy, G. A.: The preparation and properties of β-glucuronidase. 4. Inhibition by sugar acids and their lactones. Biochem. J. 52, 464–472 (1952)

    Google Scholar 

  • Levvy, G. A., Conchie, J.: Mammalian glycosidases and their inhibition by aldonolactones. In: Methods in enzymology (Colowick, S. P., Kaplan, N. O., eds.), vol. VIII. Complex carbohydrates, p. 571–584. New York-London: Academic Press 1966

    Google Scholar 

  • Levvy, G. A., Hay, A. J., Conchie, J.: Inhibition by aldonolactones of corresponding configuration. 4. Inhibitors of mannosidase and glucosidase. Biochem. J. 91, 378–384 (1964)

    Google Scholar 

  • Livingston, D. C., Coombs, M. M., Pranks, L. M., Maggi, V., Gahan, P. B.: A lead phthalocyanin method for the demonstration of acid hydrolases in plant and animal tissues. Histochemie 18, 48–60 (1969)

    Google Scholar 

  • Lojda, Z.: Some remarks concerning the histochemical detection of disaccharidases and glucosidases. Histochemie 5, 339–360 (1965)

    Google Scholar 

  • Lojda, Z.: Indigogenic methods for glycosidases. I. An improved method for β-D-glucosidase and its application to localization studies of intestinal and renal enzymes. Histochemie 22, 347–361 (1970a)

    Google Scholar 

  • Lojda, Z.: Indigogenic methods for glycosidases. II. An improved method for β-D-galactosidase and its application to localization studies of the enzymes in the intestine and in other tissues. Histochemie 23, 266–288 (1970b)

    Google Scholar 

  • Lojda, Z.: Indigogenic methods for glycosidases. IV. An improved method for β-glucuronidase. Histochemie 27, 182–192 (1971)

    Google Scholar 

  • Lojda, Z.: Aktuelle Probleme der Cytochemie der lysosomalen Hydrolasen. Acta morph. Acad. Sci. hung. (1972, im Druck)

  • Lojda, Z., Kraml, J.: Indigogenic methods for glycosidases. III. An improved method with 4-Cl-5-Br-3-indolyl-β-D-fucoside and its application in studies of enzymes in the intestine, kidney, and other tissues. Histochemie 25, 195–207 (1971)

    Google Scholar 

  • Lojda, Z., Slabý, J., Kraml, J., Kolinská, J.: Synthetic substrates in the histochemical demonstration of intestinal disaccharidases. Histochemie 34, 361–369 (1973)

    Google Scholar 

  • Lojda, Z., Večerek, B., Pelichova, H.: Some remarks concerning the histochemical detection of acid phosphatase by azo coupling reactions. Histochemie 3, 428–454 (1964)

    Google Scholar 

  • Meijer, A. E. F. H.: Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. I. Acid phosphatase. Histochemie 30, 31–39 (1972)

    Google Scholar 

  • Meijer, A. E. F. H., Vloedman, A. H. T.: Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. II. Nonspecific esterase and β-glucuronidase. Histochem. 34, 127–134 (1973)

    Google Scholar 

  • Monis, B., Tsou, K. C., Seligman, A. M.: Development of a histochemical method for α-D-galactosidase and its distribution in the rat. J. Histochem. Cytochem. 11, 653–664 (1963)

    Google Scholar 

  • Pearse, A. G. E.: Azo dye methods in enzyme histoehemistry. Int. Rev. Cytol. 3, 329–358 (1954)

    Google Scholar 

  • Pearse, A. G. E.: Histoehemistry. Theoretical and applied, 3rd ed., vol. I, II. Churchill: London 1968, 1972

    Google Scholar 

  • Price, R. G., Dance, N.: The cellular distribution of some rat glycosidases. Bioehem. J. 105, 877–889 (1967)

    Google Scholar 

  • Pugh, D.: The fine structural localization of N-acetyl-β-glucosaminidase in rat tissues using an indoxyl substrate. Ann. Histochim. 17, 55–64 (1972a)

    Google Scholar 

  • Pugh, D.: The cytochemical localization of β-galactosidase. Ann. Histochim. 17, 89–90 (1972b)

    Google Scholar 

  • Pugh, D., Walker, P. G.: The localization of N-acetyl-β-glucosaminidase in tissues. J. Histochem. 9, 242–250 (1961)

    Google Scholar 

  • Bath, F. W., Otto, L.: Zur histochemischen Darstellung der β-Glucuronidase. Acta histochem. (Jena) 25, 355–362 (1966)

    Google Scholar 

  • Robinson, D., Price, R. G., Dance, N.: Seperation and properties of β-galactosidase, β-glucuronidase and N-acetyl-β-glucosaminidase from rat kidney. Bioehem. J. 102, 525–532 (1967)

    Google Scholar 

  • Seligman, A. M., Nachlas, M. M., Mannheimer, L. H., Friedman, O. M., Wolf, G. (1949), zit. nach Pearse (1972)

  • Seligman, A. M., Tsou, K.-C., Rutenburg, S. H., Cohen, R. B.: Histochemical demonstration of β-D-glucuronidase with a synthetic substrate. J. Histochem. Cytochem. 2, 209–229 (1954)

    Google Scholar 

  • Smith, R. E., Fishman, W. H.: p-(acetoxymercuric) aniline diazotate, a reagent for visualizing the naphthol AS-BI product of acid hydrolase action at the level of light and electron microscopy. J. Histochem. Cytochem. 17, 1–22 (1969)

    Google Scholar 

  • Snaith, S. M., Levvy, G. A.: α-Mannosidase as a zinc-dependent enzyme. Nature (Lond.) 218, 91–92 (1968)

    Google Scholar 

  • Spiro, R. G.: Glycoproteins. Ann. Rev. Bioehem. 39, 599–638 (1970)

    Google Scholar 

  • Tappel, A. L.: Lysosomal enzymes and other components. In: Lysosomes (Dingle, J. T., Fell, H. B., eds.), vol. I, p. 207–244. Amsterdam-New York: North-Holland Publishing Co. 1969

    Google Scholar 

  • Verity, M. A., Caper, V. R., Brown, W. J.: Spectrofluorometric determination of β-glucuronidase activity. Arch. Bioehem. Biophys. (Amst.) 106, 386–393 (1964)

    Google Scholar 

  • Winckler, J.: Zum Einfrieren von Gewebe in Stickstoff-gekühltem Propan. Histochemie 23, 44–50 (1970)

    Google Scholar 

  • Woessner, F. J.: The physiology of the uterus and mammary gland. In: Lysosomes (Dingle, J. T., Fell, H. B., eds.), vol. II, p. 299–329. Amsterdam-New York: North-Holland Publishing Co. 1969

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Anatomisches Institut der Universität Würzburg, Bundesrepublik Deutschland

    R. Gossrau

Authors
  1. R. Gossrau
    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. Über den Histochemischen Nachweis der β-Glucuronidase, α-Mannosidase und α-Galactosidase mit 1-Naphthylglykosiden. Histochemie 36, 367–381 (1973). https://doi.org/10.1007/BF00305715

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00305715

Search

Navigation