Summary
5′-Nucleotidase (EC 3.1.3.5) activity was demonstrated in cryostat sections of rat liver using the Wachstein—Meisel medium and polyvinyl alcohol as tissue stabilizer. Optimum activity was obtained using an incubation medium containing 5mm AMP, 10mm magnesium chloride, 7.2mm lead nitrate, 0.1m Tris—maleate buffer, pH 7.2, and 17% (w/v) polyvinyl alcohol (Sigma, type III). The activity was localized at the bile canalicular and sinusoidal side of the plasma membranes of liver parenchymal cells as well as in the plasma membranes of endothelial cells of central veins and in fibroblasts surrounding portal tracts. The reaction was specific for 5′-nucleotidase because it was inhibited by ADP. Alkaline phosphatase did not interfere in the reaction. Cytophotometric analysis revealed a linear relationship between the formation of the final reaction product and incubation times up to 20 min and section thicknesses up to 8εm. The activity in pericentral zones was 1.35 times the activity in periportal zones. The Michaelis constant for AMP was 1.4mm in pericentral zones and 0.8mm in periportal zones, suggesting that the bile canalicular and sinusoidal enzymes differ in their kinetic characteristics.
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References
ABRAHAMSON, D. E., RIGATUSO, J. L. & LAZAROW, A. (1969) Cytophotometric quantitation of glucose-6-phosphatase activity in rat liver.J. Histochem. Cytochem. 17, 107–9.
ALTMAN, F. P. (1980) Tissue stabilizer methods in histochemistry. InTrends in Enzyme Histochemistry and Cytochemistry, pp. 81–101. Amsterdam: Excerpta Medica.
ALTMAN, F. P. & CHAYEN, J. (1965) Retention of nitrogenous material in unfixed sections during incubations for histochemical demonstration of enzymes.Nature 207, 1205–6.
ARCH, J. R. S. & NEWSHOLME, E. A. (1978) The control of the metabolism and the hormonal role of adenosine.Essays Biochem. 14, 82–123.
BARON, M. D., POPE, B. & LUZIO, J. P. (1986) The membrane topography of ecto-5′-nucleotidase in rat hepatocytes.Biochem. J. 236, 495–502.
BERMAN, J. J., TONG, C. & WILLIAMS, G. M. (1980) 5′-Nucleotidase activities in cultured rat liver epithelial and fibroblast cells.J. Histochem. Cytochem. 28, 174–7.
BORGERS, M. & THONÉ, F. (1975) The inhibition of alkaline phosphatase byl-p-bromotetramisole.Histochemistry 44, 277–80.
BURGER, R. M. & LOWENSTEIN, J. M. (1970) Preparation and properties of 5′-nucleotidase from smooth muscle of small intestine.J. Biol. Chem. 245, 6274–80.
BURGER, R. M. & LOWENSTEIN, J. M. (1975) 5′-Nucleotidase from smooth muscle of small intestine and from brain. Inhibition by nucleotides.Biochemistry 14, 2362–6.
BUTCHER, R. G. (1971) Tissue stabilisation during histochemical reactions: The use of collagen polypeptides.Histochemie 28, 231–5.
CABRINI, R. L., FRASCH, A. C. C. & ITOIZ, M. E. (1975) A quantitative microspectrophotometric study of the lead precipitation reaction for the histochemical demonstration of acid phosphatase.Histochem. J. 7, 419–26.
CAMPBELL, D. M. (1962) Determination of 5′-nucleotidase in blood serum.Biochem. J. 82, 34p.
CHAYEN, J., FROST, G. T. B., DODDS, R. A., BITENSKY, L., PITCHFORK, J., BAYLISS, P. H. & BARNETT, R. J. (1981) The use of a hidden metal-capture reagent for the measurement of Na+-K+-ATPase activity: a new concept in cytochemistry.Histochemistry 71, 533–41.
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.
FARQUHAR, M. G., BERGERON, J. J. M. & PALADE, G. E. (1974) Cytochemistry of Golgi fractions prepared from rat liver.J. Cell Biol. 60, 8–25.
FREDERIKS, W. M., MARX, F., BOSCH, K. S. & VAN NOORDEN, C. J. F. (1987) Diurnal variation in 5′-nucleotidase activity in rat liver. A quantitative histochemical study.Histochemistry 87, 439–43.
FRITZSON, P., HAUGEN, T. B. & TJERNSHAUGEN, H. (1986) The presence and activity in normal and regenerating rat liver postmicrosomal supernatant fraction of an enzyme with properties similar to those of membranebound 5′-nucleotidase.Biochem. J. 239, 185–90.
HARDONK, M. J. (1968) 5′-Nucleotidase. I. Distribution of 5′-nucleotidase in tissues of rat and mouse.Histochemie 12, 1–17.
HARDONK, M. J. & DE BOER, H. G. A. (1968) 5′-Nucleotidase. III. Determinations of 5′-nucleotidase isoenzymes in tissues of rat and mouse.Histochemie 12, 29–41.
HARDONK, M. J., HAARSMA, T. J., DIJKHUIS, F. W. J., POEL, M. & KOUDSTAAL, J. (1977) Influence of fixation and buffer treatment on the release of enzymes from the plasma membrane.Histochemistry 54, 57–66.
HENDERSON, B., JOHNSTONE, J. J. & CHAYEN, J. (1980) 5′-Nucleotidase activity in the human synovial lining in rheumatoid arthritis.Ann. Rheum. Dis. 39, 248–52.
HENDERSON, B., LOVERIDGE, N. & ROBERTSON, W. R. (1978) A quantitative study of the effects of different grades of polyvinyl alcohol on the activities of certain enzymes in unfixed tissue sections.Histochem. J. 10, 453–63.
JAMES, J. BOSCH, K. S., FRONIK, G. M. & HOUTKOOPER, J. M. (1986) Naphthol Yellow S protein content of individual hepatocytes as related to food intake and shortterm starvation.Cell Tissue Res.,243, 165–9.
KAULEN, H. D., HENNING, R. & STOFFEL, W. (1970) Comparison of some enzymes of the lysosomal and the plasma membranes of the rat liver cell.Hoppe-Seyler's Z. Physiol. Chem. 351, 1555–63.
KLAUSHOFER, K. & VON MAYERSBACH, H. (1977) 5′-Nucleotidase der Rattenleber. Spezifizitätsprobleme und rhythmische Veränderungen.Acta Histochem., Suppl. 18, 159–68.
KLAUSHOFER, K. & VON MAYERSBACH, H. (1979) Freeze substituted tissue in 5′-nucleotidase histochemistry. Comparative histochemical and biochemical investigations.J. Histochem. Cytochem. 27, 1582–7.
MCMILLAN P. J. (1967) Differential demonstration of muscle and heart type lactic dehydrogenase of rat muscle and kidney.J. Histochem. Cytochem. 15, 21–31.
LOJDA, Z., GOSSRAU, R. & SCHIEBLER, T. H. (1979)Enzyme Histochemistry. A Laboratory Manual. Berlin, Heidelberg, New York: Springer Verlag.
MEIJER, A. E. F. H. (1972) Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. I. Acid phosphatase.Histochemie 30, 31–9.
NAKAMURA, S. (1976) Effect of sodium deoxycholate on 5′-nucleotidase.Biochem. Biophys. Acta 426, 339–47.
SONG, C. S. & BODANSKY, O. (1967) Subcellular localization and properties of 5′-nucleotidase in the rat liver.J. Biol. Chem. 242, 694–9.
STANLEY, K. K., EDWARDS, M. R. & LUZIO, J. P. (1980) Subcellular distribution and movement of 5′-nucleotidase in rat cells.Biochem. J. 186, 59–69.
STOWARD, P. J. (1980) Criteria for the validation of quantitative histochemical enzyme techniques. InTrends in Enzyme Histochemistry and Cytochemistry, pp. 11–31. Amsterdam: Excerpta Medica.
TÄLJEDAL, I. (1969) Kinetics of glucose-6-phosphatase in pancreatic islets as revealed by staining histochemistry.Histochemie 19, 355–62.
TUCZEK, H. V., FRITZ, P., GRAU, A., MISCHLINSKY, A., WAGNER, T. & WEGNER, G. (1985) Distribution of albumin and regenerating livers of mice.Histochemistry 83, 165–9.
UUSITALO, R. J. (1981) 5′-Nucleotidase activity in lymphocytes.Histochem. J. 13, 525–34.
VAN DEN BOSCH, R., GEUZE, H. J., DU MAINE, A. P. M. & STROUS, G. J. (1986) Transport and metabolism of 5′-nucleotidase in rat hepatoma cell line.Eur. J. Biochem. 160, 49–54.
VAN NOORDEN, C. J. F. & BUTCHER, R. G. (1986) The ‘out of range’ error in microdensitometry.Histochem. J. 18, 397–8.
VAN NOORDEN, C. J. F. & BUTCHER, R. G. (1988) Quantitative enzyme histochemistry. InHistochemistry: Theoretical and Applied, 4th edn. (edited by STOWARD, P. J. & PEARSE, A. G. E.), Vol. III. Edinburgh: Churchill Livingstone.
VAN NOORDEN, C. J. F., VOGELS, I. M. C. & HOUTKOOPER, J. M. (1987) Cytophotometric analysis of alkaline phosphatase and 5′-nucleotidase activity in regenerating rat liver after partial hepatectomy.Cell Biochem. Function 6, 53–60.
WACHSTEIN, M. & MEISEL, E. (1957) Histochemistry of hepatic phosphatase at a physiologic pH.Amer. J. Clin. Pathol. 27, 13–23.
WADA, I., ETO, S., HIMERO, M. & KATO, K. (1987) 5′-Nucleotidase in rat liver lysosomes.J. Biochem. 101, 1077–85.
WATTIAUX, R., WATTIAUX-DE CONINCK, S., RONVEAUX-DUPAL, M. F. & DUBOIS, F. (1978) Isolation of rat liver lysosomes by isopycnic centrifugation in a metrizamide gradient.J. Cell Biol. 78, 349–68.
WIDNELL, C. C. (1972) Cytochemical localization of 5′-nucleotidase in subcellular fractions isolated from rat liver. I. The origin of 5′-nucleotidase activity in microsomes.J. Cell Biol. 52, 542–58.
WIDNELL, C. C. (1976) Soluble and membrane forms of 5′-nucleotidase in rat liver lysosomes.J. Cell Biol. 70, 98a.
WILKINSON, G. N. (1961) Statistical estimations in enzyme kinetics.Biochem. J. 80, 324–32.
YANNARELL, A. & ARONSON, N. N. (1973) Localization of an RNA-degrading system of enzymes on the rat liver plasma membrane.Biochim. Biophys. Acta 311, 191–204.
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Frederiks, W.M., Marx, F. A quantitative histochemical study of 5′-nucleotidase activity in rat liver using the lead salt method and polyvinyl alcohol. Histochem J 20, 207–214 (1988). https://doi.org/10.1007/BF01747465
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DOI: https://doi.org/10.1007/BF01747465