Abstract
A simple separation method enabling the quantification of alkaline phosphatase activity in unfixed, isolated, individual, duodenal epithelial cells has been presented. The activity of intestinal brush border-bound alkaline phosphatase has been demonstrated using naphthol AS-BI phosphate as a substrate and hexazotized New Fuchsin as a simultaneous coupling agent. The amount of final reaction product, as measured cytophotometrically, increases linearly with incubation time (up to 10 min) and with substrate concentration (up to 0.4 mM). Maximum enzyme activity was obtained at pH 8.9. Variation of the substrate concentration revealed the kinetic parameters for naphthol AS-BI phosphate as Km = 0.17 ± 0.015 and Vmax = 13.9 ± 1.38. The specificity of the enzyme reaction was confirmed by the complete inhibition of the enzyme activity in the presence of l-cysteine (10 mm) and 80% inhibition with L - phenylalanine (30 mM). Comparison of alkaline phosphatase activity in 8-μm cryostat sections (beginning at the tip and proceeding to the cryptal part) along the villus axis, with the activity of individual cells obtained by successive separation, revealed similar values of the percentage quotient derived from the entire activities in these two different methods. This suggests that the presented separation procedure gives rise to isolation of the respective cells from the corresponding areas of the villus. Finally, the isolated cells can be used as a valuable tool for the quantitative analysis of alkaline phosphatase activity along the length of the villus.
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Alpers, D.H., Zhang, Y. & Ahnen, D.J. (1995) Synthesis and parallel secretion of rat intestinal alkaline phosphatase and surfactant-like particle protein. Am. J. Physiol. 268, E1205-14.
Bader, C.A., Ben nasr, L., Monet, J.-D., Bachlet, M., Assaily, J. & Ulmann, A. (1984) In situ biochemical studies of intestinal alkaline phosphatase in normal and phosphate-depleted rats by microdensitometry. J. Biol. Chem. 259, 11658-61.
Ben nasr, L., Monet, J.-D. & Lucas, A. (1988) Rapid (10-minute) stimulation of rat duodenal alkaline phosphatase activity by 1,25-dihydroxyvitamin D3. Endocrinology 123, 1778-82.
Bikle, D.D., Zolock, D.T. & MUNSON, S. (1984) Differential response of duodenal epithelial cells to 1,25-dihydroxyvitamin D3 according to position on the villus: a comparison of calcium uptake, calcium-binding protein, and alkaline phosphatase activity. Endocrinology 115, 2077-84.
Burstone, M.S. (1962) Enzyme Histochemistry and its Application in the Study of Neoplasm. New York: Academic Press.
Buts, J.-P., Dkeyser, N., Kolanowski, J. & Van Hoof, F. (1990) Hormonal regulation of the rat small intestine: responsiveness of villus and crypt cells to insulin during the suckling period and unresponsiveness after weaning. Pediatr. Res. 27, 161-4.
Chayen, J., Bitensky, L. & Butcher, R.G. (1973) Practical Histochemistry, pp. 106-10. London, New York: John Wiley & Sons.
Fernley, H.N. & Walker, P.G. (1970) Inhibition of alkaline phosphatase by L-phenylalanine. Biochem. J. 116, 543-50.
Frederiks, W.M., Marx, F., Jonges, G.N. & Van Noorden, C.J.F. (1987) Quantitative histochemical study of acid phosphatase activity in rat liver using a semipermeable membrane technique. J. Histochem. Cytochem. 35, 175-80.
Gutschmidt, S., Kaul, W. & Riecken, E.O. (1979) A quantitative histochemical technique for the characterisation of á-glucosidases in the brush-border membrane of rat jejunum. Histochemistry 63, 81-101.
Gutschmidt, S., Lange, U. & Riecken, E.O. (1980) Kinetic characterization of unspecific alkaline phosphatase at different villus sites of rat jejunum. Histochemistry 69, 189-220.
Gutschmidt, S., Lange, U. & Riecken, E.O. (1981) ‘In situ’ - measurements of protein contents in the brush border region along rat jejunal villi and their correlations with four enzyme activities. Histochemistry 72, 467-79.
Karasaki, S. (1975) Cell proliferation and subcellular localization of alkaline phosphatase activity in rat liver parenchyma during azo dye carcinogenesis. Cancer Res. 35, 482-91.
Hugon, J. & Borgers, M. (1966) Ultrastructural localisation of alkaline phosphatase activity in the absorbing cells of the duodenum of mouse. J. Histochem. Cytochem. 14, 629-34.
Lojda, Z., Ploeg, M. & Duijn, P. (1967) Phosphatase of the naphthol AS series in the quantitative determination of alkaline and acid phosphatase activities ‘in situ’ studied in polyacrylamide membrane model system and by cytospectrophotometry. Histochemie 11, 13-32.
Lojda, Z., Grossrau, R. & Schibler, T.H. (1979) Enzyme Histochemistry, pp. 59-70. Berlin, Heidelberg, New York: Springer-Verlag.
Lojda, Z., Petrasko, M., HavrÁnkovÁ, E. & Lojda, L. (1984) Early postnatal development of the brush-border enzymes of enterocytes in the rat and mini-pig. Histochem. J. 16, 364-9.
Mayahara, H., Hirano, H., Saito, T. & Ogawa, K. (1967) The new lead citrate method for the ultra-cytochemical demonstration of activity of non-specific alkaline phosphatase (orthophosphoric monoester phosphohydrolase). Histochemie 11, 88-96.
Miura, S., Asokura, H., Miyairy, M., Morishita, T., Nagatha, H. & Tsuchiya, M. (1982a) Effect of colchicine on intestinal alkaline phosphatase activity during linoleic acid absorption in rat. Digestion 23, 224-31.
Miura, S., Asakura, H., Morishita, T., Hibi, T, Munakata, Y., Kobayashi, K. & Tsuchiya, M. (1982b) Changes in intestinal alkaline phosphatase activity in cholera toxine-treated rats. Gut 23, 507-12.
Nakae, Y. & Stoward, P. J. (1992) Initial reaction kinetics of succinate dehydrogenase in mouse liver studied with a real-time image analyser system. Histochemistry 98, 7-12.
Nakae, Y. & Stoward, P. J. (1993) Kinetic analysis of lactate dehydrogenase in situ in mouse liver determined with a quantitative histochemical technique. Histochem. J. 25, 206-12.
Nakae, Y. & Stoward, P. J. (1994) The diverse Michaelis constants and maximum velocities of lactate dehydrogenase in situ in various types of cell. Histochem. J. 26, 292-7.
Nakae, Y. & Stoward, P. J. (1995) The kinetics of enzymes in situ, with special reference to lactate and succinate dehydrogenases. Histol. Histopathol. 10, 463-79.
Nisselbaum, J.S. & Bodansky, O. (1963) Purification, kinetic, and immunochemical studies of the major variants of lactic dehydrogenase from human liver, hepatoma, and erythrocytes; comparison with the major variant of human heart lactic dehydrogenase. J. Biol. Chem. 238, 969-74.
Ono, K. (1979) Ultrastructural localization of alkaline phosphatase activity in small intestinal absorptive cell of adult rats. Histochem. J. 62, 113-19.
Raul, F., Simon, P., Kedinger, M. & Haffen, K. (1977) Intestinal enzymes activities in isolated villus and crypt cells during postnatal development of the rat. Cell. Tissue Res. 176, 167-78.
Saini, P.K. & Done, J. (1972) The diversity of alkaline phosphatase from rat intestine. Isolation and purification of the enzyme(s). Biochim. Biophys. Acta 258, 147-53.
Stigelmair-Herb, M.T., Pospischil, A., Hess, G., Bachmann, P.A. & Baljer, G. (1986) Enzyme histochemistry of the small intestinal mucous in experimental infections of calves with rotavirus and enterotoxigenic Escherichia coli. Vet. Pathol. 23, 125-31.
Susheela, L., Venkatesan, K. & Ramasarma, T. (1977) Structural and kinetic studies on the activators of succinate dehydrogenase. Biochim. Biophys. Acta 480, 47-55.
Vallet, J., Ronanet, J.-M. & Besancon, P. (1994) Dietary grape seed tannins: effects on nutritional balance and on some enzymatic activities along the cryptal-villus axis of rat small intestine. Ann. Nutr. Metab. 38, 75-84.
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.
Van Noorden, C.J.F. & Vogels, I.M.C. (1989) Polyvinyl alcohol and other tissue protectants in enzyme histochemistry: a consumer's guide. Histochem. J. 21, 373-9.
Van Noorden, C. J.F., Vogels, I.M.C. & Houtkooper, J.M. (1988) Cytophotometric analysis of alkaline phosphatase and 5'-nucleotidase activity in regenerating rat liver after partial hepatectomy. Cell. Biochem. Funct. 6, 53-60.
Weiser, M.M. (1973) Intestinal epithelial cell surface membrane glycoprotein synthesis. I. An indication of cell differentiation. J. Biol. Chem. 248, 2536-41.
Zhang, Y., Shao, J.-S., Xie, Q.-M. & Alpers, D. (1996) Immunolocalisation of alkaline phosphatase and surfactant-like particle proteins in rat duodenum during fat absorption. Gastroenterology 110, 478-88.
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Mozeš, Š., Lenhardt, Ľ. & Martinková, A. A Quantitative Histochemical Study of Alkaline Phosphatase Activity in Isolated Rat Duodenal Epithelial Cells. Histochem J 30, 583–589 (1998). https://doi.org/10.1023/A:1003231100654
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DOI: https://doi.org/10.1023/A:1003231100654