Histochemistry

, Volume 69, Issue 2, pp 189–202 | Cite as

Kinetic characterization of unspecific alkaline phosphatase at different villus sites of rat jejunum

A quantitative histochemical study
  • S. Gutschmidt
  • U. Lange
  • E. O. Riecken
Article

Summary

A quantitative histochemical method to determine the apparent Km and Vmax values of rat intestinal unspecific alkaline phosphatase at different sites of the villi is described. Naphthol-As-Bi-phosphate (0.025–1.5 mM) is employed as substrate and Fast Blue B as coupling reagent, and the resulting azo-dye in the brush border membrane has an absorbance maximum at λ550 nm. The ratio between the absorbance at λ550 and λ500 nm is constant as calculated from automatically recorded spectra at different intense dye deposits. Its absorbance is a linear function of incubation time up to 3 min and thickness of the slices up to 10 μm both with medium (0.5 mM) and high (1.5 mM) substrate concentrations. Using the histochemical assay under comparable conditions in test tube experiments with homogenates of intestinal mucosa an app. Km of 0.26±0.081 mM (weighted regression analysis) and 0.28–0.084 mM (direct linear plotting) is determined, demonstrating an affinity to the histochemical substrate, which is about 10 times higher than for p-Nitro-phenyl-phosphate with the purified enzyme.

The results obtained by scanning the total dye deposits along jejunal villi show considerable differences in enzymatic activity between single villi and an increase from the villus base up to the transition between medium and apical villus third. As well in the apical region as at the villus base saturation curves are obtained by determining the relationship between the absorbance and the substrate concentration under standard conditions (slice thickness 10 μm, incubation time 3 min, 37°C, pH 8.3). Calculated by weighted regression analysis and direct linear plotting from the absorbance data of six female rats the medium app. kinetic data ±SD from the jejunal villi read as follows. Apical: Km=0.81±0.438 mM, Vmax=3.99±1.217 absorbance units (A) and Km=0.87±0.428 mM, Vmax=4.02±1.191 A, respectively. Basal: Km=0.82±0.261 mM, Vmax=3.26±0.719 A and Km=0.77±0.184 mM, Vmax=3.04±0.518 AU, respectively. As demonstrated by factorial analysis of variance only Vmax is influenced by the villus position.

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References

  1. Alpers DH (1972) The relation of size to the relative rates of degradation of intestinal brush border proteins. J Clin Invest 51:2621–2630Google Scholar
  2. Batt RM, Peters TJ (1978) Analytical subcellular fractionation studies on enterocytes from the jejunum and ileum of the rat and some properties of brush border alkaline phosphatase. Clin Sci Mol Med 55:157–165Google Scholar
  3. de Both J, Dongen JM van, Hofwegen B van, Keulemans J, Visser WJ, Galjaard H (1974) The influence of various cell kinetic conditions on functional differentation in the small intestine of the rat. A study of enzymes bound to subcellular organelles. Dev Biol 38:119–137Google Scholar
  4. Burstone MS (1962) Enzyme histochemistry and its application in the study of neoplasms. Academic Press, New York LondonGoogle Scholar
  5. Cleland WW (1967) The statistical analysis of enzyme kinetica data. Adv Enzymol 29:1–32Google Scholar
  6. Davies NT, Flett AA (1978) The similarity between alkaline phosphatase EC. 3.1.3.1.) and phytase (EC. 3.1.3.8.) activities in rat intestine and their importance in phytase-induced zinc deficiency. Br J Nutr 39:307–316Google Scholar
  7. Dongen JM van, Kooyman J, Visser WJ, Holt SJ, Galjaard H (1977) The effect of increased crypt cell proliferation in the activity and subcellular localisation of esterases and alkaline phosphatase in the rat small intestine. Histochem J 9:61–75Google Scholar
  8. Duijn P van, Pascoe E, Ploeg M van der (1967) Theoretical and experimental aspects of enzyme determination in a cytochemical model system of polyacrylamide films containing alkaline phosphatase. J Histochem Cytochem 15:631–645Google Scholar
  9. Eisenthal R, Cornish-Bowden A (1974) The direct linear plot. A new graphical procedure for estimating enzyme kinetic parameters. Biochem J 139:715–720Google Scholar
  10. Fernley HN (1971) Mammalian alkaline phosphatase. In: Boyer PD (ed) The enzymes, Vol IV. Academic Press, New York London, pp 417–447Google Scholar
  11. Glickman RM, Alpers DH, Drummey GD, Isselbacher KJ (1970) Increased lymph alkaline phosphatase after fat feeding. Effects of medium chain triglycerides and inhibition of protein synthesis. Biochim Biophys Acta 201:226–235Google Scholar
  12. Gratecos D, Knibiehler M, Benoit V, Sémériva M (1978) Plasma membranes from rat intestinal epithelial cells at different stages of maturation. I. Preparation and characterization of plasma membrane subfractions originating from crypt cells and from villous cells. Biochim Biophys Acta 512:508–524Google Scholar
  13. Gutschmidt S, Kaul W, Riecken EO (1979) A quantitative histochemical technique for the characterization of α-glucosidases in the brush-border membrane of rat jejunum. Histochemistry 63:81–101Google Scholar
  14. Gutschmidt S, Emde C, Riecken EO (1980). Quantification of α-glucosidases along the villus of the small intestine in man. Introduction of a computerized histochemical method. Histochemistry 67:85–97Google Scholar
  15. Hopsu VK, McMillan PJ (1964) Quantitative characterization of a histochemical enzyme system. J Histochem Cytochem 12:315–324Google Scholar
  16. Hugon J, Borgers M (1967) Submicroscopic localization of the alkaline phosphatase activity in the duodenum of the rat. Exp Cell Res 45:698–702Google Scholar
  17. Humphreys MH, Chou LYN (1979) Anion-stimulated ATPase activity of brush border from rat small intestine. Am J Physiol 236:E70-E76Google Scholar
  18. Imre R, Pétro E (1979) Video scanning for the quantitative determination of alkaline phosphatase activity in the rat kidney. Acta Histochem 65:108–115Google Scholar
  19. Kaplan MM (1972) Progress in hepatology. Alkaline phosphatase. Gastroenterology 62:452–468Google Scholar
  20. Kaysen GA, Chou LY, Humphreys MH (1979) Requirement of Zn to demonstrate HCO3 -stimulated ATPase activity of rat small intestinal brush border. J Cell Biol 82:780–782Google Scholar
  21. Lison I (1968) Statistique appliquée à la biologie expérimentale. Gauthier-Villars, Paris, p 155Google Scholar
  22. Lojda Z, Ploeg M van der, Duijn P van (1967) Phosphates of the Naphthol-AS-series in the quantitative determination of alkaline and acid phosphatase activities “in situ” studied in polyacrylamide membrane model systems and by cytospectrophotometry. Histochemie 11:13–32Google Scholar
  23. Malik N, Butterworth PJ (1976) Molecular properties of rat intestinal alkaline phosphatase. Biochim Biophys Acta 446:105–114Google Scholar
  24. Moog F, Grey RD (1968) Alkaline phosphatase isoenzymes in the duodenum of the mouse: Attainment of a pattern of spatial distribution in the normal development and under the influence of cortisone or actinomycin D. Dev Biol 18:481–500Google Scholar
  25. Nakasaki H, Matsushima T, Sato S, Kawachi T (1979) Purification and properties of alkaline phosphatase from the mucosa of rat small intestine. J Biochem 86:1225–1231Google Scholar
  26. Neafsey PJ, Schwartz R (1977) Serum and duodenal alkaline phosphatase levels in fed and fasted magnesium deficient rats. J Nutr 107:1061–1067Google Scholar
  27. Nordström C, Dahlquist A, Josefsson L (1968) Quantitative determination of enzymes in different parts of the villi and crypts of the rat small intestine. Comparison of alkaline phosphatase, disaccharidases and dipeptidases. J Histochem Cytochem 15:713–721Google Scholar
  28. Ploeg Mvd, Duijn P van (1968) Cytophotometric determination of alkaline phosphatase activity of individual neutrophilic leucocytes with a biochemically calibrated model system. J Histochem Cytochem 16:693–706Google Scholar
  29. Raul F, Simon P, Kedinger M, Haffen K (1977) Intestinal enzymes activities in isolated villus and crypt cells curing postnatal development of the rat. Cell Tissue Res 176:167–178Google Scholar
  30. Righetti ABB, Kaplan MM (1971) The, origin of the serum alkaline phosphatase in normal rats. Biochim Biophys Acta 230:504–509Google Scholar
  31. Saini PK, Done J (1972) The diversity of alkaline phosphatase from rat intestine. Isolation and purification of the enzyme(s). Biochim Biophys Acta 258:147–153Google Scholar
  32. Simon PM, Kedinger M, Raul F, Grenier JF (1979) Developmental pattern of rat intestinal brushborder enzymatic proteins along the villus-crypt axis. Biochem J 178:407–413Google Scholar
  33. Wachsmuth ED, Tohorst A (1974) Possible precursors of aminopeptidase and alkaline phosphatase in the proximal tubules of kidney and the crypts of small intestine of mice. Histochemistry 38:43–56Google Scholar
  34. Wachsmuth ED (1976) Differentiation of epithelial cells in human jejunum: Localization and quantification of aminopeptidase, alkaline phosphatase and aldolase isoenzymes in tissue sections. Histochemistry 48:101–109Google Scholar
  35. Wilkinson GN (1961) Statistical estimations in enzyme kinetics. Biochem J 80:324–332Google Scholar
  36. Young GP, Friedman S, Alpers DH (1980) Effect of fat feeding on intestinal alkaline phosphatase. Gastroenterology 78:1297. (Abstract)Google Scholar

Copyright information

© Springer-Verlag 1980

Authors and Affiliations

  • S. Gutschmidt
    • 1
  • U. Lange
    • 1
  • E. O. Riecken
    • 1
  1. 1.Abteilung für Innere Medizin mit Schwerpunkt GastroenterologieKlinikum Steglitz der Freien Universität BerlinBerlin 45Germany

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