The demonstration of adenosinetriphosphatase activity with the electronmicroscope

  • J. -P. Persijn
  • W. Th. Daems
  • J. C. H. de Man
  • A. E. F. H. Meijer
Article

Summary

A new method for the demonstration of adenosine triphosphatase (ATP'ase) activity in tissue blocks with the electron-microscope has been developed. This method consists of short fixation (2–3 minutes,ph 7.3) in a OsO4-Tris-HCl mixture, incubation with a medium containing Tris-HCl buffer, tartrate, lead and magnesium ions, ATP and sucrose (ph 6.8) and routine postfixation. The method combines satisfactory histochemical specificity with good preservation of tissue fine structure. Estimations of enzymatic activity of ATP'ase and alkaline phosphatase, as observed with the electronmicroscope, were compared with activity determinations in tissue homogenates. The occurence of a positive histochemical reaction in swollen mitochondria is discussed.

Keywords

Sucrose Enzymatic Activity Magnesium Adenosine Alkaline Phosphatase 

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Literature

  1. Barnett, R. J.: The demonstration with the electron microscope of the endproducts of histochemical reactions in relation to the fine structure of cells. Exp. Cell. Res. Suppl.7, 65–89 (1959).Google Scholar
  2. ——, andG. E. Palade: Histochemical demonstration of the sites of activity of dehydrogenase systems with the electron microscope. J. biophys. biochem. Cytol.3, 577–589 (1957).PubMedGoogle Scholar
  3. —— —— Enzymatic activity in the M. band. J. biophys. biochem. Cytol.6, 163–170 (1959).PubMedGoogle Scholar
  4. Boyer, P. D., H. Lardy andK. Myrbäck (eds.): The Enzymes, vol. 5. New York: Academic Press 1961.Google Scholar
  5. Bullivant, S.: The staining of this sections of mouse pancreas prepared by the Fernandez-Moran Helium II freeze-substitution method. J. biophys. biochem. Cytol.8, 639–647 (1960).Google Scholar
  6. Caulfield, J. B.: Effects of varying the vehicle for OsO4 in tissue fixation. J. biophys. biochem. Cytol.5, 827–834 (1957).Google Scholar
  7. Colowick, S. P., andN. O. Kaplan (eds.): Methods in Enzymology, vol. 2. New York: Academic Press 1957.Google Scholar
  8. Deimling, O. v., E. Mölbert andF. Duspiva: Elektronenmikroskopischer Nachweis eines Glucose-1-phosphat spaltenden Enzyms im Herzmuskel der Albinoratte. Beitr. path. Anat.123, 127–143 (1960).Google Scholar
  9. Dounce, A. L.: Enzyme systems of isolated cell nuclei. Ann. N.Y. Acad. Sci.50, 982–999 (1950).Google Scholar
  10. Essner, E., A. B. Novikoff andB. Masek: Adenosinetriphosphatase and 5-nucleotidase activities in the plasma membrane of liver cells as revealed by electronmicroscopy. J. biophys. biochem. Cytol.4, 711–715 (1958).PubMedGoogle Scholar
  11. Kielley, W. W., andR. K. Kielley: Myokinase and Adenosinetriphosphatase in oxidative phosphorylation. J. biol. Chem.191, 485–500 (1951).PubMedGoogle Scholar
  12. Lindberg, O., andL. Ernster: Methods of phosphate analysis. In: Biochemical analysis, edit byD. Glick, vol. 3. New York: Interscience Publ. 1956.Google Scholar
  13. Martin, J. H., andD. M. Doty: Determination of inorganic phosphate. Analyt. Chem.21, 965–967 (1949).Google Scholar
  14. Mölbert, E., F. Duspiva u.O. v. Deimling: Die histochemische Lokalisation der Phosphatase in den Tubulus-Zellen der Mausniere im elektronenmikroskopischen Bild. Histochem.2, 5–22 (1960).Google Scholar
  15. Novikoff, A. B., D. H. Hausman andE. Podber: The localization of Adenosinetriphosphatase in liver: in situ staining and cell fractionation studies. J. Histochem. Cytochem.6, 61–71 (1958).PubMedGoogle Scholar
  16. Palade, G. E.: A study of fixation for electronmicroscopy. J. exp. Med.95, 285–293 (1952).PubMedGoogle Scholar
  17. Raaflaub, J.: a) Die Schwellung isolierter Leberzellmitochondrien und ihre physikalischehemische Beeinflußbarkeit. Helv. physiol. Acta11, 142–156 (1953);Google Scholar
  18. ——: b) Uber den Wirkungsmechanismus von Adenosinetriphosphat als Cofaktor isolierter Mitochondrien. Helv. physiol. Acta11, 157–165 (1953).Google Scholar
  19. Sheldon, H., H. Zetterquist andD. Brandes: Histochemical reactions for electron microscopy. Exp. Cell Res.9, 592–596 (1955).PubMedGoogle Scholar

Copyright information

© Springer-Verlag 1961

Authors and Affiliations

  • J. -P. Persijn
    • 1
  • W. Th. Daems
    • 1
  • J. C. H. de Man
    • 1
    • 2
  • A. E. F. H. Meijer
    • 1
    • 2
  1. 1.From the Laboratory for ElectronmicroscopyUniversity of LeidenThe Netherlands
  2. 2.Laboratory for PathologyUniversity of LeidenThe Netherlands

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