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Histochemistry

, Volume 73, Issue 2, pp 311–319 | Cite as

The usefulness of the analytical electrofocusing in a thin-layer polyacrylamide gel (PAG) in the histochemistry of enzymes cleaving peptide bonds

  • Z. Lojda
  • J. Kulich
Article

Summary

The usefulness of the analytical electrofocusing in a thin-layer polyacrylamide (PAG) plate is shown on the basis of experiments with 10%–20% homogenates of various rat, rabbit and human organs as well as in lysates of isolated human lymphocytes and leucocytes in 2% Triton X100. 0.1–0.3 μl of 12000 g supernatants were applied on LKB Ampholine PAG plates pH range 3.5–9.5 and subjected to electrofocusing. Afterwards portions of PAG plates were processed in optimized histochemical media for the demonstration of enzymes cleaving peptide bonds using various substrates. The same media were used in the histochemical detection of enzymes in sections on slides or semipermeable membranes. Electrofocused zymograms display species and organ differences. Ala-MNA, Leu-MNA and Met-MNA furnish similar zymograms. Bands obtained with Ala-MNA are most intense. Zymograms with Gly-Pro-MNA and Lys-Pro-MNA at pH 7.2 are not entirely identical. The majority of bands is more intense when Gly-Pro-MNA is used as the substrate and is due to the activity of DAP IV. The anodal band(s) focusing around pH 4.9 (rat) or 5.5 (man) is (are) much stronger with Lys-Pro-MNA and DAP II is responsible for it (them). Zymograms with His-Ser-MNA and Lys-Ser-MNA are similar. However, they differ form those revealed with Gly-Pro-MNA and Lys-Pro-MNA. Zymograms of lysates of leucocytes obtained with naphthol AS-D-chloroacetate and Ac-Ala-l-naphthyl ester are not identical showing that more than one enzyme is responsible for the bands. Results obtained with closely related substrates such as Ac-Ala-l-naphthyl ester and Ac-Met-l-naphtyl ester are not identical either. Zymograms of lysates of human lymphocytes revealed with Gly-Pro-MNA at pH 7.2 and Lys-Ala-MNA or Lys-Pro-MNA at pH 5.5 or 5.3 respectively show clearly the presence of DAP IV and DAP II in these cells. The analytical electrofocusing in PAG plates is a very useful tool in the histochemistry (and biochemistry) of enzymes cleaving peptide bonds. It helps very much in the evaluation of the substrate specificity, choosing of the discriminating substrate and enables a quick and reliable testing of the quality of various batches of commercially supplied substrates, diazonium salts and other reagents. The correlation of zymograms with the “in situ” pattern helps in the elucidation of the origin of individual bands in zymograms and suggests different molecular forms of peptidases in different localizations.

Keywords

Triton X100 Naphthol Human Lymphocyte Molecular Form Diazonium 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. Bishop R (1979) Current major application areas in electrofocusing. Sci Tools 26:2–8Google Scholar
  2. Gossrau R (1979) Peptidasen II. Zur Lokalisation der Dipeptidylpeptidase IV (DPP IV). Histochemische und biochemische Untersuchung. Histochemistry 60:231–248Google Scholar
  3. Gossrau R (1981) Investigation of proteinases in the digestive tract using 4-methoxy-2-naphthylamine (MNA) substrates. J Histochem Cytochem 29:464–480Google Scholar
  4. Gossrau R, Lojda Z (1980) Study on dipeptidylpeptidase II (DPP II) Histochemistry 70:53–76Google Scholar
  5. Huseby RM, Smith RE (1980) Synthetic oligopeptide substrates: their diagnostic application in blood coagulation, fibrinolysis, and other pathologic states. Semin in Thromb Hemostas 6:173–314Google Scholar
  6. Lojda Z (1977a) Studies on glycyl-proline naphtylamidase. I. Lymphocytes. Histochemistry 54:300–312Google Scholar
  7. Lojda Z (1977b) New data on dipeptidylaminopeptidase IV. Sb Cesk Spol Histochem Cytochem 6:59–69 (in Czech)Google Scholar
  8. Lojda Z (1979a) Studies on dipeptidyl(amino)peptidase IV (glycylroline naphthylamidase). II. Blood vessels. Histochemistry 59:153–166Google Scholar
  9. Lojda Z (1979b) Progress in the histochemistry of peptidases. Sb Cesk Spol Histochem Cytochem 7:42–43Google Scholar
  10. Lojda Z (1981) Proteinases in pathology. Usefulness of histochemical methods. J Histochem Cytochem 29:481–493Google Scholar
  11. Lojda Z, Gossrau R, Schiebler TH (1979a) Enzyme histochemistry. A laboratory manual. Springer, Berlin Heidelberg New YorkGoogle Scholar
  12. Lojda Z, Heřmanský F, Benešová E, Šálková J, Lodrová V (1979b) Dipeptidylaminopeptidase IV in lymphocytes of human peripheral blood and the significance of its demonstration in patients with malignant lymphomas. Sb Lék 81:200–207 (in Czech)Google Scholar
  13. Ornstein L, Janoff A, Sweetman FR, Ansley HR (1973) Histochemical demonstration of an elastase-like neutrophil esterase. J Histochem Cytochem 21:411Google Scholar
  14. Ornstein L, Ansley H, Saunders A (1976) Improving manual differential white cell counts with cytochemistry. Blood Cells 2:557–585Google Scholar
  15. Starkey PM (1977) Elastase and cathepsin G; the serine proteinases of human neutrophil leucocytes and spleen. In: Barrett AJ (ed) Proteinases in mammalian cells and tissues. North-Holland, Amsterdam, pp 57–89Google Scholar

Copyright information

© Springer-Verlag 1981

Authors and Affiliations

  • Z. Lojda
    • 1
    • 2
  • J. Kulich
    • 1
    • 2
  1. 1.Laboratory of Histochemistry, Faculty of MedicineCharles UniversityPrahaČSSR
  2. 2.Department of Anatomy, Faculty of MedicineCharles UniversityHradec KrálovéČSSR

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