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Thromb Haemost 1977; 38(02): 0536-0544
DOI: 10.1055/s-0038-1651860
Original Article
Schattauer GmbH

Preparation (Agar Zone Electrophoresis) and Immunological Characterisation of Urokinase

K Andrassy   *
1   Medizinische Universitätsklinik, Heidelberg, Germany
,
E Ritz
1   Medizinische Universitätsklinik, Heidelberg, Germany
› Author Affiliations
Further Information

Publication History

Received 27 August 1976

Accepted 02 April 1977

Publication Date:
04 July 2018 (online)

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Summary

Urokinase was highly purified by electrophoretical and immunological methods starting with a commercial urokinase preparation (UK-Leo). Contaminating serum proteins and enzyme activities migrated into opposite directions in agar gel electrophoresis which proved to be a valuable preparative method. The final purification achieved was 80,000 Ploug units/mg protein. Traces of albumin, α2 HS-glycoprotein and α2-macroglobulin migrated towards the cathode together with UK in a multimolecular complex. Urokinase antibodies (rabbit) gave with the cathodic fraction 2 precipitation lines (Ouchterlony technique): the one precipitation line corresponded to urokinase (molecular weight on gel chromatography 32,000 daltons), the other corresponded to UK complexed with serum proteins. Urokinase antibodies completely suppressed UK activity in various commercial preparations. All these preparations showed immunological identity; on disc electrophoresis pure urokinase (32,000 daltons, 80,000 Ploug units/mg protein) still gave 2-3 bands suggesting the presence of isoenzymes.

This paper is dedicated to Professor G. Schettler on behalf of his 60th birthday

* with support of DFG


 

  • References

  • 1 Andrassy K, Buchholz L, Bleyl U, Ritz E. 1976; Topography of human uroldnase activity in renal tissue. Nephron 16: 213.
    CrossrefPubMedGoogle Scholar
  • 2 Astrup T, Mullertz S. 1952; The fibrin plate method for estimating fibrinolytic activity. Archives of Biochemistry and Biophysics 40: 346.
    CrossrefPubMedGoogle Scholar
  • 3 Ball A, Day E. 1970; Immunological identification of two urokinases. Thrombosis et Diathesis Haemorrhagica 24: 475.
    PubMedGoogle Scholar
  • 4 Bernik M, Kwaan H. 1969; Plasminogen activator activity in cultures from human tissues. An immunological and histological study. Journal of Clinical Investigation 48: 1740.
    CrossrefPubMedGoogle Scholar
  • 5 Burges R, Brammer K, Coombes J. 1965; Molecular weight of urokinase. Nature 208: 894.
    PubMedGoogle Scholar
  • 6 Davis B. 1964; Disc electrophoresis. Method and application to human serum proteins. Annals of the New York Academy of Sciences 121: 404.
    PubMedGoogle Scholar
  • 7 Doleschel W, Auerswald W. 1967; Determination of the molecular weights of uroprotein fractions with urokinase activity by means of molecular sieving. Medical and Pharmacology Experiments 16: 225.
    PubMedGoogle Scholar
  • 8 Day E, Ball A. 1970; Immunological identification of three urokinases. Thrombosis et Diathesis Haemorrhagica 24: 487.
    PubMedGoogle Scholar
  • 9 Day E, Ball A, Jeffords D, Woodgard W, Silver D. 1969; The immunoassay of human urokinase. Thrombosis et Diathesis Haemorrhagica 21: 273.
    PubMedGoogle Scholar
  • 10 Grabar P, Williams C. 1955; Méthodes immuno-électrophorétiques d’analyses de mélanges de substances antigéniques. Biochemica Biophysica Acta 17: 67.
    CrossrefPubMedGoogle Scholar
  • 11 Hamberg U, Salvolainen M. 1966; Studies on plasminogen activators: a method for separation of purified urokinase from small samples of urine. Acta chemica scandinavica 20: 2551.
    CrossrefPubMedGoogle Scholar
  • 12 Heimburger N, Schwick G. 1962; Die Fibrinagarelektrophorese. Thrombosis et Diathesis Haemorrhagica 7: 432.
    PubMedGoogle Scholar
  • 13 Kok P, Astrup T. 1969; Isolation and purification of a tissue plasminogen activator and its comparison with urokinase. Biochemistry 8: 79.
    CrossrefPubMedGoogle Scholar
  • 14 Lesuk A, Terminiello L, Traver J. 1965; Crystalline human urokinase. Some properties, Science 147: 880.
    PubMedGoogle Scholar
  • 15 Levy H, Sober H. 1960; A simple chromatographic method for preparation of gamma-globulins. Proceedings of the Society of Experimental Biology and Medicine 103: 250.
    PubMedGoogle Scholar
  • 16 Lowry O, Rosebrough J, Farr A, Randall R. 1951; Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193: 265.
    PubMedGoogle Scholar
  • 17 Ouchterlony O. 1958; Diffusion in gel-methods for immunological analysis. In: Progress Allergy 5: 1 S. Karger, Basel.
    PubMedGoogle Scholar
  • 18 Ogawa N, Yamamoto H, Karamine R, Tajima H. 1975; Purification and some properties of urokinase. Thrombosis et Diathesis Haemorrhagica 34: 194.
    PubMedGoogle Scholar
  • 19 Ploug J, Kjeldgaard N. 1956; Isolation of a plasminogen activator (urokinase) from urine. Archives of Biochemistry and Biophysics 62: 500.
    CrossrefPubMedGoogle Scholar
  • 20 Ploug J, Kjeldgaard N. 1957; Urokinase, an activator of plasminogen from human urine. I. Isolation and properties. Biochemica Biophysica Acta 24: 279.
    PubMedGoogle Scholar
  • 21 Sgouris J, Storey R, McGall K, Anderson H. 1962; The purification, assay, sterilisation and removal of human urokinase. Vox. Sang 7: 739.
    CrossrefPubMedGoogle Scholar
  • 22 Whitaker J. 1963; Determination of molecular weights of proteins by gel filtration on Sephadex. Analytical Chemistry 35: 1950.
    CrossrefPubMedGoogle Scholar
  • 23 White W, Barlow G, Mozen M. 1966; The isolation and characterisation of plasminogen activators (urokinase) from human urine. Biochemistry 5: 2160.
    CrossrefPubMedGoogle Scholar