Download PDF
Thromb Haemost 1985; 53(01): 036-041
DOI: 10.1055/s-0038-1661232
Original Article
Schattauer GmbH Stuttgart

Formation of Stable Complexes Between Urokinase and a Human Urinary Component

Witold Cieplak
1   The Department of Microbiology, University of Texas Health Science Center at Dallas, Canada
,
David A Hart
2   The Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Centre, Canada
› Author Affiliations
Further Information

Publication History

Received 05 June 1984

Accepted 22 October 1984

Publication Date:
18 July 2018 (online)

Also available at
  PDF Download  Permissions and Reprints

Summary

Human urine was found to contain multiple species of urokinase (UK)-like plasminogen activator (PA) activity when subjected to concentration and/or dialysis and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymography. Untreated, freshly voided urine contained only Mr = 52,000 and 35,000 UK while dialyzed or undialyzed urine concentrates contained additional PA activity at Mr = 80,000 and 95.000. SDS-PAGE of incubation mixtures of radioiodinated UK (Mr = 52,000 or 35,000) and urine concentrates revealed the presence of radiolabeled complexes with MrS of 80,000 and 95.000. The bond(s) involved in complex formation was also relatively resistant to heat and reduction. Treatment of radioiodinated UK with the serine proteinase inhibitor, p-nitrophenyl guanidinobenzoate, prior to incubation with dialyzed, concentrated urine prevented formation of the complexes. In addition, the enzymatic activity of the Mr = 80,000 and 95,000 species was unaffected by diisopropyl fluorophosphate. These results indicate that UK forms SDS-stable complexes with a urinary component that has a Mr of approximately 40,000. The results further suggest that these complexes express PA activity when analyzed by SDS- PAGE and zymography.

Key words

Urokinase - Zymography - Complexes - Urinary binding component
 

  • References

  • 1 Christman JK, Silverstein SC, Acs G. Plasminogen activators. In: Proteinases in Mammalian Cells and Tissues. Barrett AJ. (Ed.) North-Holland Pub Co; Amsterdam: 1978. pp 91-149
    Google Scholar
  • 2 Castellino FJ. Biochemistry of human plasminogen. Semin Thromb Haemostas 1984; 10: 18-23
    Article in Thieme ConnectPubMedGoogle Scholar
  • 3 Collen D. On the regulation and control of fibrinolysis. Thromb Haemostas 1980; 43: 77-89
    PubMedGoogle Scholar
  • 4 Bachmann F, Kruithof EK O. Tissue plasminogen activator: chemical and physiological aspects. Semin Thromb Haemostas 1984; 10: 6-17
    Article in Thieme ConnectPubMedGoogle Scholar
  • 5 Soberano ME, Ong EB, Johnson AJ, Schoellmann G. Purification and characterization of two forms of urokinase. Biochim Biophys Acta 1976; 445: 763-773
    CrossrefPubMedGoogle Scholar
  • 6 Schaller J, Nick H, Rickli EE, Gillessen D, Lergier W, Studer RO. Human low-molecular weight urinary urokinase. Partial characterization and preliminary sequence data of the two polypeptide chains Eur J Biochem 1982; 125: 251-257
    CrossrefPubMedGoogle Scholar
  • 7 Steffens GJ, Gunzler WA, Otting F, Frankus E, Flohe L. The complete amino acid sequence of low molecular mass urokinase from human urine. Hoppe-Seyler’s Z Physiol Chem 1982; 363: 1043-1058
    PubMedGoogle Scholar
  • 8 Salerno G, Verde P, Nolli ML, Corti A, Szots H, Meo T, Johnson J, Bullock S, Cassani G, Blasi F. Monoclonal antibodies to human urokinase identify the single-chain pro-urokinase precursor. Proc Natl Acad Sri 1984; 81: 110-114
    PubMedGoogle Scholar
  • 9 Astedt B, Barlow G, Holmberg L. Time-related release of various molecular forms of urokinase in tissue culture. Thromb Res 1977; 11: 149-153
    CrossrefPubMedGoogle Scholar
  • 10 Vetterlein R, Young PL, Bell TE, Roblin R. Immunological characterization of multiple molecular weight forms of human cell plasminogen activators. J Biol Chem 1979; 254: 575-578
    PubMedGoogle Scholar
  • 11 Bobbitt JL, Clavin SA, Hutchins JF, Arnett GC, Hull RN. Studies on plasminogen activator from pig kidney cell cultures. III. Purification and characterization. Thromb Res 1980; 18: 315-332
    CrossrefPubMedGoogle Scholar
  • 12 Holmberg L, Astedt B. Radioimmunoassays of high and low molecular weight urokinase. In: Urokinase: Basic and Clinical Aspects, Serono Symposium No48. Mannucci PM, D’Angelo A. (Eds) Academic Press; New York: 1982. pp 33-42
    Google Scholar
  • 13 Schneider Ph, Bachmann F, Sauser D. Urokinase.A short review of its properties and of its metabolism. In: Urokinase: Basic and Clinical Aspects, Serono Symposium No 48. Mannucci PM, D’Angelo A. (Eds) Academic Press; New York: 1982. pp 1-15
    Google Scholar
  • 14 Hart DA, Kramer R, Cieplak W. Identification of subpopulations of human urinary plasminogen activators. Thromb Haemostas 1984; 51: 212-216
    PubMedGoogle Scholar
  • 15 Goding JW. Use of Staphylococcal protein A as an immunological reagent. J Immunol Methods 1978; 20: 241-253
    CrossrefPubMedGoogle Scholar
  • 16 Fraker PJ, Speck JC. Protein and cell membrane iodinations with a sparingly soluble chloroamide l,3,4,6-tetrachloro-3α,6α-diphenyl-gly- coluril. Biochem Biophys Res Commun 1978; 80: 849-857
    CrossrefPubMedGoogle Scholar
  • 17 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature (London) 1970; 227: 680-682
    CrossrefPubMedGoogle Scholar
  • 18 Goding JW, Harris AW. Subunit structure of cell surface proteins: disulfide bonding in antigen receptors, Ly-2,3 antigens, and transferrin receptors of murine T and B lymphocytes. Proc Natl Acad Sci 1981; 78: 4530-4534
    CrossrefPubMedGoogle Scholar
  • 19 GraneUi-Pipemo A, Reich E. A study of proteases and protease inhibitor complexes in biological fluids. J Exp Med 1978; 148: 223-224
    CrossrefPubMedGoogle Scholar
  • 20 Hart DA, Kramer R, Cieplak W. Evidence for heterogenous glycosylation of human urinary plasminogen activators. Fed Proc 1983; 42: 1859 (Abstr)
    PubMedGoogle Scholar
  • 21 Goldberg AR, Wolf BA, Lefevbre PA. Plasminogen activators of transformed and normal cells. In: Proteases in Biological Control. Reich E, Rifkin DB, Shaw E. (Eds) Cold Spring Harbor Laboratory; 1975. pp 857-868
    Google Scholar
  • 22 Baker JB, Low DA, Simmer RL, Cunningham DD. Protease-nexin: a cellular component that links thrombin and plasminogen activator and mediates their binding to cells. Cell 1980; 21: 37-45
    CrossrefPubMedGoogle Scholar
  • 23 Scott RW, Eaton DL, Duran N, Baker JB. Regulation of extracellular plasminogen activator by human fibroblasts. J Biol Chem 1983; 258: 4397-4403
    PubMedGoogle Scholar
  • 24 Clemmensen I. Inhibition of urokinase by complex formation with human antithrombin III in absence and presence of heparin. Thromb Haemostas 1978; 39: 616-623
    PubMedGoogle Scholar
  • 25 Murano G, Aronson D, Williams L, Brown L. The inhibition of high and low molecular weight urokinase in plasma. Blood 1980; 55: 430-436
    PubMedGoogle Scholar
  • 26 Waller EK, Schleuning W-D, Reich E. Complex-formation and inhibition of urokinase by blood plasma proteins. Biochem J 1983; 215: 123-131
    CrossrefPubMedGoogle Scholar
  • 27 Hoal EG, Wilson EL. Dowdle. The regulation of tissue plasminogen activator activity by human fibroblasts. Cell 1983; 34: 273-279
    CrossrefPubMedGoogle Scholar
  • 28 Levin EG. Latent tissue plasminogen activator produced by human endothelial cell in culture: Evidence for an enzyme-inhibitor complex. Proc Natl Acad Sci 1983; 80: 6804-6808
    CrossrefPubMedGoogle Scholar
  • 29 Philips M, Thosen S. Binding of plasminogen activator (PA) to a component (X) of endothelial cells (EC) may explain the 95,000-MrPA in human plasma. Thromb Haemostas 1983; 50: 193 (Abstr.)
    PubMedGoogle Scholar
  • 30 Kruithof EK O, Ransijn A, Tran-Thang C, Bachmann F. Characteristics of a fast acting inhibitor of plasminogen activator in human plasma. Thromb Haemostas 1983; 50: 193 (Abstr)
    PubMedGoogle Scholar
  • 31 Owen WG, Penick GD, Yoder E, Poole BL. Evidence for an ester bond between thrombin and heparin cofactor. Thromb Haemostas 1975; 35: 87-95
    PubMedGoogle Scholar
  • 32 Wiman B, Collen D. On the mechanism of the reaction between human α2-antiplasmin and plasmin. J Biol Chem 1979; 254: 9291-9297
    PubMedGoogle Scholar
  • 33 Wilson EL, Becker ML B, Hoal EG, Dowdle EB. Molecular species of plasminogen activators secreted by normal and neoplastic cells. Cancer Res 1980; 40: 933-938
    PubMedGoogle Scholar
  • 34 Wun T-C, Ossowski L, Reich E. A proenzyme form of urokinase. J Biol Chem 1982; 257: 7262-7268
    PubMedGoogle Scholar
  • 35 Booyse FM, Scheinbuks J, Radek J, Oskiowicz G, Feder S, Quarfoot AJ. Immunological identification and comparison of plasminogen activator forms in cultured normal human endothelial cells and smooth muscle cells. Thromb Res 1981; 24: 495-504
    CrossrefPubMedGoogle Scholar
  • 36 Wun T-C, Schleuning W-D, Reich E. Isolation and characterization of urokinase from human plasma. J Biol Chem 1982; 257: 3276-3283
    PubMedGoogle Scholar
  • 37 Low DA, Baker JB, Koonce WC, Cunningham DD. Released protease-nexin regulates cellular binding, internalization, and degradation of serine proteases. Proc Natl Acad Sci 1981; 78: 2340-2344
    CrossrefPubMedGoogle Scholar