Skip to main content
SpringerLink
Log in

Osteoblast low-molecular-weight proteinase inhibitor I. Isolation and characterization of activity from osteoblastic cells and bone

  • Molecular and Cellular Biology
  • Published:
Calcified Tissue International Aims and scope Submit manuscript

Summary

Isolated mouse calvarial cells having phenotypic characteristics of osteoblasts, mouse parietal bone segments, mouse serum, and control mouse lung fibroblasts were extracted in NaCl and ultrafiltered to recover final concentrates having nominal molecular weights between 50,000 and 1000 daltons. Final concentrates of osteoblasts and bone but not of serum or control fibroblasts were positive for the inhibition of trypsin degradation of fibrin. Osteoblast final concentrates inhibited trypsin hydrolysis of the synthetic substratep-tosyl-l-arginine methyl ester. Osteoblast and bone final concentrates comigrated with Trasylol but were electrophoretically distinct from alphal-antiproteinase. Final concentrates of osteoblast and bone extracts did not inhibit tadpole collagenase using the [3H]glycine-labeled diffuse chick collagen fibril assay. High-performance liquid chromatography (HPLC) of osteoblast final concentrates after purification using immobilized trypsin affinity chromatography revealed the presence of a major peak that was positive for the inhibition of trypsin. Molecular weight determination by HPLC indicated that the inhibitor(s) range in nominal molecular weight from 4300 to 5100 daltons. The presence of low-molecular-weight serine proteinase inhibitory activity in bone suggests its participation in the regulation of bone resorption through the regulation of enzyme activation of collagenase, and possibly its role in defense against bone matrix enzymatic degradation during tumor cell invasion.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Vaes G (1980) Collagenase, lysosomes and osteoclastic bone resorption. In: Wolley D, Evanson J (eds) Collagenase in normal and pathological connective tissues. John Wiley & Sons, New York, p 185

    Google Scholar 

  2. Delaisse J, Eeckhout Y, Vaes G (1984) In vivo and in vitro evidence for the involvement of cysteine proteinases in bone resorption. Biochem Biophys Res Commun 125:441–447

    Article  PubMed  CAS  Google Scholar 

  3. Partridge N, Jeffrey J, Ehlich L, Teitelbaum S, Fliszar C, Welgus H, Kahn A (1987) Hormonal regulation of the production of collagenase and a collagenase inhibitor activity by rat osteogenic sarcoma cells. Endocrinology 120:1956–1962

    PubMed  CAS  Google Scholar 

  4. Heath S, Atkinson S, Meikle M, Reynolds J (1984) Mouse osteoblasts synthesize collagenase in response to bone resorbing agents. Biochem Biophys Acta 802:151–154

    PubMed  CAS  Google Scholar 

  5. Fritz H, Tschesche L, Greene J, Truscheit E (eds) (1974) Proteinase inhibitors Proceedings Second International Research Conference. Springer-Verlag, New York.

    Google Scholar 

  6. Holzer H (1980) Control of proteolysis. Annu Rev Biochem 49:63–91

    Article  PubMed  CAS  Google Scholar 

  7. Laskowski M, Kato I (1980) Protein inhibitors of proteinases. Annu Rev Biochem 49:593–626

    Article  PubMed  CAS  Google Scholar 

  8. Murphy G, Cartwright E, Sellers A, Reynolds J (1977) The detection and characterization of collagenase inhibitors from rabbit tissues in culture. Biochem Biophys Acta 483:493–498

    PubMed  CAS  Google Scholar 

  9. Sellers A, Cartwright E, Murphy G, Reynolds J (1977) Evidence that latent collagenases are enzyme-inhibitor complexes. Biochem J 163:303–307

    PubMed  CAS  Google Scholar 

  10. Vaes G (1972) The release of collagenase as an inactive proenzyme by bone explants in culture. Biochem J 126:275–289

    CAS  Google Scholar 

  11. Sellers A, Murphy G, Meikle M, Reynolds J (1979) Rabbit bone collagenase inhibitor blocks the activity of other neutral metalloproteinases. Biochem Biophys Res Commun 87:581–587

    Article  PubMed  CAS  Google Scholar 

  12. Sellers A, Meikle M, Reynolds J (1980) Collagenase and collagenase inhibitor levels following changes in bone resorption in vitro. Calcif Tissue Int 31:35–43

    Article  PubMed  CAS  Google Scholar 

  13. Sakamoto M, Sakamoto S, Goldhaber P, Glimcher M (1978) Immunocytochemical studies of collagenase localization in normal mouse tissues with special reference to cartilage and bone. Trans Orthop Res Soc 3:141

    Google Scholar 

  14. Sakamoto M, Sakamoto S (1985) Immunocytochemical localization of collagenase in isolated mouse bone cells. Biomed Res 5:29–33

    Google Scholar 

  15. Puzas J, Brand J (1979) Parathyroid hormone stimulation of collagenase secretion by isolated bone cells. Endocrinology 104:559–562

    Article  PubMed  CAS  Google Scholar 

  16. Cawston T, Galloway W, Mercer E, Murphy G, Reynolds J (1981) Purification of rabbit bone inhibitor of collagenase. Biochem J 195:159–165

    PubMed  CAS  Google Scholar 

  17. Otsuka K, Sodek J, Limeback H (1984) Synthesis of collagenase and collagenase inhibitors of osteoblast-like cells in culture. Eur J Biochem 145:123–129

    Article  PubMed  CAS  Google Scholar 

  18. Hamilton J, Lingelbach S, Partridge N, Martin T (1984) Stimulation of plasminogen activator in osteoblast-like cells by bone-resorbing hormones. Biochem Biophys Res Commun 122:230–236

    Article  PubMed  CAS  Google Scholar 

  19. Hamilton J, Lingelbach S, Patridge N, Martin T (1985) Regulation of plasminogen activator production by bone-resorbing hormones in normal and malignant osteoblasts. Endocrinology 116:2186–2193

    PubMed  CAS  Google Scholar 

  20. Eeckhout Y, Vaes G (1977) Further studies on the activation of procollagenase, the latent precursor of bone collagenase. Biochem J 166:21–31

    PubMed  CAS  Google Scholar 

  21. Waxler B, Wezeman F (1983) Low molecular weight proteinase inhibitors: I. Extraction and identification of activity in normal and malignant human breast tissues. Br J Exp Pathol 64:252–260

    PubMed  CAS  Google Scholar 

  22. Waxler B, Wezeman F (1983) Low molecular weight proteinase inhibitors: II. Extraction and identification of activity from infiltrating ductal carcinoma during lactation. Br J Exp Pathol 64:626–632

    PubMed  CAS  Google Scholar 

  23. Waxler B, Corey J, Wezeman F (1984) Low molecular weight proteinase inhibitors: III. quantification and isolation of activity from benign and malignant human breast tissue. J Lab Clin Med 104:528–537

    PubMed  CAS  Google Scholar 

  24. Yagiela J, Woodbury S (1977) Enzymatic isolation of osteoblasts from fetal rat calvaria. Anat Rec 188:287–305

    Article  PubMed  CAS  Google Scholar 

  25. Wong G, Cohn D (1975) Target cells in bone for parathormone and calcitonin are different: enrichment for each cell type by sequential digestion of mouse calvaria and selective adhesion to polymeric surfaces. Proc Natl Acad Sci USA 72:3167–3171

    Article  PubMed  CAS  Google Scholar 

  26. Kuettner K, Croxen R, Eisenstein R, Sorgente N (1975) Proteinase inhibitor activity in connective tissue. Experientia 30:595–597

    Article  Google Scholar 

  27. Monson J, Click E, Bornstein P (1975) Further characterization of procollagen. Purification and analysis of the pro alpha-1 chain of chick bone procollagen. Biochemistry 14:4088–4092

    Article  CAS  Google Scholar 

  28. Kassel B (1970) Bovine trypsin-kallikrein inhibitor (Kunitz inhibitor, basic pancreatic trypsin inhibitor, polyvalent inhibitor from bovine organs). Methods Enzymol 19:844–852

    Article  Google Scholar 

  29. Rifkin D, Crowe R (1977) Isolation of a protease inhibitor from tissue resistant to tumor invasion. Hoppe-Seyler's Z Physiol Chem 358:1525–1531

    PubMed  CAS  Google Scholar 

  30. Kunitz M (1947) Crystalline soybean trypsin inhibitor. J Gen Physiol 30:291–320

    Article  CAS  Google Scholar 

  31. Kozlowski K, Wezeman F, Schultz R (1984) Tumor cell proteinase visualization and quantification using a fluorescent transition-state analog probe. Proc Natl Acad Sci USA 81:1135–1139

    Article  PubMed  CAS  Google Scholar 

  32. Steven F, Griffin M, Itzhaki S, Al-Habib A (1980) A trypsinlike neutral protease on Ehrlich ascites cell surfaces, its role in the activation of tumor cell zymogen of collagenase. Br J Cancer 42:712–721

    PubMed  CAS  Google Scholar 

  33. Rose G, Robertson P (1977) Collagenolysis by human gingival fibroblast cell lines. J Dent Res 56:416–423

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biology, Loyola University of Chicago, 6525 N. Sheridan Road, 60626, Chicago, Illinois, USA

    Frederick H. Wezeman Ph.D. & Janice Corey

  2. Department of Anesthesiology, Cook County Hospital, 6525 N. Sheridan Road, 60626, Chicago, Illinois, USA

    Beverly Waxler

Authors
  1. Frederick H. Wezeman Ph.D.
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Janice Corey
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Beverly Waxler
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wezeman, F.H., Corey, J. & Waxler, B. Osteoblast low-molecular-weight proteinase inhibitor I. Isolation and characterization of activity from osteoblastic cells and bone. Calcif Tissue Int 46, 263–269 (1990). https://doi.org/10.1007/BF02555006

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02555006

Key words

Search

Navigation