Inhibition of recombinant human neutrophil collagenase by doxycycline is pH dependent

Gerald N. Smith, Kenneth D. Brandt, Elizabeth A. Mickler, Karen Hasty

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Objective. To examine, as part of an evaluation of the role of matrix metalloproteinase (MMP) inhibition in the amelioration of cartilage damage by doxycycline, the effect of pH on the inhibition of activity and reduction in stability of recombinant human neutrophil collagenase (rhMMP-8) by doxycycline in vitro. Methods. After activation with trypsin, rhMMP-8 was assayed using a peptolide substrate and a colorimetric assay. The rate of hydrolysis in the presence and absence of 30 μM doxycycline was measured over a pH range of 6.5-7.9. The molecular weight changes that accompanied activation of the proenzyme by acetylphenylmercuric acetate (APMA) in the presence and absence of doxycycline at pH 6.9 and 7.5 were studied by Western blotting. Results. At pH values above 7.1, doxycycline inhibited the activity of the enzyme. At pH values below 7.1, no inhibition was observed. When doxycycline was present during activation with APMA at pH 7.5, significant amounts of small (< 30 kDa) fragments were generated. In contrast, when doxycycline was present during activation with APMA at pH 6.9, no small fragments were detected. Conclusion. The ability of doxycycline to inhibit matrix rhMMP-8 activity or to promote its degradation is lost at pH values lower than 7. Although relatively high pH values may exist in adult articular cartilage in some pathological situations, at lower pH the effect of doxynycline on proenzyme levels in the extracellular matrix may be due to an effect on the regulation of synthesis of the proenzyme, rather than to direct inhibition of the active enzyme or reduction in the level of enzyme by proteolysis.

Original languageEnglish (US)
Pages (from-to)1769-1773
Number of pages5
JournalJournal of Rheumatology
Volume24
Issue number9
StatePublished - Sep 1997
Externally publishedYes

Fingerprint

Matrix Metalloproteinase 8
Doxycycline
Enzyme Precursors
Acetates
Enzymes
Articular Cartilage
Matrix Metalloproteinases
Trypsin
Proteolysis
Cartilage
Extracellular Matrix
Hydrolysis
Molecular Weight
Western Blotting

All Science Journal Classification (ASJC) codes

  • Immunology
  • Rheumatology

Cite this

Inhibition of recombinant human neutrophil collagenase by doxycycline is pH dependent. / Smith, Gerald N.; Brandt, Kenneth D.; Mickler, Elizabeth A.; Hasty, Karen.

In: Journal of Rheumatology, Vol. 24, No. 9, 09.1997, p. 1769-1773.

Research output: Contribution to journalArticle

Smith, Gerald N. ; Brandt, Kenneth D. ; Mickler, Elizabeth A. ; Hasty, Karen. / Inhibition of recombinant human neutrophil collagenase by doxycycline is pH dependent. In: Journal of Rheumatology. 1997 ; Vol. 24, No. 9. pp. 1769-1773.
@article{916d48b66db941c9b10820f76d21e611,
title = "Inhibition of recombinant human neutrophil collagenase by doxycycline is pH dependent",
abstract = "Objective. To examine, as part of an evaluation of the role of matrix metalloproteinase (MMP) inhibition in the amelioration of cartilage damage by doxycycline, the effect of pH on the inhibition of activity and reduction in stability of recombinant human neutrophil collagenase (rhMMP-8) by doxycycline in vitro. Methods. After activation with trypsin, rhMMP-8 was assayed using a peptolide substrate and a colorimetric assay. The rate of hydrolysis in the presence and absence of 30 μM doxycycline was measured over a pH range of 6.5-7.9. The molecular weight changes that accompanied activation of the proenzyme by acetylphenylmercuric acetate (APMA) in the presence and absence of doxycycline at pH 6.9 and 7.5 were studied by Western blotting. Results. At pH values above 7.1, doxycycline inhibited the activity of the enzyme. At pH values below 7.1, no inhibition was observed. When doxycycline was present during activation with APMA at pH 7.5, significant amounts of small (< 30 kDa) fragments were generated. In contrast, when doxycycline was present during activation with APMA at pH 6.9, no small fragments were detected. Conclusion. The ability of doxycycline to inhibit matrix rhMMP-8 activity or to promote its degradation is lost at pH values lower than 7. Although relatively high pH values may exist in adult articular cartilage in some pathological situations, at lower pH the effect of doxynycline on proenzyme levels in the extracellular matrix may be due to an effect on the regulation of synthesis of the proenzyme, rather than to direct inhibition of the active enzyme or reduction in the level of enzyme by proteolysis.",
author = "Smith, {Gerald N.} and Brandt, {Kenneth D.} and Mickler, {Elizabeth A.} and Karen Hasty",
year = "1997",
month = "9",
language = "English (US)",
volume = "24",
pages = "1769--1773",
journal = "Journal of Rheumatology",
issn = "0315-162X",
publisher = "Journal of Rheumatology",
number = "9",

}

TY - JOUR

T1 - Inhibition of recombinant human neutrophil collagenase by doxycycline is pH dependent

AU - Smith, Gerald N.

AU - Brandt, Kenneth D.

AU - Mickler, Elizabeth A.

AU - Hasty, Karen

PY - 1997/9

Y1 - 1997/9

N2 - Objective. To examine, as part of an evaluation of the role of matrix metalloproteinase (MMP) inhibition in the amelioration of cartilage damage by doxycycline, the effect of pH on the inhibition of activity and reduction in stability of recombinant human neutrophil collagenase (rhMMP-8) by doxycycline in vitro. Methods. After activation with trypsin, rhMMP-8 was assayed using a peptolide substrate and a colorimetric assay. The rate of hydrolysis in the presence and absence of 30 μM doxycycline was measured over a pH range of 6.5-7.9. The molecular weight changes that accompanied activation of the proenzyme by acetylphenylmercuric acetate (APMA) in the presence and absence of doxycycline at pH 6.9 and 7.5 were studied by Western blotting. Results. At pH values above 7.1, doxycycline inhibited the activity of the enzyme. At pH values below 7.1, no inhibition was observed. When doxycycline was present during activation with APMA at pH 7.5, significant amounts of small (< 30 kDa) fragments were generated. In contrast, when doxycycline was present during activation with APMA at pH 6.9, no small fragments were detected. Conclusion. The ability of doxycycline to inhibit matrix rhMMP-8 activity or to promote its degradation is lost at pH values lower than 7. Although relatively high pH values may exist in adult articular cartilage in some pathological situations, at lower pH the effect of doxynycline on proenzyme levels in the extracellular matrix may be due to an effect on the regulation of synthesis of the proenzyme, rather than to direct inhibition of the active enzyme or reduction in the level of enzyme by proteolysis.

AB - Objective. To examine, as part of an evaluation of the role of matrix metalloproteinase (MMP) inhibition in the amelioration of cartilage damage by doxycycline, the effect of pH on the inhibition of activity and reduction in stability of recombinant human neutrophil collagenase (rhMMP-8) by doxycycline in vitro. Methods. After activation with trypsin, rhMMP-8 was assayed using a peptolide substrate and a colorimetric assay. The rate of hydrolysis in the presence and absence of 30 μM doxycycline was measured over a pH range of 6.5-7.9. The molecular weight changes that accompanied activation of the proenzyme by acetylphenylmercuric acetate (APMA) in the presence and absence of doxycycline at pH 6.9 and 7.5 were studied by Western blotting. Results. At pH values above 7.1, doxycycline inhibited the activity of the enzyme. At pH values below 7.1, no inhibition was observed. When doxycycline was present during activation with APMA at pH 7.5, significant amounts of small (< 30 kDa) fragments were generated. In contrast, when doxycycline was present during activation with APMA at pH 6.9, no small fragments were detected. Conclusion. The ability of doxycycline to inhibit matrix rhMMP-8 activity or to promote its degradation is lost at pH values lower than 7. Although relatively high pH values may exist in adult articular cartilage in some pathological situations, at lower pH the effect of doxynycline on proenzyme levels in the extracellular matrix may be due to an effect on the regulation of synthesis of the proenzyme, rather than to direct inhibition of the active enzyme or reduction in the level of enzyme by proteolysis.

UR - http://www.scopus.com/inward/record.url?scp=1842405998&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=1842405998&partnerID=8YFLogxK

M3 - Article

C2 - 9292802

AN - SCOPUS:1842405998

VL - 24

SP - 1769

EP - 1773

JO - Journal of Rheumatology

JF - Journal of Rheumatology

SN - 0315-162X

IS - 9

ER -