Activation of recombinant human neutrophil procollagenase in the presence of doxycycline results in fragmentation of the enzyme and loss of enzyme activity

Gerald N. Smith, Kenneth D. Brandt, Karen Hasty

Research output: Contribution to journalArticle

68 Citations (Scopus)

Abstract

Objective. To determine if reduction of collagenase activity in vitro by doxycycline (doxy) is related to activation of the proenzyme, and to determine how exogenous Ca++ and Zn++ affect the reduction. Methods. Recombinant human neutrophil procollagenase was activated with trypsin or APMA. Activity was assayed on a small peptolide substrate or on 14C- acetylated collagen fibers. The molecular weight of the proenzyme, active enzyme, and enzyme fragments was determined by Western blotting, using a polyclonal antiserum raised against the recombinant proenzyme. Similar experiments were performed in the presence of EDTA, EGTA, 1,10- phenanthroline, or doxy. The effects of exogenous Ca++ and Zn++ were also tested. Results. Doxy inhibited activity of the enzyme against both substrates. If the drug was present during activation, the yield of activity was lower than when it was added after activation of the proenzyme. Western blotting showed that activation in the presence of doxy resulted in the appearance of lower molecular weight fragments and accumulation of less active enzyme. APMA generated prominent 28- and 26-kd fragments, while trypsin cleavage yielded 40- and 30-kd fragments. Fragmentation of the enzyme also occurred in the presence of EDTA or EGTA, but not 1,10-phenanthroline. It was prevented by Ca++ concentrations greater than 50 mM, but was not altered by addition of Zn++ in concentrations as high as 500 μM. Inhibition of collagenase activity by doxy could be overcome by 100 mM Ca++, but addition of Zn++ had no effect. Conclusion. These data suggest that doxy alters the conformation of procollagenase or collagenase by binding enzyme-associated Ca++, rendering the proteins more susceptible to proteolysis and resulting in irreversible loss of enzyme protein.

Original languageEnglish (US)
Pages (from-to)235-244
Number of pages10
JournalArthritis and rheumatism
Volume39
Issue number2
DOIs
StatePublished - Feb 1 1996

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Doxycycline
Neutrophils
Enzyme Precursors
Enzymes
Collagenases
Egtazic Acid
Edetic Acid
Trypsin
Molecular Weight
Western Blotting
procollagenase
Proteolysis
Immune Sera
Proteins
Collagen
Pharmaceutical Preparations

All Science Journal Classification (ASJC) codes

  • Immunology and Allergy
  • Rheumatology
  • Immunology
  • Pharmacology (medical)

Cite this

Activation of recombinant human neutrophil procollagenase in the presence of doxycycline results in fragmentation of the enzyme and loss of enzyme activity. / Smith, Gerald N.; Brandt, Kenneth D.; Hasty, Karen.

In: Arthritis and rheumatism, Vol. 39, No. 2, 01.02.1996, p. 235-244.

Research output: Contribution to journalArticle

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abstract = "Objective. To determine if reduction of collagenase activity in vitro by doxycycline (doxy) is related to activation of the proenzyme, and to determine how exogenous Ca++ and Zn++ affect the reduction. Methods. Recombinant human neutrophil procollagenase was activated with trypsin or APMA. Activity was assayed on a small peptolide substrate or on 14C- acetylated collagen fibers. The molecular weight of the proenzyme, active enzyme, and enzyme fragments was determined by Western blotting, using a polyclonal antiserum raised against the recombinant proenzyme. Similar experiments were performed in the presence of EDTA, EGTA, 1,10- phenanthroline, or doxy. The effects of exogenous Ca++ and Zn++ were also tested. Results. Doxy inhibited activity of the enzyme against both substrates. If the drug was present during activation, the yield of activity was lower than when it was added after activation of the proenzyme. Western blotting showed that activation in the presence of doxy resulted in the appearance of lower molecular weight fragments and accumulation of less active enzyme. APMA generated prominent 28- and 26-kd fragments, while trypsin cleavage yielded 40- and 30-kd fragments. Fragmentation of the enzyme also occurred in the presence of EDTA or EGTA, but not 1,10-phenanthroline. It was prevented by Ca++ concentrations greater than 50 mM, but was not altered by addition of Zn++ in concentrations as high as 500 μM. Inhibition of collagenase activity by doxy could be overcome by 100 mM Ca++, but addition of Zn++ had no effect. Conclusion. These data suggest that doxy alters the conformation of procollagenase or collagenase by binding enzyme-associated Ca++, rendering the proteins more susceptible to proteolysis and resulting in irreversible loss of enzyme protein.",
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N2 - Objective. To determine if reduction of collagenase activity in vitro by doxycycline (doxy) is related to activation of the proenzyme, and to determine how exogenous Ca++ and Zn++ affect the reduction. Methods. Recombinant human neutrophil procollagenase was activated with trypsin or APMA. Activity was assayed on a small peptolide substrate or on 14C- acetylated collagen fibers. The molecular weight of the proenzyme, active enzyme, and enzyme fragments was determined by Western blotting, using a polyclonal antiserum raised against the recombinant proenzyme. Similar experiments were performed in the presence of EDTA, EGTA, 1,10- phenanthroline, or doxy. The effects of exogenous Ca++ and Zn++ were also tested. Results. Doxy inhibited activity of the enzyme against both substrates. If the drug was present during activation, the yield of activity was lower than when it was added after activation of the proenzyme. Western blotting showed that activation in the presence of doxy resulted in the appearance of lower molecular weight fragments and accumulation of less active enzyme. APMA generated prominent 28- and 26-kd fragments, while trypsin cleavage yielded 40- and 30-kd fragments. Fragmentation of the enzyme also occurred in the presence of EDTA or EGTA, but not 1,10-phenanthroline. It was prevented by Ca++ concentrations greater than 50 mM, but was not altered by addition of Zn++ in concentrations as high as 500 μM. Inhibition of collagenase activity by doxy could be overcome by 100 mM Ca++, but addition of Zn++ had no effect. Conclusion. These data suggest that doxy alters the conformation of procollagenase or collagenase by binding enzyme-associated Ca++, rendering the proteins more susceptible to proteolysis and resulting in irreversible loss of enzyme protein.

AB - Objective. To determine if reduction of collagenase activity in vitro by doxycycline (doxy) is related to activation of the proenzyme, and to determine how exogenous Ca++ and Zn++ affect the reduction. Methods. Recombinant human neutrophil procollagenase was activated with trypsin or APMA. Activity was assayed on a small peptolide substrate or on 14C- acetylated collagen fibers. The molecular weight of the proenzyme, active enzyme, and enzyme fragments was determined by Western blotting, using a polyclonal antiserum raised against the recombinant proenzyme. Similar experiments were performed in the presence of EDTA, EGTA, 1,10- phenanthroline, or doxy. The effects of exogenous Ca++ and Zn++ were also tested. Results. Doxy inhibited activity of the enzyme against both substrates. If the drug was present during activation, the yield of activity was lower than when it was added after activation of the proenzyme. Western blotting showed that activation in the presence of doxy resulted in the appearance of lower molecular weight fragments and accumulation of less active enzyme. APMA generated prominent 28- and 26-kd fragments, while trypsin cleavage yielded 40- and 30-kd fragments. Fragmentation of the enzyme also occurred in the presence of EDTA or EGTA, but not 1,10-phenanthroline. It was prevented by Ca++ concentrations greater than 50 mM, but was not altered by addition of Zn++ in concentrations as high as 500 μM. Inhibition of collagenase activity by doxy could be overcome by 100 mM Ca++, but addition of Zn++ had no effect. Conclusion. These data suggest that doxy alters the conformation of procollagenase or collagenase by binding enzyme-associated Ca++, rendering the proteins more susceptible to proteolysis and resulting in irreversible loss of enzyme protein.

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