Gelatinase a and b inhibition kinetics using inhibitors which cause glaucoma in a model system

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Abstract

The matrix metalloproteinases (MMPs) and their inhibitors (TTMPs) play a vital role in remodeling extracellular matrix (ECM). We hypothesize that defective ECM turnover, initiated by trabecular MMPs, causes increased intraocular pressure and ultimately, glaucoma. To test this hypothesis, we utilized specific MMP inhibitors in a glaucoma model system. In the present study, we evaluate the efficacy of these inhibitors on purified gelatinase A and B using two fluorescent substrates. Inhibition was analyzed directly using an FITC-labeled gelatin and using an Mca- and DNP-labeled peptide. Inhibitors included minocycline, tryptophan-hydroxamate, a mercapto-peptide inhibitor, a propeptide inhibitor and purified TIMP3. We observed competitive inhibition with tryptophan-hydroxamate (K, = 123 nM) and mixed inhibition with the other synthetic inhibitors for gelatinase A and B (K, = 250-500 uM). TIMP3 shows mixed inhibition on gelatinase A (Ki = 25 nM). Thus, inhibitors used previously to cause glaucoma in our model system are shown here to specifically inhibit gelatinase A and B in two assays via complex kinetic mechanisms at concentrations where they cause glaucoma in the human model system. This further supports a role for the MMPs in glaucoma.

Original languageEnglish (US)
JournalFASEB Journal
Volume10
Issue number6
StatePublished - 1996

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Gelatinases
glaucoma
Matrix Metalloproteinase 2
gelatinase A
Glaucoma
metalloproteinases
Matrix Metalloproteinase 9
gelatinase B
kinetics
Kinetics
Matrix Metalloproteinase Inhibitors
Matrix Metalloproteinases
extracellular matrix
tryptophan
Extracellular Matrix
Peptides
Minocycline
minocycline
Fluorescein-5-isothiocyanate
peptides

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

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title = "Gelatinase a and b inhibition kinetics using inhibitors which cause glaucoma in a model system",
abstract = "The matrix metalloproteinases (MMPs) and their inhibitors (TTMPs) play a vital role in remodeling extracellular matrix (ECM). We hypothesize that defective ECM turnover, initiated by trabecular MMPs, causes increased intraocular pressure and ultimately, glaucoma. To test this hypothesis, we utilized specific MMP inhibitors in a glaucoma model system. In the present study, we evaluate the efficacy of these inhibitors on purified gelatinase A and B using two fluorescent substrates. Inhibition was analyzed directly using an FITC-labeled gelatin and using an Mca- and DNP-labeled peptide. Inhibitors included minocycline, tryptophan-hydroxamate, a mercapto-peptide inhibitor, a propeptide inhibitor and purified TIMP3. We observed competitive inhibition with tryptophan-hydroxamate (K, = 123 nM) and mixed inhibition with the other synthetic inhibitors for gelatinase A and B (K, = 250-500 uM). TIMP3 shows mixed inhibition on gelatinase A (Ki = 25 nM). Thus, inhibitors used previously to cause glaucoma in our model system are shown here to specifically inhibit gelatinase A and B in two assays via complex kinetic mechanisms at concentrations where they cause glaucoma in the human model system. This further supports a role for the MMPs in glaucoma.",
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AU - Acott, Ted

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N2 - The matrix metalloproteinases (MMPs) and their inhibitors (TTMPs) play a vital role in remodeling extracellular matrix (ECM). We hypothesize that defective ECM turnover, initiated by trabecular MMPs, causes increased intraocular pressure and ultimately, glaucoma. To test this hypothesis, we utilized specific MMP inhibitors in a glaucoma model system. In the present study, we evaluate the efficacy of these inhibitors on purified gelatinase A and B using two fluorescent substrates. Inhibition was analyzed directly using an FITC-labeled gelatin and using an Mca- and DNP-labeled peptide. Inhibitors included minocycline, tryptophan-hydroxamate, a mercapto-peptide inhibitor, a propeptide inhibitor and purified TIMP3. We observed competitive inhibition with tryptophan-hydroxamate (K, = 123 nM) and mixed inhibition with the other synthetic inhibitors for gelatinase A and B (K, = 250-500 uM). TIMP3 shows mixed inhibition on gelatinase A (Ki = 25 nM). Thus, inhibitors used previously to cause glaucoma in our model system are shown here to specifically inhibit gelatinase A and B in two assays via complex kinetic mechanisms at concentrations where they cause glaucoma in the human model system. This further supports a role for the MMPs in glaucoma.

AB - The matrix metalloproteinases (MMPs) and their inhibitors (TTMPs) play a vital role in remodeling extracellular matrix (ECM). We hypothesize that defective ECM turnover, initiated by trabecular MMPs, causes increased intraocular pressure and ultimately, glaucoma. To test this hypothesis, we utilized specific MMP inhibitors in a glaucoma model system. In the present study, we evaluate the efficacy of these inhibitors on purified gelatinase A and B using two fluorescent substrates. Inhibition was analyzed directly using an FITC-labeled gelatin and using an Mca- and DNP-labeled peptide. Inhibitors included minocycline, tryptophan-hydroxamate, a mercapto-peptide inhibitor, a propeptide inhibitor and purified TIMP3. We observed competitive inhibition with tryptophan-hydroxamate (K, = 123 nM) and mixed inhibition with the other synthetic inhibitors for gelatinase A and B (K, = 250-500 uM). TIMP3 shows mixed inhibition on gelatinase A (Ki = 25 nM). Thus, inhibitors used previously to cause glaucoma in our model system are shown here to specifically inhibit gelatinase A and B in two assays via complex kinetic mechanisms at concentrations where they cause glaucoma in the human model system. This further supports a role for the MMPs in glaucoma.

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