Kinetics of inhibition of purified bovine neutrophil matrix metalloproteinase 9 by low-molecular-weight inhibitors.

OBJECTIVE

To measure the effects of lowmolecular-weight inhibitors on the activity of bovine neutrophil matrix metalloproteinase 9 (MMP-9).

SAMPLE POPULATION: Bovine MMP-9 purified from bovine neutrophilconditioned medium.

PROCEDURES: Neutrophils were degranulated by stimulation with phorbol ester. Enzyme purification was performed by use of gelatin affinity and gel-filtration chromatography. Activated enzyme was incubated with inhibitors prior to addition of substrate (gelatin fluorescein conjugate or fluorogenic peptide). Rates of enzymatic cleavage were determined by monitoring fluorescence as the reactions progressed. Values of IC(50) (molar concentration of compound that inhibits specific activity by 50%) and K(I) (in vitro inhibition constant) were determined.

RESULTS

Rates of enzymatic activity of monomeric and dimeric bovine MMP-9 measured by use of gelatin and peptide substrates were linear with respect to time and concentrations of enzyme and substrate. The MMP-9 was potently inhibited by hydroxamic acids (IC(50) for gelatin, 29.2 to 55.7 nM; IC(50) for peptide, 4.8 to 24.6 nM; K(I), 0.2 to 0.5 nM), whereas tetracyclines (IC(50) for gelatin, 30.1 to 112.7 MM; IC(50) for peptide, 48.0 to 123.8 MM; K(I), 25.2 to 61.4 microM) and chlorhexidine (IC(50) for gelatin, 139.1MM; IC(50) for peptide, 672.5MM to 1.7 mM; K(I), 495.0 to 663.0 MM) had limited inhibition. Gelatinase-specific inhibitor SB-3CT had intermediate potency (IC(50) for peptide, 185.0 to 290.0 nM; K(I), 66.5 to 86.0 nM).

CONCLUSIONS AND CLINICAL RELEVANCE: Bovine MMP-9 was potently inhibited by hydroxamic acids and gelatinase inhibitor. These compounds may be useful as modulators of neutrophil-mediated protease activity in cattle.