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).


METHODS
Bovine MMP-9 purified from bovine neutrophilconditioned medium.


METHODS
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
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.
Bannikov
Gregory A
GA
Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210, USA.
Lakritz
Jeffrey
J
Premanandan
Christopher
C
Mattoon
John S
JS
Abrahamsen
Eric J
EJ
D000818:Animals; D002138:Calibration; D002417:Cattle / Q000502:physiology*; D004789:Enzyme Activation / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D007700:Kinetics; D020780:Matrix Metalloproteinase 9 / Q000737:chemistry; D061965:Matrix Metalloproteinase Inhibitors; D008970:Molecular Weight; D009504:Neutrophils / Q000201:enzymology*; D013997:Time Factors
D004791:Enzyme Inhibitors; D061965:Matrix Metalloproteinase Inhibitors; D020780:Matrix Metalloproteinase 9