Inhibition of recombinant human neutrophil collagenase by doxycycline is pH dependent.
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 microM 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.


CONCLUSIONS
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 in some pathological situations, at lower pH the effect of doxycycline 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.
Smith
G N
GN
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202-5103, USA.
Brandt
K D
KD
Mickler
E A
EA
Hasty
K A
KA
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D015153:Blotting, Western; D017364:Collagenases / Q000378:metabolism; D004318:Doxycycline / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020784:Matrix Metalloproteinase 8; D061965:Matrix Metalloproteinase Inhibitors; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology; D010662:Phenylmercuric Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D011994:Recombinant Proteins
D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D061965:Matrix Metalloproteinase Inhibitors; D011994:Recombinant Proteins; C010024:4-aminophenylmercuriacetate; D017364:Collagenases; D020784:Matrix Metalloproteinase 8; D004318:Doxycycline; D010662:Phenylmercuric Acetate