INHIBITION OF RECOMBINANT HUMAN NEUTROPHIL COLLAGENASE BY DOXYCYCLINEIS PH DEPENDENT

Objective. To examine, as part of an evaluation of the role of matrix metalloproteinase (MMP) inhibition in the amelioration of cartilage da mage by doxycycline, the effect of pH on the inhibition of activity an d reduction in stability of recombinant human neutrophil collagenase ( rhMMP-8) by doxycycline in vitro. Methods. After activation with tryps in, rhMMP-8 was assayed using a peptolide substrate and a colorimetric assay. The rate of hydrolysis in the presence and absence of 30 mu M doxycycline was measured over a pH range of 6.5-7.9. The molecular wei ght changes that accompanied activation of the proenzyme by acetylphen ylmercuric acetate (APMA) in the presence and absence of doxycycline a t pH 6.9 and 7.5 were studied by Western blotting. Results. At pH valu es above 7.1, doxycycline inhibited the activity of the enzyme. At pH values below 7.1, no inhibition was observed. When doxycycline was pre sent during activation with APMA at pH 7.5, significant amounts of sma ll (< 30 kDa) fragments were generated. In contrast, when doxycycline was present during activation with APMA at pH 6.9, no small fragments were detected. Conclusion. The ability of doxycycline to inhibit matri x 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 ar ticular cartilage in some pathological situations, at lower pH the eff ect 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.