To determine if reduction of collagenase activity in vitro by doxycycline (doxy) is related to activation of the proenzyme, and to determine how exogenous Ca++ and Zn++ affect the reduction.
Methods. Recombinant human neutrophil procollagenase was activated with trypsin or APMA. Activity was assayed on a small peptolide substrate or on 14C-acetylated collagen fibers. The molecular weight of the proenzyme, active enzyme, and enzyme fragments was determined by Western blotting, using a polyclonal antiserum raised against the recombinant proenzyme. Similar experiments were performed in the presence of EDTA, EGTA, 1,lO-phenanthroline, or doxy. The effects of exogenous Ca++ and Zn++ were also tested.
Results. Doxy inhibited activity of the enzyme against both substrates. If the drug was present during activation, the yield of activity was lower than when it was added after activation of the proenzyme. Western blotting showed that activation in the presence of doxy resulted in the appearance of lower molecular weight fragments and accumulation of less active enzyme. APMA generated prominent 28-and 26-kd fragments, while trypsin cleavage yielded 40-and 30-kd fragments. Fragmentation of the enzyme also occurred in the presence of EDTA or EGTA, but not 1,lO-phenanthroline. It was prevented by Ca++ concentrations greater than 50 mM, but was not altered by addition of Zn++ in concentrations as high as ..