ACTIVATION OF RECOMBINANT HUMAN NEUTROPHIL PROCOLLAGENASE IN THE PRESENCE OF DOXYCYCLINE RESULTS IN FRAGMENTATION OF THE ENZYME AND LOSS OFENZYME-ACTIVITY

Objective. To determine if reduction of collagenase activity in vitro by doxycycline (doxy) is related to activation of the proenzyme, and t o determine how exogenous Ca++ and Zn++ affect the reduction. Methods. Recombinant human neutrophil procollagenase was activated with trypsi n or APMA. Activity was assayed on a small peptolide substrate or on C -14-acetylated collagen fibers. The molecular weight of the proenzyme, active enzyme, and enzyme fragments was determined by Western blottin g, using a polyclonal antiserum raised against the recombinant proenzy me. Similar experiments were performed in the presence of EDTA, EGTA, 1, 10-phenanthroline, or doxy. The effects of exogenous Ca++ and Zn+were also tested. Results. Doxy inhibited activity of the enzyme again st both substrates. If the drug was present during activation, the yie ld of activity was lower than when it was added after activation of th e proenzyme, Western blotting showed that activation in the presence o f doxy resulted in the appearance of lower molecular weight fragments and accumulation of less active enzyme, APMA generated prominent 28- a nd 26-kd fragments, while trypsin cleavage yielded 40- and 30-kd fragm ents. Fragmentation of the enzyme also occurred in the presence of EDT A or EGTA, but not 1,10-phenanthroline. It was prevented by Ca++ conce ntrations greater than 50 mM, but was not altered by addition of Zn+in concentrations as high as 500 mu M. Inhibition of collagenase activ ity by doxy could be overcome by 100 mM Ca++, but addition of Zn++ had no effect. Conclusion. These data suggest that doxy alters the confor mation of procollagenase or collagenase by binding enzyme-associated C a++, rendering the proteins more susceptible to proteolysis and result ing in irreversible loss bf enzyme protein.