Effects of calcium and magnesium on a 41-kDa serine-dependent protease possessing collagen-cleavage activity

We report here the continued characterization of a 41-kDa protease expresse d in the early stage of the sea urchin embryo. This protease was previously shown to possess both a gelatin-cleavage activity and an echinoderm-specif ic collagen-cleavage activity. In the experiments reported here, we have ex plored the biochemical nature of this proteolytic activity. Pepstatin A (an acidic protease inhibitor), 1,10-phenanthroline (a metalloprotease inhibit or), and E-64 (a thiol protease inhibitor) were without effect on the gelat in-cleavage activity of the 41-kDa species. Using a gelatin substrate gel z ymographic assay, the serine protease inhibitors phenylmethylsulfonyl fluor ide and benzamide appeared to partially inhibit gelatin-cleavage activity. This result was confirmed in a quantitative gelatin-cleavage assay using th e water soluble, serine protease inhibitor [4-(2-aminoethyl)benzenesulfonyl fluoride]. The biochemical character of this protease was further explored by examining the effects of calcium and magnesium, the major divalent catio ns present in sea water, on the gelatin-cleavage activity. Calcium and magn esium competed for binding to the 41-kDa collagenase/gelatinase, and prebou nd calcium was displaced by magnesium. Cleavage activity was inhibited by m agnesium, and calcium protected the protease against this inhibition. These results identify calcium and magnesium as antagonistic agents that may reg ulate the proteolytic activity of the 41-kDa species. (C) 2000 Wiley-Liss, Inc.