Identification and characterization of gelatin-cleavage activities in the apically located extracellular matrix of the sea urchin embryo

We have identified and partially characterized several gelatinase activitie s associated with the sea urchin extraembryonic matrix, the hyaline layer. A previously identified 41-kDa collagenase/gelatinase activity was generall y not found to be associated with isolated hyaline layers but was dissociat ed from the surface of 1-h-old embryos in the absence of Ca2+ and Mg2+. Whi le hyaline layers, freshly prepared from 1-h-old embryos, were devoid of an y associated gelatinase activities, upon storage at 4 degrees C for 4 days, a number of gelatin-cleavage activities appeared. Comparative analysis of these activities with the 41-kDa collagenase/gelatinase revealed that all s pecies were inhibited by ethylenediamine tetraacetic acid but were refracto ry to inhibition with the serine protease inhibitors, phenylmethyl sulfonyl fluoride and benzamidine. In contrast, the largely Zn2+ specific chelator 1,10-phenanthroline had markedly different effects on the gelatinase activi ties. While several of the storage-induced, hyaline-layer-associated gelati nase activities were inhibited, the 41-kDa collagenase/gelatinase was refra ctory to inhibition as was a second gelatinase species with an apparent mol ecular mass of 45 kDa. We also examined the effects of a series of divalent metal ions on the gelatin-cleavage activities. In both qualitative and qua ntitative assays, Ca2+ was the most effective activator while Mn2+ Cu2+, Cd 2+, and Zn2+ were all inhibitory. In contrast, Mg2+ had a minimal inhibitor y effect on storage-induced gelatinase activities but significantly inhibit ed the 41-kDa collagenase/gelatinase. These results identify several distin ct gelatin-cleavage activities associated with the sea urchin extraembryoni c hyaline layer and point to diversity in the biochemical nature of these s pecies.