Characterization of matrix metalloprotease activities induced in the sea urchin extraembryonic matrix, the hyaline layer

Hyaline layers, freshly prepared from one-hour-old embryos, were devoid of gelatin-cleavage activity. However, upon storage at 4 degreesC, gelatin-cle avage activities appeared; three species of apparent mol mass 94 right arro w 117-, 90-, and 45-kDa were seen. All three species required zinc for acti vity. Using gel-exclusion chromatography we separated the 94 right arrow 11 7-, and 90-kDa species from the 45-kDa activity. The two higher mol mass sp ecies were inhibited by ethylenebis (oxyethylenenitrilo) tetraacetic acid a nd the lost activity was restored by calcium. Reconstitution of activity oc curred with an apparent dissociation constant (calcium) of 5 muM. The prese nce of millimolar concentrations of magnesium had a minimal inhibitory effe ct on activity. The thermal denaturation profile of the higher mol mass gel atin- cleavage activity was significantly different in the presence and abs ence of calcium. Stabilization of these activities against thermal denatura tion at 60 degreesC occurred with an apparent dissociation constant (calciu m) of 0.6 mM. Magnesium had no significant effect on the thermal denaturati on profile. Collectively, these results suggest at least two different mode s of interaction between calcium and the higher mol mass gelatinases. These conclusions are discussed in the context of the high calcium and magnesium concentrations present in the sea water environment of the sea urchin embr yo.