Comparative analysis of the kinetic parameters and thermal stability of two matrix metalloproteinases expressed in the developing sea urchin embryo

Abstract

The extracellular matrix is now recognized as a dynamic structure which influences cellular properties. Many matrix metalloproteinase activities have been identified and characterized in vertebrates and constitute important agents in controlling the composition of the extracellular matrix. We have begun a study of matrix metalloproteinase activities in the developing sea urchin embryo. Using sea urchin peristome collagen or gelatin as physiological substrates we have determined the kinetic parameters, K m and V max, for an 87 kDa gelatinase activity expressed in late stage sea urchin embryos. We also determined the kinetic parameters K m, V max and k cat, for a 41 kDa species, expressed in the early sea urchin embryo, which possesses both collagenase and gelatinase activities. All values determined were similar to those reported in the literature for vertebrate collagenases and gelatinases and K m values in the micromolar range suggest that both species possess physiologically relevant activities. Both activities have previously been shown to require Ca 2+ for activity. Using an assay for quantitating the cleavage of gelatin into trichloroacetic acid soluble peptides we report here markedly different effects of Ca 2+ on the thermal denaturation profiles of the gelatinases. This latter finding may be indicative of different modes of action for this activating cation. Collectively, these results demonstrate both similarities and differences between vertebrate and invertebrate sea urchin gelatinases.