Characterization and immunolocalization of a 41 kDa collagenase/gelatinase activity in the sea urchin embryo and its effect(s) on development

Mayne, Janice Ella (2001) Characterization and immunolocalization of a 41 kDa collagenase/gelatinase activity in the sea urchin embryo and its effect(s) on development. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Using gelatin substrate gel zymography, several activities were detected in sea urchin eggs and in various stage embryos which ranged in apparent molecular masses from 27.5 k to 86.9 k. We have purified and characterized the prominent 41 kDa collagenase/gelatinase activity from the sea urchin egg. The native molecular mass was 160 kDa, suggesting a tetrameric structure. Upon dissociation of calcium, enzyme activity was lost and this inhibition was found to be reversible. Reconstitution of activity occurred with an apparent dissociation constant of 3.7 mM. Up to 200 mols of calcium bound per mol of the 41 kDa subunit. Two classes of calcium binding sites were detected with dissociation constants of 0.5 and 5 mM. Amino acid analysis of the 41 kDa collagenase/gelatinase revealed that it contained twenty-four mol percent carboxy and carboxyamide residues which can function as weak calcium-binding sites. In the presence of calcium no secondary or quaternary structural changes were detected in the collagenase/gelatinase. The 41 kDa species had collagen-cleavage activity which appeared to be specific for echinoderm collagen, and a less specific gelatin-cleavage activity toward both invertebrate and vertebrate gelatin. The kinetic parameters, KmVmax and the ratio kcat/Km determined for the collagenase and gelatinase activities using peristome collagen and gelatin, were similar to those reported in the literature for vertebrate collagenase and gelatinase activities. Immunolocalization was carried out to determine the subcellular localization of the 41 kDa collagenase/gelatinase in the unfertilized egg and in various stage embryos. The 41 kDa collagenase/gelatinase was found to be stored in several compartments in the egg: cortical granules (located along the periphery of the egg), and yolk granules and yolk granule-like structures (dispersed throughout the cytoplasm). Following fertilization, the 41 kDa species was detected in both the apical and basal extracellular matrices, the hyaline layer and basal lamina, respectively. In addition, the cytoplasmic storage compartment(s) retained a portion of their label into the gastrula stage. We followed the development of embryos in the presence of the 41 kDa collagenase/gelatinase and in the presence of the anti-41 kDa collagenase/gelatinase antiserum. In both studies embryos developed normally to the mesenchyme blastula stage, while subsequent development was retarded. The process of gastrulation was delayed and embryos failed to develop into plutei with well developed arms.

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