Developmentally regulated protease expression during sea urchin embryogenesis.
A temporal study of protease expression employing the technique of SDS-PAGE gelatin substrate zymography revealed a definitive appearance of proteases during early development in the sea urchins, Lytechinus pictus and Strongylocentrotus purpuratus. The levels of these proteases increase substantially during gastrulation in each species. The two major proteases with relative molecular masses of 57 and 50 kDa were found to be inhibited by the zinc chelator, 1,10-phenanthroline, the more nonspecific metal chelator, EDTA, and the reducing agent, dithiothreitol. The serine protease inhibitor, benzamidine, exerted no effects on the activities of these proteases, and both enzymes exhibited activity in the neutral to slightly basic pH range. Treatment of embryos with actinomycin D, an inhibitor of transcription, beginning up to 9 hr after fertilization, inhibited the subsequent appearances of the two proteases 48 hr after fertilization, as well as any morphological changes associated with gastrulation. Treatments beginning 15 and 21 hr after fertilization resulted in increased levels of proteases that correlate with arrests at successively more advanced stages of gastrulation. SDS-PAGE zymographic analyses of five different embryo fractions indicated that the 57- and 50-kDa proteases are localized in the blastocoel, and blastocoelic protease activity was further confirmed microscopically by in situ zymography. Hence, the 57- and 50-kDa proteases are characterized as metalloproteases. Their expression is dependent on transcription of the embryonic genome, and their spatiotemporal appearance suggests an involvement in blastocoelic matrix remodeling during gastrulation.
Vafa
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O
Department of Biology, Georgetown University, Washington, DC 20057.
Nishioka
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D
D000818:Animals; D003609:Dactinomycin / Q000494:pharmacology; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005260:Female; D005775:Gastrula / Q000187:drug effects; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006651:Histocytochemistry; D008297:Male; D008970:Molecular Weight; D012617:Sea Urchins / Q000196:embryology / Q000201:enzymology* / Q000235:genetics*
D003609:Dactinomycin; D010450:Endopeptidases