Identification and partial characterization of two inducible gelatin-cleavage activities localized to the sea urchin extraembryonic matrix, the hyaline layer.
We have identified two inducible, gelatin-cleaving activities in the sea urchin extraembryonic matrix, the hyaline layer. Isolated hyaline layers, incubated in the presence of benzamidine, were devoid of gelatin-cleavage activities with apparent molecular mass less then 80k. However, when layers were incubated for 9-11 h in the absence of benzamidine, gelatin-cleavage activities, with apparent molecular mass 40- and 50k, were detected. Induction required the presence of NaCl and CaCl(2) at concentrations similar to those found in seawater and readdition of the reversible serine protease inhibitor benzamidine prevented induction. Both gelatin-cleaving activities were activated by calcium at a concentration similar to the calcium concentration found in seawater. Magnesium, also a major cationic species present in seawater, could not replace calcium as the activating ion. In addition, magnesium could not compete with calcium for binding to the gelatinases. Both cleavage activities showed substrate specificity and each failed to cleave bovine serum albumin, bovine hemoglobin or casein. Cleavage activity towards gelatin was inhibited by benzamidine and aminoethyl benzenesulfonyl fluoride, indicating that both activities belonged to the serine class of proteases. The induced 40-kDa activity displayed similar properties to those of a comigrating, gelatin-cleaving activity present in 69-h-old embryos.
Robinson
John J
JJ
Department of Biochemistry, Memorial University of Newfoundland, A1B 3X9, St. John's, NF, Canada. johnro@mun.ca
Sharpe
Christopher
C
Calloway
Carolyn
C
D000818:Animals; D001550:Benzamidines / Q000494:pharmacology; D002122:Calcium Chloride / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000201:enzymology; D047108:Embryonic Development; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D005780:Gelatin / Q000378:metabolism*; D006818:Hyalin / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure; D008858:Microscopy, Phase-Contrast; D012617:Sea Urchins / Q000196:embryology* / Q000201:enzymology / Q000378:metabolism*; D012623:Seawater; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013450:Sulfones / Q000494:pharmacology
D001550:Benzamidines; D002135:Calcium-Binding Proteins; D016326:Extracellular Matrix Proteins; D015842:Serine Proteinase Inhibitors; D013450:Sulfones; C076585:hyalin protein, Echinoidea; C002010:4-(2-aminoethyl)benzenesulfonylfluoride; D005780:Gelatin; C032157:benzamidine; D002122:Calcium Chloride