Identification and characterization of gelatin-cleavage activities in the apically located extracellular matrix of the sea urchin embryo.
We have identified and partially characterized several gelatinase activities associated with the sea urchin extraembryonic matrix, the hyaline layer. A previously identified 41-kDa collagenase/gelatinase activity was generally not found to be associated with isolated hyaline layers but was dissociated from the surface of 1-h-old embryos in the absence of Ca2+ and Mg2+. While hyaline layers, freshly prepared from 1-h-old embryos, were devoid of any associated gelatinase activities, upon storage at 4 degrees C for 4 days, a number of gelatin-cleavage activities appeared. Comparative analysis of these activities with the 41-kDa collagenase/gelatinase revealed that all species were inhibited by ethylenediamine tetraacetic acid but were refractory to inhibition with the serine protease inhibitors, phenylmethyl sulfonyl fluoride and benzamidine. In contrast, the largely Zn2+ specific chelator 1,10-phenanthroline had markedly different effects on the gelatinase activities. While several of the storage-induced, hyaline-layer-associated gelatinase activities were inhibited, the 41-kDa collagenase/gelatinase was refractory to inhibition as was a second gelatinase species with an apparent molecular mass of 45 kDa. We also examined the effects of a series of divalent metal ions on the gelatin-cleavage activities. In both qualitative and quantitative assays, Ca2+ was the most effective activator while Mn2+, Cu2+, Cd2+, and Zn2+ were all inhibitory. In contrast, Mg2+ had a minimal inhibitory effect on storage-induced gelatinase activities but significantly inhibited the 41-kDa collagenase/gelatinase. These results identify several distinct gelatin-cleavage activities associated with the sea urchin extraembryonic hyaline layer and point to diversity in the biochemical nature of these species.
Flood
J
J
Department of Biochemistry, Memorial University of Newfoundland, St. John's, Canada.
Mayne
J
J
Robinson
J J
JJ
D000818:Animals; D001550:Benzamidines / Q000494:pharmacology; D002021:Buffers; D002413:Cations, Divalent / Q000378:metabolism; D016764:Cell Polarity; D017364:Collagenases / Q000378:metabolism*; D004586:Electrophoresis; D004625:Embryo, Nonmammalian / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology; D005109:Extracellular Matrix / Q000737:chemistry / Q000201:enzymology*; D005780:Gelatin / Q000378:metabolism; D018093:Gelatinases / Q000378:metabolism*; D010618:Phenanthrolines / Q000494:pharmacology; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D012617:Sea Urchins / Q000196:embryology* / Q000201:enzymology; D013379:Substrate Specificity
D001550:Benzamidines; D002021:Buffers; D002413:Cations, Divalent; D010618:Phenanthrolines; D011480:Protease Inhibitors; D010664:Phenylmethylsulfonyl Fluoride; D005780:Gelatin; D017364:Collagenases; D018093:Gelatinases; C025205:1,10-phenanthroline