Characterisation of a 41 kDa collagenase/gelatinase activity expressed in the sea urchin embryo

Zygote ◽  
1999 ◽  
Vol 8 (S1) ◽  
pp. S37-S38
Author(s):  
John J. Robinson ◽  
Janice Mayne
Keyword(s):  
Extracellular Matrix ◽  
Sea Urchin ◽  
High Concentration ◽  
Gelatinase Activity ◽  
Cleavage Activity ◽  
Physiological Processes ◽  
Sea Urchin Egg ◽  
Sea Urchin Embryo ◽  
The Matrix ◽  
High Concentrations

Protease activities have been recognised as important elements in controlling the composition of the extracellular matrix. Regulated remodelling of the matrix is required for a number of physiological processes including embryonic development. Excessive and unregulated remodelling has been associated with a number of pathological conditions including the metastatic phenotype of malignant cancer (Kim et al., 1998). We have begun a search for protease activities which utilise components of the sea urchin extracellular matrix as substrates. We have identified and purified a 41 kDa protease which is present in the sea urchin egg and embryo. This species possesses a non-specific gelatin-cleavage activity as well as a collagen cleavage activity which appears to be specific for echinoderm collagen (Mayne & Robinson, 1996, 1999).The 41 kDa collagenase/ gelatinase was inhibited by EGTA and reactivated by calcium. The calcium-concentration dependence for reactivation indicated an apparent kd of 3.7 mM and was coincident with the binding of 80 moles calcium/mole of protein. These results are interpretable in terms of the high concentration of calcium (10 mM) present in seawater. In addition to calcium, seawater also contains 50 mM magnesium. The substantial amounts of calcium bound to the 41 kDa protease suggest the existence of binding sites with both low affinity and specificity for binding metal ions. To determine whether high concentrations of magnesium could influence the interaction of calcium with the 41 kDa species we used both qualitative and quantitative gelatin-cleavage assays to examine protease activity in the presence of both calcium and magnesium.

Identification and characterization of gelatin-cleavage activities in the apically located extracellular matrix of the sea urchin embryo

2000 ◽  
Vol 78 (4) ◽  
pp. 455-462 ◽  
Author(s):  
Justin Flood ◽  
Janice Mayne ◽  
John J Robinson
Keyword(s):  
Extracellular Matrix ◽  
Sea Urchin ◽  
Inhibitory Effect ◽  
Divalent Metal Ions ◽  
Gelatinase Activity ◽  
Sea Urchin Embryo ◽  
Phenylmethyl Sulfonyl Fluoride ◽  
Sulfonyl Fluoride ◽  
Identification And Characterization

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°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.Key words: gelatinase, sea urchin, extracellular matrix.

Purification and metal ion requirements of a candidate matrix metalloproteinase: a 41 kDa gelatinase activity in the sea urchin embryo

1996 ◽  
Vol 74 (2) ◽  
pp. 211-218 ◽  
Author(s):  
Janice Mayne ◽  
John J. Robinson
Keyword(s):  
Matrix Metalloproteinase ◽  
Sea Urchin ◽  
Metal Ion ◽  
Divalent Cations ◽  
Chelating Agent ◽  
Ion Binding ◽  
Metal Ion Binding ◽  
Gelatinase Activity ◽  
Sea Urchin Embryo ◽  
The Matrix

Using substrate gel zymography, the sea urchin embryo was found to express a dynamic pattern of gelatinase activities with a 41 kDa species persisting throughout the course of embryonic development. We have purified to near homogeneity the 41 kDa gelatinase in the sea urchin egg. In both qualitative and quantitative assays, the 41 kDa gelatinase activity was inhibited by ethylenediaminetetracetic acid but not the serine protease inhibitor, phenylmethylsulfonylfluoride, or the chelating agent, 1,10-phenanthroline. Activity could be restored to the inactivated gelatinase by each of several divalent cations: Ca2+ > Mn2+ > Mg2+ > Cu2+. Cadmium and Zn2+ were largely ineffective at reconstituting the inactivated enzyme. In metal ion binding assays, the relative apparent affinities of the metal ions for binding to the gelatinase were determined to be Zn2+ ≥ Cd2+ ≥ Ca2+ > Mn2+ > Mg2+ > Cu2+. While the gelatinase is clearly a metalloproteinase, metal ion binding per se is not sufficient for activity. The 41 kDa gelatinase exhibited selective substrate utilization, being most active with gelatin, substantially less active with casein, and inactive towards bovine haemoglobin and bovine serum albumin as substrates. The substrate specificity and metal ion requirements suggest that this species is a member of the matrix metalloproteinase class of extracellular matrix remodelling enzymes.Key words: gelatinase, metalloproteinase, sea urchin.

Comparative studies of rat liver and sea urchin embryo nuclear matrices: partial fractionation and protein kinase activity distribution

1982 ◽  
Vol 55 (1) ◽  
pp. 189-198
Author(s):  
L. Sevaljevic ◽  
M. Petrovic ◽  
M. Konstantinovic ◽  
K. Krtolica
Keyword(s):  
Protein Kinase ◽  
Sea Urchin ◽  
Rat Liver ◽  
Kinase Activity ◽  
Matrix Proteins ◽  
Protein Kinase Activity ◽  
Structural Elements ◽  
Activity Distribution ◽  
Sea Urchin Embryo ◽  
The Matrix

Rat liver and sea urchin embryo nuclear matrices were found to differ in composition and in the strength of the association of their structural elements. Apart from the qualitative differences in composition, the embryonic matrices retained greater amounts of nuclear proteins and DNA, and were less susceptible to ultrasonic treatment than those of rat liver. They were essentially resistant to mild sonication, by which the rat liver matrix structure was resolved into two distinct fractions, referred to by Berezney (1980) as matricin and ribonucleoprotein (RNP). Both sub-fractions exhibited a protein kinase activity; the phosphorylating capacity of the RNP-associated protein kinases was found to be higher than that of the matricin-bound enzyme. The preferred substrate was among the secondary matrix proteins. In sea urchin embryos, sonication introduced no change in the type and lesion of the matrix proteins phosphorylated by the associated enzyme.

The organic matrix of the sea urchin embryo spicule visualized by electron microscopic immunocytochemistry

1986 ◽  
Vol 44 ◽  
pp. 158-159
Author(s):  
N.C. Benson ◽  
S.C. Benson ◽  
F. Wilt
Keyword(s):  
Sea Urchin ◽  
Organic Matrix ◽  
Electron Microscopic ◽  
Electron Microscopic Immunocytochemistry ◽  
Molecular Weights ◽  
Stage Of Development ◽  
Sea Urchin Embryo ◽  
The Matrix ◽  
Mesenchyme Cells ◽  
Intracellular Vacuole

The embryonic spicule of the sea urchin, Strongylocentrotus purpuratus is first detected at the late gastrula stage of development. The calcite spicule elongates within an intracellular vacuole of a syncytium of primary mesenchyme cells (PMC) originating at the ventral end of the blastocoel cavity and continues to form upward within a cytoplasmic cable until fully formed at the prism larval stage. The calcite is laid down as concentric lamallae on an organic matrix that extends within and through the spicule. The matrix has been characterized as a glycoprotein with major bands of molecular weights of about 47, 50, 57 and 64 kd. The PMCs surrounding the spicule are presumably the sites of synthesis and glycosylation of the organic matrix.

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