Effects of calcium and magnesium on a 41-kDa serine-dependent protease possessing collagen-cleavage activity

Abstract

We report here the continued characterization of a 41-kDa protease expressed in the early stage of the sea urchin embryo. This protease was previously shown to possess both a gelatin-cleavage activity and an echinoderm-specific collagen-cleavage activity. In the experiments reported here, we have explored the biochemical nature of this proteolytic activity. Pepstatin A (an acidic protease inhibitor), 1,10-phenanthroline (a metalloprotease inhibitor), and E-64 (a thiol protease inhibitor) were without effect on the gelatin-cleavage activity of the 41-kDa species. Using a gelatin substrate gel zymographic assay, the serine protease inhibitors phenylmethylsulfonyl fluoride and benzamide appeared to partially inhibit gelatin-cleavage activity. This result was confirmed in a quantitative gelatin-cleavage assay using the water soluble, serine protease inhibitor [4-(2-aminoethyl)benzenesulfonylfluoride]. The biochemical character of this protease was further explored by examining the effects of calcium and magnesium, the major divalent cations present in sea water, on the gelatin-cleavage activity. Calcium and magnesium competed for binding to the 41-kDa collagenase/gelatinase, and prebound calcium was displaced by magnesium. Cleavage activity was inhibited by magnesium, and calcium protected the protease against this inhibition. These results identify calcium and magnesium as antagonistic agents that may regulate the proteolytic activity of the 41-kDa species.

Characterization of metalloproteinase-like activities in barnacle (Balanus amphitrite) nauplii

“…The gelatinase activities were dependent on the presence of Zn and Ca and were significantly ions, as previously reported for other marine invertebrate MMPs (Robinson, 1997;Ziegler et al, 2002). Also, addition of Mg partly activated the enzymes, suggesting a 2q broad, but weak metal ion binding capacity and a probable participation of these cations in barnacle gelatinase activities, similarly to what observed in the sea urchin embryo (Robinson, 1997(Robinson, , 2000 and the mussel Mytilus galloprovincialis . All naupliar gelatinase activities were strongly inhibited by heavy metal ions (Hg )Cu )Cd )Pb ).…”

“…MMP activity was evaluated in larval extracts with the quantitative substrate assay previously described by Robinson (1997), with slight modifications. Briefly, the quantitative assay was based on measurement of gelatinase-dependent cleavage of gelatin 90 Bloom into trichloroacetic acid (TCA) soluble peptides, according to the 'end-point method' described in detail elsewhere (Robinson, 2000). One unit of gelatinase activity was defined as the amount of gelatinase required to cleave 0.1 mg of gelatin 90 Bloomymin into TCA soluble peptides at 25 8C.…”

in situ Fabrication of Biological Chitosan and Gelatin-Based Hydrogels Loading Biphasic Calcium Phosphate Nanoparticles for Bone Tissue Regeneration

“…The INgel prepared at 1 PCD: 5 PGD and 1 PCD: 2.5 PGD ratios performed a lowest degradation rate approximately 60 wt/wt % of its weight at the end of this survey. The different behaviours could be derived from binding of calcium ions (released from BCPNPs) and collagenase leading to inhibition of its proteolytic activity [43]. These studies clarified that gelatin-based materials have a fast biodegradable characteristic in a collagenase containing media while the biodegradation of chitosan-formulated hydrogel could be modulated.…”

Changes in the urinary proteome of rats after short-term intake of magnesium threonate

“…Magnesium and zinc ions activate and inhibit protein tyrosine phosphatase 1B (PTP1B), respectively [18] . Magnesium inhibits the collagen-cleaving activity of the 41-kDa serine-dependent protease, and calcium protects the protease from this inhibition [19] . Magnesium sulfate inhibits LPS-macrophage binding by decreasing CD14 expression, and these mechanisms may involve the activation of serine proteases [20] .…”