Effects of calcium and magnesium on a 41-kDa serine-dependent protease possessing collagen-cleavage activity.
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.
Robinson
J J
JJ
Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland, Canada. johnro@morgan.ucs.mun.ca
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D003094:Collagen / Q000378:metabolism*; D017364:Collagenases / Q000378:metabolism*; D006868:Hydrolysis; D008274:Magnesium / Q000378:metabolism*; D012617:Sea Urchins / Q000196:embryology; D012694:Serine / Q000378:metabolism*
D012694:Serine; D003094:Collagen; D017364:Collagenases; D008274:Magnesium; D002118:Calcium