Purification and metal ion requirements of a candidate matrix metalloproteinase: a 41 kDa gelatinase activity in the sea urchin embryo.
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: Ca(2+) > Mn(2+) > Mg(2+) > Cu(2+). Cadmium and Zn(2+) 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 Zn(2+) > or = Cd(2+) > or = Ca(2+) > Mn(2+) > Mg(2+) > Cu(2+). 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.
Mayne
J
J
Department of Biochemistry, Memorial University of Newfoundland, St. John's, Canada.
Robinson
J J
JJ
D000494:Allosteric Regulation; D000818:Animals; D002413:Cations, Divalent / Q000378:metabolism*; D002417:Cattle; D002614:Chelating Agents / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004625:Embryo, Nonmammalian / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005109:Extracellular Matrix / Q000378:metabolism; D005780:Gelatin / Q000378:metabolism; D018093:Gelatinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism; D008970:Molecular Weight; D010618:Phenanthrolines / Q000494:pharmacology; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D012617:Sea Urchins / Q000196:embryology / Q000201:enzymology*; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013379:Substrate Specificity
D002413:Cations, Divalent; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D006454:Hemoglobins; D010618:Phenanthrolines; D012710:Serum Albumin, Bovine; D010664:Phenylmethylsulfonyl Fluoride; D005780:Gelatin; D004492:Edetic Acid; D018093:Gelatinases; C025205:1,10-phenanthroline