Calcium-protein interactions in the extracellular environment: calcium binding, activation, and immunolocalization of a collagenase/gelatinase activity expressed in the sea urchin embryo.
We have purified and characterized a collagenase/gelatinase activity expressed during sea urchin embryonic development. The native molecular mass was determined to be 160 kDa, while gelatin substrate gel zymography revealed an active species of 41 kDa, suggesting that the native enzyme is a tetramer of active subunits. Incubation in the presence of EGTA resulted in nearly complete loss of activity and this effect could be reversed by calcium. Calcium-induced reactivation appeared to be cooperative and occurred with an apparent kd value of 3.7 mM. Two modes of calcium binding to the 41-kDa subunit were detected; up to 80 moles of calcium bound with a kd value of 0.5 mM, while an additional 120 moles bound with a kd value of 5 mM. Amino acid analysis revealed a carboxy plus carboxyamide content of 24.3 mol/100 mol, indicating the availability of substantial numbers of weak Ca2+-binding sites. Calcium binding did not result in either secondary or quaternary structural changes in the collagenase/gelatinase, suggesting that Ca2+ may facilitate activation through directly mediating the binding of substrate to the enzyme. The collagenase/gelatinase activity was detected in blastocoelic fluid and in the hyalin fraction dissociated from 1-h-old embryos. Immunolocalization studies revealed two storage compartments in the egg; cortical granules and small granules/vesicles dispersed throughout the cytoplasm. After fertilization, the antigen was detected in both the apical and basal extracellular matrices, the hyaline layer, and basal lamina, respectively.
Mayne
J
J
Department of Biochemistry, Memorial University of Newfoundland, St. John's, Canada.
Robinson
J J
JJ
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D017364:Collagenases / Q000276:immunology / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism*; D005260:Female; D018093:Gelatinases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D006818:Hyalin / Q000737:chemistry / Q000378:metabolism; D007106:Immune Sera; D010063:Ovum / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D012617:Sea Urchins / Q000196:embryology*
D000596:Amino Acids; D007106:Immune Sera; D017364:Collagenases; D018093:Gelatinases; D002118:Calcium