Purification and characterization of the sea urchin embryo hatching enzyme.
The sea urchin hatching enzyme provides an interesting model for the control of gene expression during early development. In order to study its properties and developmental regulation, the hatching enzyme of the species Paracentrotus lividus has been purified. The fertilization envelopes of the embryos were digested before hatching by a crude culture supernatant previously made. The enzyme was then solubilized by 1 M NaCl and 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and purified by hydrophobic chromatography on Procion-agarose. A 470-fold increase in specific activity was obtained. The kinetic parameters of the proteolytic activity using dimethylcasein as substrate are: Km = 120 micrograms x ml-1, Vm = 200 mumol x min-1 x mg-1, and kcat = 180 s-1 at 500 mM NaCl, 10 mM CaCl2, pH 8.0, at 35 degrees C. The purified enzyme is highly active on fertilization envelopes: at 20 degrees C and 500 mM NaCl, 10 mM CaCl2, pH 8.0, 100 ng of enzyme completely denudes embryos in about 20 min under standard conditions. The molecular mass of the enzyme was estimated as 57 kDa by gel filtration, 51 kDa by gel electrophoresis, and 52 kDa by amino acid analysis. The hatching enzyme was shown to be a glycoprotein which autolyzes to a 30-kDa inactive form. Antibodies raised against the 51- or 30-kDa forms reacted with both these forms. Immunoblotting experiments showed that the hatching supernatants contain important amounts of the autolyzed species.
Lepage
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Unité de Biologie Cellulaire Marine, Centre National de la Recherche Scientifique, Villefranche-sur-Mer, France.
Gache
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D000596:Amino Acids / Q000302:isolation & purification; D000818:Animals; D001329:Autolysis; D002474:Cell-Free System; D002847:Chromatography, Agarose; D004625:Embryo, Nonmammalian / Q000201:enzymology*; D006023:Glycoproteins / Q000302:isolation & purification; D007700:Kinetics; D008666:Metalloendopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D012617:Sea Urchins / Q000201:enzymology*
D000596:Amino Acids; D006023:Glycoproteins; D010446:Peptide Fragments; D008666:Metalloendopeptidases; C024344:envelysin