Abstract
The hatching enzyme secreted by the blastula stage sea urchin embryo proteolyzes the fertilization envelope, thereby allowing the embryo to hatch. Using an assay that measures fertilization envelope degradation, we have purified the hatching enzyme by ion-exchange and affinity chromatography from the hatching medium of Strongylocentrotus purpuratus embryos. The hatching enzyme was found to be a 33-kDa metalloprotease that exhibited a substrate preference for only a minor subset of proteins in the fertilization envelope. Secretion of the hatching enzyme by blastula stage embryos occurred during the 2-h period prior to hatching. The hatching enzyme was initially secreted as a 57-kDa protein, but during purification it was converted to the 33-kDa form. Biosynthesis of the hatching enzyme began at the late morula/early blastula stage up to 6 h before secretion. Experiments using the ionophore monensin suggest that the lag between synthesis and secretion of the hatching enzyme by the blastula stage embryos may be a result of a slow constitutive secretory process.
Highlights
The hatching enzyme was initially secreted as a 57-kDa protein, but during purification it was converted to the 33-kDa form
Other cortical granule proteins are incorporated into the maturing fertilization envelope, some being cross-linked by peroxidase-mediated dityrosine formation or an egg cell surface transglutaminase
Characterization of HatchLng Enzyme Activity-To ensure that the secreted protease purified in these studies was, the hatching enzyme, fertilization envelopes isolated from fertilized eggs were labeled with Na]“I, and the hatching enzyme activity was assayed by release of nonsedimentable fragment of “‘I-fertilization envelopes in a centrifugation assay
Summary
The “‘I-fertilization envelopes were washed by centrifugation (4240 X g for 1.5 min) 10 times with stop buffer, washed for 16 h (4 “C) in 70 mM NaCl, 10 mM CaC12, 10 mM Tris.HCl, pH. Solid urea was added to give a final concentration of 6 M, and the hatched blastula medium was incubated at 4 “C for 30 min followed bv incubation at 24 “C for 5.5 h. The column was washed with 10 column volumes of HET buffer, pH 8.2, followed by elution of bound proteins with 2 M NaCl, 10 mM CaC&, 10 mM Tris.HCl, pH 8.2. Embryos were allowed to settle by gravity, washed in ASW, and resuspended in ASW, 10 mM Tris, pH 8.0,5 pg/ml gentamycin sulfate Because it arrested development, excess unlabeled methionine was not added to cultures of early stages. Pellet\ were bolled in Laemmli sample buffer for 2 mm and sedImented, and the gupernatant fluld was analyzed by SDS-PAGE and radloautograph?
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
