Assembly of the fertilization membrane of the sea urchin: isolation of a divalent cation-dependent intermediate and its crosslinking in vitro.
To analyze the mechanism of assembly of the fertilization membrane of the sea urchin Strongylocentrotus purpuratus, we inhibited the ovoperoxidase that catalyzes dityrosine formation to isolate an uncrosslinked, soft fertilization membrane (SFM). The SFM intermediates were stabilized by divalent cation-dependent interactions: in the absence of divalent cations, the SFM became amorphous and less refractile and released proteins into the surrounding medium. We term the remaining structures "wraiths." The rate of this disaggregation was increased in solutions of low ionic strength, but 5-10 mM divalent cations (Ca2+, Mg2+, Mn2+ or Ba2+) prevented disaggregation. Wraiths could be reassembled into structures that resembled SFM by readdition of divalent cations. The SFM contained active ovoperoxidase and could be hardened in vitro by washing away the ovoperoxidase inhibitor and adding H2O2. After hardening, certain proteins of over 100 kd were excluded from SDS-polyacrylamide gels, suggesting that these proteins contain the substrates for crosslinking. We propose that the SFM is a divalent cation-dependent intermediate on the pathway of fertilization membrane assembly containing tyrosyl residues that are appropriately juxtaposed for crosslinking.
D002118:Calcium / Q000502:physiology*; D004220:Disulfides; D005306:Fertilization; D006023:Glycoproteins / Q000502:physiology; D008855:Microscopy, Electron, Scanning; D008970:Molecular Weight; D009994:Osmolar Concentration; D010544:Peroxidases / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000032:analysis; D012617:Sea Urchins / Q000502:physiology*; D014443:Tyrosine
D004220:Disulfides; D006023:Glycoproteins; D011506:Proteins; D014443:Tyrosine; D010544:Peroxidases; D002118:Calcium