Ovoperoxidase assembly into the sea urchin fertilization envelope and dityrosine crosslinking.
Ovoperoxidase, the enzyme implicated in hardening the extracellular coat of the fertilized sea urchin egg, is inserted into the assembling uncrosslinked (soft) fertilization membrane via specific interactions with a protein, proteoliaisin (P. Weidman, E. Kay, and B. M. Shapiro (1985). J. Cell. Biol. 100, 938-946), and the vitelline scaffold. Dityrosine crosslinks introduced by ovoperoxidase have been postulated to harden the assembled structure from such indirect data as the discovery of dityrosine in hard fertilization membranes (Foerder and B. M. Shapiro (1977). Proc. Natl. Acad. Sci. USA 74, 4214-4128; H. G. Hall (1978). Cell 15, 343-355). In this report, we show directly that soft fertilization membranes (SFM) contain no dityrosine residues but acquire these crosslinks in vitro only during hardening. In vitro hardening alters the susceptibility of the fertilization membrane to disruption in cation-depleted solutions and in detergent; the kinetics of these phenomena are all similar to those of hardening in vivo. Ovoperoxidase substrates were identified as a class of high-molecular-weight proteins of SFM by polyacrylamide gel electrophoresis after in vitro hardening or after an ovoperoxidase-catalyzed radioiodination reaction. The specificity of ovoperoxidase for particular substrates decreased once it was no longer associated with these polypeptides within the SFM. Moreover, after disruption of the SFM, ovoperoxidase had an increased capacity to iodinate an exogenous protein, myoglobin. These data suggest that assembly of ovoperoxidase into a specific locus within the soft fertilization membrane provides a regulatory mechanism to guarantee the crosslinking of only certain appropriately juxtaposed tyrosyl residues in the assembled structure.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005109:Extracellular Matrix / Q000201:enzymology*; D005306:Fertilization; D007454:Iodides / Q000378:metabolism; D008970:Molecular Weight; D010063:Ovum / Q000201:enzymology*; D010544:Peroxidases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D012617:Sea Urchins; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D007454:Iodides; D011506:Proteins; D014443:Tyrosine; D004492:Edetic Acid; C007543:dityrosine; D010544:Peroxidases; D002118:Calcium