Release of ovoperoxidase from sea urchin eggs hardens the fertilization membrane with tyrosine crosslinks

Abstract

One feature of fertilization is the alteration of the vitelline layer, by components released from the egg, to produce an elevated, covalently crosslinked, hard, insoluble, fertilization membrane. Evidence indicates that crosslinking and hardening [in Strongylocentrotus purpuratus] are caused by the production of di- and trityrosyl residues, by oxidation of protein-bound tyrosyl residues in the presence of peroxidase. Hardening of the fertilization membrane, as evidenced by its loss of solubility in 50 mM dithiothreitol, is inhibited by compounds known to inhibit many peroxidases. A peroxidase termed ovoperoxidase is released from eggs at fertilization. This enzyme is inhibited by the same compounds that inhibit hardening and at similar concentrations. Inhibitors of the ovoperoxidase and the hardening reaction include KCN, 3-amino-1,2,4-triazole, NaN3, phenylhydrazine, K4Fe(CN)6, sodium sulfite and glycine ethyl ester. Tyramine and N-acetyltyrosine both inhibit hardening but O-methyltyrosine does not. Di- and trityrosyl residues are found in acid hydrolysates of isolated, hardened fertilization membranes. These residues were identified by cellulose phosphate column chromatography, TLC and amino acid analysis. The amino acid data were used to estimate that there is 1 dityrosine crosslink/55,000 daltons of protein. By catalyzing the crosslinking of tyrosyl residues, the ovoperoxidase leads to the production of a hard fertilization membrane that blocks entry of additional sperm. Because peroxidases are spermicidal, a secondary function of the enzyme could be to kill sperm in the vicinity of the fertilized egg.