Ovoperoxidase is a cortical granule-derived enzyme that hardens the sea urchin fertilization envelope by catalyzing the formation of dityrosine residues. Ovoperoxidase works in concert with a second protein, proteoliaisin, which anchors ovoperoxidase to the nascent fertilization envelope in a divalent cation-dependent manner. In this study, we examined the Ca(2+)-dependent interaction of proteoliaisin with ovoperoxidase in rotary-shadowed Pt replicas. Ovoperoxidase, a uniformly sized globular molecule, binds to a distal portion of rod-shaped proteoliaisin when low concentrations of Ca2+ are present. Higher Ca2+ concentrations lead to the formation of extended proteoliaisin strands that are decorated along their lengths with ovoperoxidase. Using immunogold labeling, we also examined the assimilation of these two proteins into the fertilization envelope in quick-frozen, deeply etched samples. Both proteins are abundant in the fertilization envelope as early as one minute after fertilization. Coincident with paracrystalline coating of the envelope, the labeling density is markedly reduced, suggesting that antigenic sites may be masked by the paracrystalline coat. This suggests that the ovoperoxidase-proteoliaisin complex resides within the central portion of the fertilization envelope, rather than in the paracrystalline coat.

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