Purification and properties of ovoperoxidase, the enzyme responsible for hardening the fertilization membrane of the sea urchin egg.
The ovoperoxidase from the egg of the sea urchin, Strongylocentrotus purpuratus, has been purified to apparent homogeneity. Ovoperoxidase is secreted from the egg at fertilization and is responsible, in vivo, for hardening of the fertilization membrane by forming cross-links between protein tyrosyl residues. Purification was accomplished by activation of cortical granule exocytosis with acetic acid, followed by NH4SO4 precipitation, DEAE-Sephacel chromatography in the absence of divalent cations, and CM-Sephadex chromatography. The purified enzyme is a glycoprotein of Mr 70,000, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibits a UV-visible spectrum typical of heme peroxidases (epsilon 412 = 1.19 X 10(5) M-1 cm-1). Ovoperoxidase catalyzes the oxidation of tyrosine, guaiacol, iodide, and bromide, but not chloride, and can employ either H2O2 or, with 8% relative efficiency, ethyl peroxide as an oxidative substrate. Phenylhydrazine, 3-amino-1,2,4-triazole, azide, and sulfite all inhibit purified ovoperoxidase at concentrations similar to those that inhibit hardening in vivo. Inhibition by 3-amino-1,2,4-triazole is reversible, requires H2O2, and is slow relative to substrate turnover. The purified enzyme is sensitive to protease cleavage in the native state, yielding an active product of Mr approximately 50,000 which varies slightly depending upon the protease employed. Ovoperoxidase should provide a useful tool for the study of fertilization membrane formation as a paradigm of macromolecular assembly and modification.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D003594:Cytoplasmic Granules / Q000502:physiology; D005089:Exocytosis; D005321:Extraembryonic Membranes / Q000502:physiology*; D005260:Female; D005306:Fertilization; D007700:Kinetics; D007784:Lactoperoxidase / Q000378:metabolism; D008970:Molecular Weight; D010063:Ovum / Q000201:enzymology* / Q000502:physiology; D010446:Peptide Fragments / Q000032:analysis; D010544:Peroxidases / Q000302:isolation & purification* / Q000378:metabolism; D012617:Sea Urchins; D013053:Spectrophotometry
D000596:Amino Acids; D002241:Carbohydrates; D010446:Peptide Fragments; D007784:Lactoperoxidase; D010544:Peroxidases