Sea urchin ovoperoxidase: solubilization and isolation from the fertilization envelope, some structural and functional properties, and degradation by hatching enzyme.
The soft fertilization envelope (SFE) devoid of tyrosine-derived cross-links was isolated from the sea urchin Hemicentrotus pulcherrimus embryos fertilized in the presence of 2 mM aminotriazole, a reversible inhibitor of ovoperoxidase (OPO). Most of the component proteins including OPO were solubilized from SFE by the use of a buffer containing 10 mM EDTA. The solubilized OPO was purified by chromatographies on Sepharose CL-6B, DEAE-Sephacel, and Superose 12HR columns to apparent electrophoretical homogeneity with Mr 70,000. The enzyme accounts for at least 3% of the total FE by weight and 10% by gel densitometry. Partial amino acid sequences of three proteolytic fragments of OPO, accidentally including the distal and proximal His residues, revealed up to 84% homology to mammalian thyroid peroxidases, myeloperoxidases, and lactoperoxidases (LPO), but the N-terminal sequence showed little homology to them. The uv-VIS absorption spectrum of OPO has a maximum at 279 nm, Soret band at 415 nm, and a small quartet at 490, 542, 580, and 642 nm. The tyrosine cross-link profile in the reaction products of the OPO/tyrosine/H2O2 system as analyzed by HPLC with uv and fluorescence detection was similar to those of LPO and HRPO, with dityrosine as the only major component. However, it is different from that of the HCl hydrolysate of normally hardened FE that has much higher contents of trivalent cross-links trityrosine and pulcherosine. This result reflects the difference in the accessibility of the substrates to the enzyme: free tyrosine and OPO in solution vs Tyr residues and OPO both immobilized in FE. The enzyme OPO was degraded limitedly by purified hatching enzyme (envelysin) after 24 h incubation to mainly a 47-kDa fragment with its 67% peroxidase activity retained.
Nomura
K
K
Department of Biochemistry, Tokyo Metropolitan Institute of Gerontology, Japan.
Suzuki
N
N
D000595:Amino Acid Sequence; D000818:Animals; D004625:Embryo, Nonmammalian / Q000201:enzymology; D004795:Enzyme Stability; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012617:Sea Urchins / Q000201:enzymology*; D016415:Sequence Alignment
D010544:Peroxidases; D008666:Metalloendopeptidases; C024344:envelysin