Evaluation of solubilizing methods of the egg envelope of the fish, Oryzias latipes, and partial determination of amino acid sequence of its subunit protein, ZI-3

The inner layer of most teleostean egg envelopes, especially those after hardening, is almost insoluble in ordinary solvents, and therefore the inner layer of only the unhardened egg envelope has been subjected to solubilization with some potent solvents. We comparatively evaluated the methods of solubilization of the inner layer of egg envelope of medaka, Oryzias latipes, with SDS, urea and guanidium chloride (GuHCl). Analysis of the solubilized samples by SDS-polyacrylamide gel electrophoresis, comparison of their amino acid compositions or peptide maps using high-performance liquid chromatography and partial determination of their amino acid sequences showed that SDS and GuHCl were appropriate for solubilization and characterization of the envelope. Urea solubilization resulted in some artificial modifications of lysine and/or cysteine residues of envelope proteins. Partial determination of amino acid sequence of a subunit, ZI-3, isolated from the SDS- or GuHCl-solubilized envelope strongly suggested the identity of the envelope subunit, ZI-3, and its precursor, L-SF.