Biochemical characterization of sialoprotein anti-agglutinin purified from boar epididymal and seminal plasma

摘要: Sialoprotein “anti-agglutinin,” previously shown to inhibit sperm head-to-head agglutination, is found in both boar epididymal and seminal plasma. The present report characterizes anti-agglutinin by mass spectrometry, N-terminal amino acid sequence analysis, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) Western blotting techniques assess phosphate content of the molecule. Anti-agglutinin had SDS-PAGE mobility approximately 25 kDa. By electrospray ionization-mass however, spectra were characterized two major peaks (19,379–19,382 Da 19,395–19,397 Da) several minor peaks. Mass spectrometry tryptic peptide fragments deglycosylated analysis revealed that protein has a unique peptide-mass fingerprinting (12,668 Da, 5,209 1,226 1,168 novel (KTDDY AISGA KEEEF YDYME ELYAV), respectively. Additionally blot techniques, using commercially available monoclonal antibodies, used detect presence phosphothreonine phosphoserine substituents, but different antibodies did not phosphotyrosine. Moreover, treatment with alkaline phosphotases converted molecule, as assessed detection silver stain, from parent form about kDa forms 19 (similar assigned spectrometry) and/or 15 Original antiserum generated toward, reacting native anti-agglutinin, reacted only form. These results are consistent conclusion may be phosphorylated at serine threonine residues. Mol. Reprod. Dev. 55:96–103, 2000. © 2000 Wiley-Liss, Inc.