STUDIES ON MOUSE SPERM BINDING MOLECULE (LIGAND) TO EGG ZONA-PELLUCIDA .2. PURIFICATION OF LIGAND MOLECULE FROM CAUDA EPIDIDYMAL SPERM

One of the ligand molecules was purified from mouse cauda epididymal sperm for an indication of its inhibitory activity in sperm-egg binding. It was isolated in an electrophoretically homogeneous form by ionophore A23187 treatment of sperm, sedimentation by ultracentrifugation, solubilization by 2% Nonidet P-40 (NP-40), repeated ion exchange chromatography on DEAE-Sepharose CL-6B and gel filtration on Sephacryl S-200. The purified ligand exhibited a molecular mass of 60000 dalton by gel filtration and 67000 dalton by sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE). This molecule was radioiodinated by the lactoperoxidase method and assayed the binding ability to egg zona pellucida. The labeled ligand specifically bound to both cumulus-free egg zona pellucida and isolated-zona pellucida of unfertilized egg in a similar extent, but didn't bind to 2-cell egg zona pellucida. The maximal binding site of ligand per egg zona pellucida was estimated about 660000 molecules by Scatchard plot analysis.