Abstract
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.
