Binding of2D6 Antigen to the Sperm Plasma Membrane
Secreted epididymal glycoproteins are known to bind to spermatozoa with varying tenacities. To elucidate the strength and nature of the binding of the 24-kDa form of 2D6 antigen to the sperm plasma membrane, cauda epidi-dymidal spermatozoa were washed in PBS followed by resuspension in different dissociating agents, at extremes of pH or in detergents. Plasma membrane proteins were then solubilized with 1% NP-40, and the amount of 2D6 antigen remaining was assessed by SDS-PAGE/Western blotting. asthma inhaler side effects
As an internal control, parallel blots were probed with a polyclonal antibody to PBP antigen (also known as PEPB or raf kinase inhibitory protein ). PBP is easily released from spermatozoa under mild dissociating conditions and can be quantitatively recovered in the supernatant. As shown in Figure 5, incubation of cauda spermatozoa in 1 M KCl or 4 M urea or 0.4% (3-mercaptoethanol or PBS (pH 3.0) or PBS (pH 10.0) or 0.1 M EDTA did not solubilize detectable amounts of 2D6 antigen, whereas >95% of PBP was removed with 1 M KCl, 4 M urea, PBS (pH 10.0), and 0.1 M EDTA. These observations suggest that 2D6 antigen is bound strongly to the sperm plasma membrane, possibly by hydrophobic interactions.
FIG. 5. The 24-kDa antigen recognized by 2D6 McAb on cauda spermatozoa behaves like an integral membrane protein. Washed cauda spermatozoa were incubated with various dissociating reagents as shown, followed by extraction of the remaining proteins with 1% SDS. Western blots of reduced proteins in the 1% SDS extracts were then probed with 2D6 McAb (upper panel) or a polyclonal antibody (PAb) to PBP antigen (lower panel).