Secreted Epididymal Glycoprotein: RESULTS(6)


Two methods widely used for assessing protein-lipid interactions are partitioning of the antigen into Triton X-114 and its solubilization by PI-specific PLC (PI-PLC). The latter procedure indicates the presence of a GPI anchor, although not all GPI-anchored proteins on cell surfaces are susceptible to PI-PLC cleavage owing to problems of steric hindrance. Following solubilization and partitioning of cauda sperm plasma membrane proteins with Triton X-114, the distribution of 2D6 antigen between the aqueous and detergent phases was analyzed by SDS-PAGE/Western blotting. 2B1 glycoprotein was used as an internal control, as it has been shown to contain a GPI anchor and to partition strongly into the detergent phase.

As shown in Figure 6, the 24-kDa subunit form of 2D6 antigen is present in approximately equal proportions in the aqueous and detergent layers, whereas >90% of 2B1 glycoprotein is found in the Triton X114 layer.

Digestion of washed cauda spermatozoa with PI-PLC failed to release detectable quantities of 2D6 into the medium (Fig. 7). In these experiments we used CD52 antigen as the internal control (although 2B1 glycoprotein has a GPI anchor, it is resistant to cleavage by PI-PLC, whereas CD52 antigen is released in significant amounts by the enzyme). ventolin hfa inhaler price

Fig6Secreted Epididymal Glycoprotein1-6
FIG. 6. The 2D6 antigen from cauda spermatozoa partitions equally between Triton X-114 and aqueous phases. In contrast, 2B1 antigen (the orthologue of PH20 antigen, a GPI-anchored glycoprotein) partitions completely into the Triton X-114 phase.

Fig7Secreted Epididymal Glycoprotein1-7
FIG. 7. The 24-kDa 2D6 antigen on cauda spermatozoa is unaffected by incubation with PI-PLC. In contrast, CD52 glycoprotein (which is anchored to the plasma membrane with a GPI anchor) is partially solubilized and appears in the supernatant fraction. Lane 1: detergent extract of sperm incubated with PI-PLC; lane 2: detergent extract of control sperm; lane 3: supernatant fraction from PI-PLC incubated sperm; lane 4: supernatant from control sperm.