Figure 7. Identification of acrosin- and N-acetylglucosaminidase (NAGA)-binding polypeptide(s) of outer acrosomal membrane–associated matrix complex (OMC). To identify acrosin and NAGA binding protein(s) of the OMC complex, a centrifugation assay was utilized. Purified OMC32 and OMC45 polypeptides were conjugated separately to AminoLink Plus coupling gel (Pierce) at pH 10.0 according to the manufacturer's instructions. Bovine epididymal cauda sperm proteins were extracted in high-salt Triton X-100 solution. The supernatant obtained after centrifugation that contain hydrolases activities was dialyzed against 25 mM Tris-HCl buffer, pH 7.5, incubated with OMC32- and OMC45-conjugated beads, and after centrifugation both the pellet (dark bars) and the supernatant (light bars) fractions were assayed for acrosin and NAGA. Both acrosin (A-I) and NAGA (B-I) specifically bound the OMC32 polypeptide in a concentration-dependent fashion, as evidenced by the increased fraction of sedimentable acrosin and NAGA activities. In contrast, the OMC45 polypeptide depleted acrosin activity (A-II) in a concentration-dependent manner, whereas OMC45 polypeptide binds NAGA but with weaker affinity than for acrosin (B-II), indicating a strong binding interaction between acrosin and OMC45 polypeptide. Parallel control incubation that substituted equal amounts of high-pH extracted sperm tail proteins coupled to AminoLink Plus coupling beads at pH 10.0 exhibited no significant binding of either hydrolase (A-III, B-III). These experiments demonstrate that the OMC32 polypeptide possesses specific binding sites for both acrosin and NAGA, whereas OMC45 polypeptide exhibits stronger affinity to acrosin than to NAGA. The data are representative of 3 separate experiments.