Purification and characterization of prolyl endopeptidase from the Pacificherring, Clupea pallasi, and its role in the activation of sperm motility

Protease activities with specificity toward synthetic substrates, Suc-Gly-P ro-Leu-Gly-Pro-MCA for prolyl endopeptidase or collagenase-like peptidase, and Suc-Ala-Ala-Pro-Phe-MCA for chymotrypsin were identified in the deterge nt-soluble fraction of herring spermatozoa. The enzyme activities increased in the presence of herring sperm-activating protein (HSAP). Among them a p rolyl endopeptidase [EC. 3. 4. 21. 26] was purified to near homogeneity fro m herring testis. The molecular mass of the enzyme was 79 kDa and the prope rties of the enzyme were quite similar to prolyl endopeptidase from other t issues or cells. Both the enzyme activation and the sperm motility activati on by HSAP were inhibited by benzyloxycarbonyl-L-thioproline-thioprolinal, a specific inhibitor for prolyl endopeptidase. Furthermore, the motility ac tivation by HSAP was inhibited by substrates of the prolyl endopeptidase. W estern blotting with mouse anti-prolyl endopeptidase serum revealed the pre sence of 79 kDa prolyl endopeptidase in the tail fraction of herring sperm; These results suggest that prolyl endopeptidase exists on the surface of t he sperm tail and interacts with the HSAP.