CHARACTERIZATION OF THE ACROSIN ACTIVE SITE

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Acrosin (E.C. 3.4.21.10) is a sperm protease and the acrosin precursor (proacrosin) is found in greater concentrations than all other components of the sperm proteolytic enzyme system. The proacrosinacrosin system is of major importance since sperm must have enzymatically active acrosin in order to be able to fertilize ova. The focus of this grant proposal is to affinity label, purify, sequence, and synthesize the active site region of acrosin. The first specific aim will utilize boar sperm to concentrate on the partial purification, and auto-activation of proacrosin to acrosin in the presence of an active site affinity labeling reagent. The resultant labeled acrosin will be further purified, and subjected to enzymatic hydrolysis. The peptides will be separated, the labeled regions identified, and their amino-acid sequence determined. The goal of the second specific aim will be to characterize the acrosin active site region in the human. The third specific aim focuses on applications of the active site region sequences. These sequences will be compared to each other and to other trypsin- like enzymes. The active site regions of both species will be synthesized. A rabbit system will be used to develop highly purified polyclonal antibodies to the synthesized peptides. Experiments will be performed to determine if the antibodies are able to inhibit proteolytic activity, and to see if they will inhibit fertilization. The latter will include both the zona-free hamster egg test as well as a porcine in vitro fertilization system. This would clarify the potential role of the antibodies as a male contraceptive agent. The antibodies will also be used to develop an enzyme-liked immunosorbent assay for acrosin, and a fluorescent labeled anti-rabbit IgG will be used to localize the rabbit IgG-acrosin complex to follow changes in the location of acrosin in the sperm during capacitation and the acrosome reaction. The unique strengths of this project are: 1) It allows a strongly motivated clinician the opportunity to pursue a serious academic career, 2) It will apply new technology which will enable one to identify the active site sequence of the acrosin molecule, 3) It will provide a basis on which to investigate potential uses of the active site sequence including a role as a male contraceptive agent.