ACTIVE SITE CHARACTERIZATION OF ACROSIN

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Acrosin (E.N. 3.4.21.10) is a unique trypsin-like enzyme in mammalian sperm acrosomes and is used by sperm to penetrate the ovum, a prerequisite to fertilization. This project is designed to continue the characterization of acrosin, including the definition of the topography (structure) of acrosin's active site, so that specific affinity labeling (irreversible) acrosin inhibitors can be synthesized which may be used to inhibit fertilization. Particular emphasis will be placed on determining the mechanism of acrosin's unique catalytic properties in order to define reagents that specifically bind to acrosin and not trypsin (the enzyme most similar to acrosin). Agents that specifically bind to acrosin will be modified to become affinity labeling inhibitors. These inhibitors will be tested for their specificity to acrosin and the functional amino acid residues which participate in the binding will be determined. Since acrosin is a membrane bound proteinase, artifical membranes in the form of phospholipid micelles (liposomes) will be utilized as a model system to study various parameters of membrane bound acrosin which may affect the design of the inhibitors. The biological effectiveness (inhibition of sperm bound acrosin) will be ascertained. Finally, the most significant experiments will be repeated with human acrosin and sperm to permit an accurate evaluation of the possible contraceptive usefulness of these inhibitors.