MCB 0507087 LeAnn Lindsay and Jerry Hedrick Specific objectives for the project are to: (1) complete the cloning and expression of ovochymase cDNA to determine its primary structure for structure-function correlation with other members of the serine active site protease family; this information will define its proenzyme form, active site, substrate specificity and potential mechanisms for regulation of its enzymatic activity; (2) define the cleavage site(s) of ovochymase's vitelline envelope substrate (gp69,64) by determining the glycoprotein's amino acid sequence by peptide sequencing and recombinant DNA methods, and search for homologies with other extracellular matrix glycoproteins, particularly the mammalian zona pellucida glycoproteins; (3) confirm the perivitelline space location of ovochymase and ascertain its extracellular location before and after fertilization using antibody prepared against the expressed protein and immunocytochemical methods; (4) establish if expressed and purified ovochymase will hydrolyze its substrate gp69,64 in situ in the egg envelope and effect a block to polyspermy. The results of this study will provide an increased understanding of the regulation of cell-cell interactions through proteolytic processing of the extracellular matrix. %%% This project will study the influence of cell surface molecules on sperm and egg interactions during fertilization using frog cells. The DNAs for an enzyme which hydrolyzes proteins, a protease called ovochymase, and its extracellular protein substrate, will be cloned. The extracellular location of ovochymase before and after fertilization will be determined. This information will provide a means to understand how cell-cell interactions are regulated by modification of cell surface molecules. ***
