Enzymic Characterization of Rabbit Blastocyst Proteinase with Synthetic Substrates of Trypsin-Like Enzymes

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Abstract
Certain proteinases of the trophoblast play a key role in initiation of implantation of the embryo in the uterus. Enzymic characterization of endopeptidase activity of rabbit blastocyst extracts was attempted. Tripeptide p-nitroanilide substrates allow for the first time quantitative assays of enzyme activities in this material. Substrates containing only 1 amino acid are not hydrolyzed at any measurable rates. Relative hydrolysis rates of various tripeptide p-nitroanilide substrates indicate a preference of blastocyst proteinase(s) for hydrophobic or thrombin-preferred amino acid residues in position P2 and/or P3. The pH optimum is close to 8.5. Titration experiments with various proteinase inhibitors show a particularly strong inhibition by aprotinin (Trasylol). Substrate specificity and inhibition experiments indicate that the active site of the trophoblast enzyme(s) is closely related to that of trypsin [EC 3.4.21.1] but substrate specificity is more restricted. The enzymic properties found are consistent with a close similarity of the blastocyst proteinase(s) to kallikreins [EC 3.4.21.8] and/or sperm acrosin [EC 3.4.21.10]. Enzymes of both types may be present in rabbit implantation stage blastocysts. Problems of identification with the previously described gelatinolytic proteinase are discussed.

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