Enzymic characterization of rabbit blastocyst proteinase with synthetic substrates of trypsin-like enzymes.

Since previous investigations have provided evidence that certain proteinases of the trophoblast playa key role in initiation of implantation of the embryo in the uterus, enzymic characterization of endopeptidase activity of rabbit blastocyst extracts was attempted. Recently developed tripeptide p-nitroanilide substrates now allow far the first time quantitative assays of enzyme activities in this material. Substrates containing one amino acid only are found not to be hydrolyzed at any measurable rates. Relative hydrolysis rates of various tripeptide p-nitroanilide substrates indicate a preference of blastocyst proteinase(s) for hydrophobie or thrombin-preferred amino acid residues in positi on P2 andjor P3. The pH optimum is found close to 8.5. Titration experiments with various proteinase inhibitors show a particularly strong inhibition by aprotinin (Trasylol). As judged from both substrate specificity and inhibition experiments, the active site of the trophoblast enzyme(s) is closely related to that of trypsin but substrate specificity is more restricted. The enzymic pro-' perties found are consistent with a close similarity of the blastocyst proteinase(s) to kallikreins andjor sperm acrosin. The possibility is thus indicated that enzymes of both types are present in rabbit implantation stage blastocysts. Problems of identification with the previously described gelatinolytic proteinase are discussed.