TWEETEN, K.A.*; JANIGA, A.M.; College of St. Catherine; College of St. Catherine: Analysis of Serine Proteases from Lumbriculus variegatus

The biochemical properties of serine proteases from the California blackworm, Lumbriculus variegatus were studied. Degradation of fibrin by tissue homogenates from the worms and the inhibitory effect of aprotinin on enzymes in these extracts indicated presence of plasmin-like activity. The ability of L. variegatus extracts to generate clots when incubated with rabbit plasma and partial inhibition of extract activity by PMSF and hirudin indicated presence of thrombin-like activity. Partial purification of the plasmin- and thrombin-like proteases was done using heparin, benzamidine, and lysine affinity chromatography and chromogenic substrates to monitor enzyme activity in column fractions. Because most of the serine protease activity in tissue homogenates did not bind to these columns, gel electrophoresis of extracts treated with fluorescently labeled probes which specifically bind to serine proteases was done. SDS polyacrylamide gel electrophoresis and 2-D gel electrophoresis revealed a diversity of serine proteases. This is consistent with protease inhibitor studies which showed partial inhibition of serine protease activity in the tissue extracts by diisopropyl fluorophosphate, hirudin, TLCK, aprotinin, soybean trypsin inhibitor, and PMSF. This suggests that the substrate cleavage we have measured in these experiments is due to a mixture of enzymes rather than to a single enzyme with broad range specificity. In evaluation of the potential biological role of the enzymes, we detected a fibrinogen-like molecule in L. variegatus extracts by cross-reactivity with antibodies to mammalian fibrinogen. Whether or not this protein is cleaved by any of the Lumbriculus variegatus serine proteases is not known.