CHARACTERIZATION OF A 14 KDA PLANT-RELATED SERINE-PROTEASE INHIBITOR AND REGULATION OF CYTOTOXIC ACTIVITY IN EARTHWORM CELOMIC FLUID

We have purified and characterized the serine protease inhibitor activ ity contained in the coelomic fluid of the earthworms, Eisenia. Serine protease inhibitor activity was stable between pH 3 and 9.5, not floc culable by pH 3.0 and resistant to 100 degrees C for 15 min. or to 4 d egrees C for 24 h. Ten mu L of coelomic fluid was sufficient to inhibi t in vitro the protease activity of 0.12 mu g of trypsin, Injection of living bacteria into earthworms resulted in increased serine protease activity 1-2 days post-injection, and increased serine protease inhib itor activity on day 4, suggesting that serine protease inhibitor is r esponsible for serine protease neutralization. Purified to homogeneity by affinity chromatography on trypsin, the serine protease inhibitor of Eisenia is a monomer of 14 kDa, Its partial NH2 amino acid sequence revealed a basic hydrophobic fragment which shared 68-75% homologies and 47-60% identities with several plant serine protease inhibitors, E isenia cytotoxic activity due to the two fetidins of 40 and 45 kDa was stimulable in vitro by several serine proteases, Incubation with soyb ean trypsin inhibitor variant a (STIa) resulted in less cytotoxicity, The inhibitory effect occurred only when STIa was added before cell di sruption. Interpretative cytotoxic scheme involving the release of int racellular cytotoxic proteins, intracellular trypsin-like activator an d extracellular serine protease inhibitor suggests regulatory mechanis ms for cellular/humoral immune system of earthworms. (C) 1998 Elsevier Science Ltd. All rights reserved.