Characterization of a 14 kDa plant-related serine protease inhibitor and regulation of cytotoxic activity in earthworm coelomic fluid.
We have purified and characterized the serine protease inhibitor activity contained in the coelomic fluid of the earthworms, Eisenia. Serine protease inhibitor activity was stable between pH3 and 9.5, not flocculable by pH 3.0 and resistant to 100 degrees C for 15 min. or to 4 degrees C for 24 h. Ten microL of coelomic fluid was sufficient to inhibit in vitro the protease activity of 0.12 microgram of trypsin. Injection of living bacteria into earthworms resulted in increased serine protease activity 1-2 days post-injection, and increased serine protease inhibitor activity on day 4, suggesting that serine protease inhibitor is responsible 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. Eisenia cytotoxic activity due to the two fetidins of 40 and 45 kDa was stimulable in vitro by several serine proteases. Incubation with soybean trypsin inhibitor variant a (STIa) resulted in less cytotoxicity. The inhibitory effect occurred only when STIa was added before cell disruption. Interpretative cytotoxic scheme involving the release of intracellular cytotoxic proteins, intracellular trypsin-like activator and extracellular serine protease inhibitor suggests regulatory mechanisms for cellular/humoral immune system of earthworms.
Roch
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Défense et Resistance chez les Invertébrés Marins (DRIM), IFREMER-CNRS-Université de Montpellier, France. proch@ifremer.fr
Ville
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Cooper
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D016980:Aeromonas hydrophila; D000595:Amino Acid Sequence; D000818:Animals; D001826:Body Fluids / Q000276:immunology; D016905:Gram-Negative Bacterial Infections / Q000276:immunology; D019684:Magnoliopsida; D008969:Molecular Sequence Data; D009835:Oligochaeta / Q000276:immunology*; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D015842:Serine Proteinase Inhibitors / Q000302:isolation & purification*
D015842:Serine Proteinase Inhibitors