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The Role of Amoebocytes in Endotoxin‐Mediated Coagulation in the Innate Immunity of Achatina fulica Snails
Author(s) -
Chiranjit Biswas,
Chitra Mandal
Publication year - 1999
Publication title -
scandinavian journal of immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.934
H-Index - 88
eISSN - 1365-3083
pISSN - 0300-9475
DOI - 10.1046/j.1365-3083.1999.00466.x
Subject(s) - achatina , serine protease , proteases , chemistry , sepharose , protease , serine , kallikrein , biochemistry , affinity chromatography , biology , microbiology and biotechnology , snail , enzyme , ecology
Achatina amoebocyte lysate (AAL) derived from amoebocytes of Achatina fulica was activated by Gram‐negative bacterial endotoxins in a time‐dependent manner resulting in gel formation/coagulation. The activation and maximum proliferation of amoebocytes was observed 40 min after intramuscular injection (20 μg/snail) of endotoxin. Endotoxin‐mediated proteolytic activity of AAL towards a serine‐protease‐specific chromogenic substrate was maximum at pH 8.0, 37°C and within 15 min in a divalent‐cation‐dependent manner. The AAL activity induced by the endotoxin was directly dependent on the endotoxin concentration, showed a high specificity and saturated at higher endotoxin concentrations. An endotoxin‐sensitive factor (ESF) was purified from AAL to apparent homogeneity by single‐step affinity chromatography on a heparin‐Sepharose 4B column. Native ESF of molecular weight 140 000 was composed of two identical subunits of molecular weight 70 000 attached through non‐covalent association. A strong binding to endotoxin ( Escherichia coli 055:B5) was exhibited by ESF with a 40‐fold higher biological activity than AAL. The ESF was shown to have a unique Phe–Ile active site with regard to its alternate activation by α‐chymotrypsin instead of endotoxin. The ESF was characterized as a serine protease type as evidenced by potent inhibition with specific inhibitors.