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Separation of Monomerizing and Lysozyme Activities of Destabilase from Medicinal Leech Salivary Gland Secretion

I. P. Baskova1*, L. L. Zavalova2, A. V. Basanova1, and A. V. Sass2

1School of Biology, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (095) 939-1745

2Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 119997 Russia; fax: (095) 330-6538; E-mail: leech@humgen.siobc.ras.ru

* To whom correspondence should be addressed.

Received February 19, 2001; Revision received April 11, 2001
Destabilase, endo-epsilon-(gamma-Glu)-Lys-isopeptidase, was prepared from the salivary gland secretion of the medicinal leech (Hirudo medicinalis). The secretion prepared by the known method of Rigbi et al. (1987) (secretion-K) lacks the destabilase-characteristic highly specific isopeptidase activity (the D-dimer-monomerizing activity) because of its degradation by proteolytic activity (the substrate of Glp-Ala-Ala-Leu-pNA) due to contamination with leech intestinal channel contents. Therefore, we have elaborated a new technique for preparation of a true leech secretion (secretion-I). This secretion is characterized by the complete absence of the leech intestinal channel contents and has no proteolytic activity. For the first time the destabilase-specific D-dimer-monomerizing and lysozyme activities were separated by fractionation of secretion-I by HPLC gel filtration through Superose S-12. For the purified destabilase preparation, these activities were separated by reversed-phase chromatography in an acetonitrile gradient (0-60%) in the presence of 0.1% trifluoroacetic acid. The monomerizing activity of destabilase is responsible for the ability of secretion-I to dissolve stabilized fibrin via isopeptidolysis of alpha-alpha and gamma-gamma fibrin chains bound by epsilon-(gamma-Glu)-Lys-isopeptide bonds.
KEY WORDS: medicinal leech, salivary gland secretion, destabilase, isopeptidase, lysozyme, stabilized fibrin