title: A comparison of the enzymatic properties of three recombinant isoforms of thrombolytic and antibacterial protein--Destabilase-Lysozyme from medicinal leech.
abstract: BACKGROUND
  Destabilase-Lysozyme (mlDL) is a multifunctional i-type enzyme that has been found in the secretions from the salivary glands of medicinal leeches. mlDL has been shown to exhibit isopeptidase, muramidase and antibacterial activity. This enzyme attracts interest because it expresses thrombolytic activity through isopeptidolysis of the ε-(γ-Glu)-Lys bonds that cross-link polypeptide chains in stabilised fibrin. To date, three isoforms of mlDL have been identified. The enzymatic properties of pure mlDL isoforms have not yet been described because only destabilase complexes containing other proteins could be isolated from the salivary gland secretion and because low product yield from the generation of recombinant proteins has made comprehensive testing difficult.
  
  
  RESULTS
  In the present study, we optimised the procedures related to the expression, isolation and purification of active mlDL isoforms (mlDL-Ds1, mlDL-Ds2, mlDL-Ds3) using an Escherichia coli expression system, and we detected and compared their muramidase, lytic, isopeptidase and antimicrobial activities. After optimisation, the product yield was 30 mg per litre of culture. The data obtained in our study led to the suggestion that the recombinant mlDL isoforms isolated from inclusion bodies form stable oligomeric complexes. Analyses of the tested activities revealed that all isoforms exhibited almost identical patterns of pH and ionic strength effects on the activities. We determined that mlDL-Ds1, 2, 3 possessed non-enzymatic antibacterial activity independent of their muramidase activity. For the first time, we demonstrated the fibrinolytic activity of the recombinant mlDL and showed that only intact proteins possessed this activity, suggesting their enzymatic nature.
  
  
  CONCLUSIONS
  The recombinant Destabilase-Lysozyme isoforms obtained in our study may be considered potential thrombolytic agents that act through a mechanism different from that of common thrombolytics.
authors.affiliation: Federal Research and Clinical Center of Physical-Chemical Medicine, Malaya Pirogovskaya, 1a, Moscow, 119435, Russia. aleks-kuzmaland@bk.ru.
authors.forename: Alexey S
authors.initials: AS
authors.lastname: Kurdyumov
authors.affiliation: Federal Research and Clinical Center of Physical-Chemical Medicine, Malaya Pirogovskaya, 1a, Moscow, 119435, Russia.
authors.forename: Valentin A
authors.initials: VA
authors.lastname: Manuvera
authors.affiliation: Biological Faculty, M. V. Lomonosov Moscow State University, Moscow, 119991, Russia.
authors.forename: Isolda P
authors.initials: IP
authors.lastname: Baskova
authors.affiliation: Federal Research and Clinical Center of Physical-Chemical Medicine, Malaya Pirogovskaya, 1a, Moscow, 119435, Russia.
authors.forename: Vassili N
authors.initials: VN
authors.lastname: Lazarev
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D001412:Bacillus subtilis / Q000187:drug effects / Q000254:growth & development; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004355:Drug Stability; D010450:Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000187:drug effects / Q000254:growth & development; D005343:Fibrinolytic Agents / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D047709:Hirudo medicinalis / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D009113:Muramidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D005343:Fibrinolytic Agents; D007527:Isoenzymes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D009113:Muramidase; D010450:Endopeptidases; C045348:fibrin destabilase