Activity.

We revealed that isopeptidase activity was very sensitive to the
Activity. We revealed that isopeptidase activity was very sensitive to the ionic strength. It has not been detected at NaCl concentrations above 0.3 M. For isopeptidase activity we measured Km and kkat. It was found that for all isoforms Km = 20?0 M and kkat = 5? ?10-2 s-1. So high Km and low kkat can be explained also by low specificity of substrate. Although these values are comparable to those obtained previously for mlDL-Ds2 (Km = 50 M, kkat =0.1 ?10-2 s-1) [13] and TJL (Km = 20 M, kkat = 0.2 ?10-4 s-1) [32]. For all fermentative activities we found the halfmaximal pH and I values (Table 1). These values were similar for all mlDL isoforms: for lytic activity–pH 4.5?0 and I = 0?.4 M, for isopeptidase activity–pH 4.5?.5 and I = 0?.1 M. For muramidase activity mlDL had two ranges: pH 2.5? and I = 0?.25 M, pH 4.5?2 and I = 0?0.05 M. This information allows us to suggest the similarity of their enzymatic properties. Previous studies have demonstrated the antimicrobial activity of heat-inactivated mlDL [19]. This antimicrobial effect was also observed with synthetic amphipathic fragments of mlDL [18]. We used the mlDL isoforms and their tryptic peptides to investigate antimicrobial activity. We revealed the inhibition of bacterial growth (E. coli and B. subtilis) in the presence of the mlDL isoforms and their tryptic peptides. The tryptic peptides exhibited more efficient inhibition of bacterial growth than the entire enzymes. This was confirmed by MIC determination. The MICs of tryptic peptides were lower than those of the entire PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/20460822 mlDL isoforms. The antimicrobial activity detected in the tryptic peptides of the mlDL isoforms confirmed that this activity is nonenzymatic. The antimicrobial activity that was independent of muramidase activity has been shown for other invertebrate lysozymes, but the mechanism of (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-4,4-dimethylpentanoic acid this action remains unclear. For the first time, we have revealed that recombinant mlDL isoforms exhibit fibrinolytic activity. We determined that only the intact mlDL isoforms possess fibrinolytic activity. Neither tryptic peptides nor heat-inactivated protein exhibited fibrinolytic activity, suggesting that this activity is enzymatic and requires structural organisation of the active centre.Kurdyumov et al. BMC Biochemistry (2015) 16:Page 10 ofConclusions We isolated all of the currently known mlDL isoforms from medicinal leeches and demonstrated the enzymatic activities of all of the recombinant isoforms. All of the mlDL isoforms exhibited almost identical patterns of pH and ionic strength effects on the (S)-tert-Butyl 6-(hydroxymethyl)-5-azaspiro[2.4]heptane-5-carboxylate activities. We also found that the antimicrobial activity of the mlDL isoforms is muramidase activity-independent and non-enzymatic. We found that the tryptic peptides of the mlDL PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/24083752 isoforms exhibited more efficient inhibition of bacterial growth and had lower MICs than the whole enzymes did. We also showed that only the whole mlDL isoforms possess fibrinolytic activity.Availability of data and materials(in units) was calculated relative to the reference enzyme HEWL according to formula (1). (n = 5). (PDF 106 kb) Additional file 9: Figure S8. Lytic activity of mlDL isoforms. Effects of pH (a) and ionic strength (b) on the lytic activity of mlDL-Ds1. Effects of pH (c) and ionic strength (d) on the lytic activity of mlDL-Ds2. The activity was expressed as the concentration of protein released from the cells of B.subtilis. Effects of pH (e) and ionic strength (f) on the lytic activity of mlDL-Ds1 in the presence of 5 mM EDTA. The acti.