Protein content analysis and antimicrobial activity of the crude venom of Montivipera bornmuelleri; a viper from Lebanon.

Viperidae snakes venoms represent a source of efficient bioactive components that have already led to the development of several new drugs. In this work, we analyzed the protein content of the Montivipera bornmuelleri crude venom using LC-ESI-MS, sephadex G-75 gel filtration and SDS-PAGE and demonstrated the presence of proteins with molecular masses corresponding to metalloprotease III, serine-protease and PLA2 in three fractions collected after gel filtration. Equally, we examined the antimicrobial effect of the venom that showed an important potency, as bactericidal agent, based on MBC and MIC values obtained, against Staphylococcus aureus and Morganella morganii bacteria. However, no activity was registered against Enterococcus faecalis, being the most resistant bacteria, neither against Aspergillus flavus and Penicillium digitatum fungal. Furthermore, on eleven other bacterial strains and the Candida albicans fungus, the venom has shown an intermediate efficacy by slightly reducing the growth. Our data concerning the Montivipera bornmuelleri venom give evidence of a rich and complex content aiding the exploration of new bioactive molecules for biopharmaceuticals purposes.

A multidisciplinary approach to probing enthalpy-entropy compensation and the interfacial mobility model.

In recent years, interfacial mobility has gained popularity as a model with which to rationalize both affinity in ligand binding and the often observed phenomenon of enthalpy-entropy compensation. While protein contraction and reduced mobility, as demonstrated by computational and NMR techniques respectively, have been correlated to entropies of binding for a variety of systems, to our knowledge, Raman difference spectroscopy has never been included in these analyses. Here, nonresonance Raman difference spectroscopy, isothermal titration calorimetry, and X-ray crystallography were utilized to correlate protein contraction, as demonstrated by an increase in protein interior packing and decreased residual protein movement, with trends of enthalpy-entropy compensation. These results are in accord with the interfacial mobility model and lend additional credence to this view of protein activity.

A single step purification for autolytic zinc proteinases.

We describe a novel single-step method for the purification of stromelysin-1 catalytic domain (SCD) via immobilized metal affinity chromatography under denaturing conditions that inhibit proteolytic activity followed by on-column refolding and spontaneous autolysis of the fusion peptide to yield pure, active stromelysin-1 catalytic domain. The methodology provides a general approach for the rapid purification of large quantities of zinc proteinases.

Multiple changes in gene expression in chronic human Achilles tendinopathy.

Atlas cDNA cell interaction arrays (CLONTECH) were used to examine degenerate tissue from four patients with Achilles tendon disorders, in order to identify changes in expression of genes important in cell-cell and cell-matrix interactions. The greatest difference between normal (post-mortem) and degenerate tissue samples was in the level of MMP-3 (stromelysin) mRNA, which was down-regulated in all the degenerate samples. Quantitative RT-PCR assay of RNA extracted from paired 'normal' and degenerate Achilles tendon tissue samples taken from tendons during surgery mirrored the results of the arrays. Levels of MMP-3 mRNA were lower, whereas levels of type-I and type-III collagen mRNAs were significantly higher, in the degenerate compared to the 'normal' samples. Immunoblotting of proteins extracted from the same tendon samples showed that three of four normal tissue samples taken from individuals without apparent tendon disorder had much higher levels of MMP-3 protein than 'normal' or degenerate samples from patients with tendinosis. We suggest that MMP-3 may play an important role in the regulation of tendon extracellular matrix degradation and tissue remodelling.

Differential regulation of MMP-13 (collagenase-3) and MMP-3 (stromelysin-1) in mouse calvariae.

Bone resorption in mice involves the degradation of extracellular matrix. Whereas several proteases seem to be implicated in this process, it becomes increasingly clear that matrix metalloproteinases (MMPs), amongst them especially MMP-13 and MMP-3, play an essential role. We have purified MMP-13 and MMP-3 from mouse calvariae-conditioned media by differential fractionation and analyzed their collagenolytic, caseinolytic, gelatinolytic and proteoglycanolytic activities. It could be shown that in mouse calvariae-conditioned media most of the measured enzyme activities were due to MMP-13, although zymographies revealed that MMP-3, MMP-2, MMP-9 as well as TIMPs were present too. MMP-13 and MMP-3 proteins were detected and their enzyme activities were neutralized by specific polyclonal antisera. Furthermore, it was demonstrated that in cultures of mouse calvariae the production of MMP-13 was induced by the potent MMP-stimulator heparin and by parathyroid hormone (PTH), whereas the levels of MMP-3 remained unchanged. Although PTH-induced bone resorption was inhibited by calcitonin treatment, MMP-13 mRNA and protein expression were not significantly altered by this hormone. Together with previous observations, these results indicate that PTH regulates bone resorption through MMP-13, but not by MMP-3, and that its reversion by calcitonin involves neither of the two enzymes.

Insulin-like growth factor binding protein (IGFBP) substrate zymography. A new tool to identify and characterize IGFBP-degrading proteinases.

Insulin-like growth factor binding protein (IGFBP) degrading proteinase activities have been described in biological fluids and conditioned media from numerous cell lines. To identify and characterize IGFBP-degrading proteinases, our laboratory has developed IGFBP substrate zymography. Herein, we illustrate how IGFBP substrate zymography can be used both to identify candidate IGFBP-degrading proteinases and characterize their degradative capabilities. For this purpose, human matrix metalloproteinase-3 (MMP-3), a proteinase that degrades IGFBP-3 in human fibroblast cultures, was first electrophoresed through a polyacrylamide gel containing IGFBP-3 as substrate and then analyzed for its ability to degrade the substrate into immunoreactive fragments that were absorbed onto a polyvinylidene difluoride membrane. IGFBP-3 substrate zymography was capable of detecting as little as 20 ng of human MMP-3, demonstrating a sensitivity similar to casein substrate zymography. Using the zymogram as a template, MMP-3 was identified in a standard SDS-polyacrylamide gel run in parallel with the zymogram, and the corresponding area of the gel was excised. Electroelution of the gel slice yielded active MMP-3 when examined by casein substrate zymography. Furthermore, digestion of IGFBP-3 in solution by the electroeluted MMP-3 revealed the same fragmentation pattern of the binding protein as that produced by MMP-3, which had not been electroeluted. Together, these studies demonstrate that IGFBP substrate zymography can be a useful tool for both the identification and the characterization of IGFBP-degrading proteinases.

Degradation of T-kininogen by cathepsin D and matrix metalloproteinases.

Cathepsin D, matrix metalloproteinase (MMP)-2, MMP-3 (stromelysin), and MMP-9 were isolated from rat granulomatous tissues. HT1080 human fibrosarcoma cells and rheumatoid synovial cell CM. At acidic conditions, cathepsin D cleaved T-kininogen into small peptides and released Met-T-kinin-Leu (kinin precursor), but failed to release kinin. MMP-3 cleaved T-kininogen into a 57 kDa fragment as measured by SDS-PAGE and Western blot analysis using anti-T-kininogen antiserum. On the other hand, no degradation of T-kininogen occurred during incubation with MMP-2 or MMP-9100/1) at pH 7.5 for 7 h.