Substrate-zymography: a still worthwhile method for gelatinases analysis in biological samples

Abstract

Matrix metallo-proteinases (MMPs) are a family of zinc-dependent endopeptidases, capable of degrading all the molecular components of extracellular matrix. A class of MMPs is gelatinases which includes gelatinase A or MMP-2 (72 kDa) and gelatinase B or MMP-9 (92 kDa), which have been shown to play critical roles in pathophysiology of many human disease and, in particular, cancer progression. For these reasons they obtained a great interest as potential non-invasive biomarker in providing useful clinical information in cancer diagnosis and therapy. A sensitive and unexpensive method for analysis of gelatinases is the gelatine zymography, which allows to measure the relative amounts of active and inactive enzymes in body fluids and tissue extracts. The procedure involves the electrophoretic separation of proteins under denaturing but non reducing conditions through a polyacrylamide gel containing a synthetic substrate (gelatin). The aim of this mini-review has been to describe the general principles of gelatine zymography technique, underling the main advantages and disadvantages. Even though an improvement of this method is necessary for a better applicability in laboratory medicine, gelatine zymography represents the most convenient method to detect the activity of the different gelatinases from a wide range of biological samples.

MicroRNA-194 modulates epithelial–mesenchymal transition in human colorectal cancer metastasis

“…Our study confirms that miR-194 inhibited epithelial marker E-cadherin and increased mesenchymal markers vimentin and MMP-2. Moreover, zymography assay was performed in our study, which allows one to measure the relative amounts of active and inactive enzymes,33 and results showed that miR-194 significantly increased the gelatin-degrading activity of MMP-2. These data suggest that miR-194 is an EMT promoter in CRC cells.…”

Reviewing Mechanistic Peptidomics in Body Fluids Focusing on Proteases

“…MMPs have overlapping substrate specificities. Even so, they are divided into five groups according to their preferential degradation of different matrix substrates: matrilysin (MMP7 and MMP26), collagenases (MMP1, MMP8, MMP13, MMP18), gelatinases (MMP2 and MMP9), stromelysin (MMP3, MMP10, MMP11), and membrane MMPs (MT‐MMPs, MMP14, MMP15, MMP16, MMP17, MMP23A, MMP23B, MMP24, MMP25) . These proteases not only target ECM proteins but they are also involved in the regulation of inflammatory signaling pathways .…”

Evaluation of matrix metalloproteinase type IV-collagenases in serum of patients with tumors of the central nervous system

“…Gelatinolytic activity was performed as previously described [ 16 ]. Briefly, total protein (25 µg) of each sample was mixed with sample buffer (10 mM Tris–HCl pH 6.8, 12.5 % SDS, 5 % sucrose, 0.1 % bromophenol blue) and applied directly without prior heating or reduction to 7.5 % (w/v) acrylamide gels containing 0.3 % (w/v) of gelatin.…”

“…Furthermore, the character of proteolytic bands was analyzed by incubating the identical zymograms in 0.1 mg/ml of PMSF, a serine protease inhibitor; or 2 mM Pefabloc, an irreversible serine protease inhibitor. Following zymography, the degree of gelatin digestion was quantified as previously described [ 16 ]. Briefly, we used an image analysis software (ImageQuant TL, Amersham Bioscience, Chicago, IL, USA) according to the manufacturer’s specifications.…”