An Assay for Matrix Metalloproteinases and Other Proteases Acting on Proteoglycans, Casein, or Gelatin

doi:10.1006/abio.1993.1572

Analytical Biochemistry

Volume 215, Issue 2, December 1993, Pages 171-179

https://doi.org/10.1006/abio.1993.1572 Get rights and content

Abstract

We have set up a quantitative and sensitive enzymatic assay for proteases of different classes acting on proteoglycans, casein, or gelatin. Radiolabeled substrates were covalently attached to insoluble microcarriers and assays were performed in 96-well plates. Protease activities were determined by the release of labeled degradation products. Time- and dose-response curves were linear when the solubilization of labeled substrates did not exceed 15-20% of the initially bound molecules. Results were compared to those from zymographic analyses on proteoglycan-, gelatin-, and casein-polyacrylamide gels, as well as to the results obtained with conventional assays using soluble [³H]-casein and [³H]gelatin. Our assay procedure was more sensitive than other available methods: it detected picogram amounts of trypsin as well as picogram or nanogram amounts of the purified human matrix metalloproteinases, MMP-1, MMP-2, MMP-3, and MMP-9, depending on the specific activities of these MMPs on the different substrates. Our new procedure was appropriate for assaying the MMPs present in crude culture media conditioned by chondrocytes cultivated under various conditions.

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Cited by (39)

Continuous protease assays using liquid crystal as a reporter
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To address this issue, many researchers used labeled caseins as substrates to lower LODs. For example, Manicourt and Lefebvre used [3H] and [125I]-labeled casein to detect matrix metalloproteinases (MMPs) and plasmin with LODs of 2.5 ng/mL and 5 μg/mL, respectively [23,24]. However, labeling of casein with radioisotopes is tedious, time-consuming, expensive and requires special handling of radioactive materials.
Abnormal protease activities are associated with cancers, vascular diseases and Alzheimer diseases. Therefore, detection of protease activities has become increasingly important in recent years. Herein, we report a semi-quantitative, liquid crystal (LC)-based protease assay for naked-eye detection of protease activity. In this assay, casein molecules are cleaved by proteases into small peptide fragments, which can be quantified either by using Lowry’s method or using LC. In the latter, peptide fragments adsorb on a solid surface and disrupt LC to produce a bright spot for naked-eye detection. The bright spot is observed only when the surface-adsorbed peptide density exceeds a critical value. In the assay, a major challenge is how to separate undigested casein and remaining protease from peptide fragments to prevent their interferences with LC. To overcome this issue, trichloroacetic acid (TCA) is added to precipitate casein and proteases. By using the assay, we are able to detect 10 ng/mL of protease (activity 7.23 U/mg protease, R² = 0.991) or 6.5 μg/mL of peptide fragments. Finally, a continuous protease assay is developed to minimize manual sampling and reduce errors in kinetic studies.
Inhibition of invasion by glycogen synthase kinase-3 beta inhibitors through dysregulation of actin re-organisation via down-regulation of WAVE2
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After MDA-MB-231 cells were treated with 20 μM AR-A0114418, 20 μM GSKi26 or DMSO for 24 h, cells were allowed to release matrix metalloproteinases (MMPs) into new serum-free media with or without 100 ng/ml epidermal growth factor (EGF) for 24 h. Then, the media were subjected to quantification. Gelatin zymography was performed according to a previous report [20] with minor adjustments. The loading volume was normalised by the cell count using the crystal violet method [21].
Cancer cell invasion is a critical phenomenon in cancer pathogenesis. Glycogen synthase kinase-3β (GSK-3β) has been reported to regulate cancer cell invasion both negatively and positively. Thus, the net effect of GSK-3β on invasion is unclear. In this report, we showed that GSK-3β inhibitors induced dysregulation of the actin cytoskeleton and functional insufficiency of focal adhesion, which resulted in suppressed invasion. In addition, WAVE2, an essential molecule for actin fibre branching, was down-regulated after GSK-3β inhibition. Collectively, we propose that the WAVE2-actin cytoskeleton axis is an important target of GSK-3β inhibitors in cancer cell invasion.
ARF6-Regulated Shedding of Tumor Cell-Derived Plasma Membrane Microvesicles
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Isolated microvesicles were washed in phosphate-buffered saline (PBS) and lysed, and protein content was estimated via the Bradford assay (Bio-Rad). Microvesicles isolated from the growth media were lysed in RIPA buffer and separated on SDS-PAGE gels containing gelatin to assess gelatinase activity, as previously described [44]. The gelatin degradation assay was as previously described [45].
Increased mitogen-activated protein kinase (MAPK) signaling, small GTPase activation, cytoskeletal rearrangements, and the directed targeting of proteases to sites of extracellular matrix degradation all accompany the process of tumor cell invasion. Several studies have implicated the small GTP-binding protein ARF6 in tumor cell invasion, although the molecular basis by which ARF6 facilitates this process is unclear.
We show that the ARF6 GTP/GDP cycle regulates the release of protease-loaded plasma membrane-derived microvesicles from tumor cells into the surrounding environment. To enable microvesicle shedding, ARF6-GTP-dependent activation of phospholipase D promotes the recruitment of the extracellular signal-regulated kinase (ERK) to the plasma membrane where, in turn, ERK phosphorylates and activates myosin light-chain kinase (MLCK). MLCK-mediated MLC phosphorylation is required for microvesicle release. Inhibition of ARF6 activation is accompanied by PKC-mediated phosphorylation of MLC, which blocks microvesicle shedding. Protein cargo appears to be selectively sorted into microvesicles, and adhesion to the extracellular matrix (ECM) is facilitated by microvesicle-associated integrin receptors.
Microvesicle shedding in tumor cells occurs via an actomyosin-based membrane abscission mechanism that is regulated by nucleotide cycling on ARF6. Microvesicle shedding appears to release selected cellular components, particularly those involved in cell adhesion and motility, into the surrounding environment. These findings suggest that ARF6 activation and the proteolytic activities of microvesicles, both of which are thought to correlate directly with tumor progression, could potentially serve as biomarkers for disease.
Assays of matrix metalloproteinases (MMPs) activities: A review
2005, Biochimie
Citation Excerpt :
For sensitivity and ease of detection, labeling by means of [3H] acetic anhydride is frequently performed. Assays have been developed with casein [16], collagens (native or denatured) [17] and elastin [3]. It is also possible to use [125I] gelatin [18].
Measurement of matrix metalloproteinase (MMP) activity often remains a challenge, mainly in complex media. Two sets of methods are currently used. The first one measures the hydrolysis of natural protein substrates (labeled or not) and includes the popular zymography. These techniques which are quite sensitive, cannot generally be carried out on a continuous basis. The second one takes mainly advantage of the increase of fluorescence, which is associated to the hydrolysis of initially quenched fluorogenic peptide substrates. Quite recently, another group, which is a compromise between the other two, has been developed. It measures the hydrolysis of synthetic triple-helical peptide substrates. These different methods are described and discussed.
Selective cleavage of human IgG by the matrix metalloproteinases, matrilysin and stromelysin
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We have shown that two of the matrix metalloproteinases (MMPs), matrilysin and stromelysin-1, are capable of cleaving all of the human IgG subclasses. The cleavage occurs at a conserved site in the CH₂ domain of the heavy chain of IgG, releasing a single chain Fc-like fragment. We have not been able to demonstrate cleavage of IgA, IgD, IgM or IgE classes, which lack the cleavage site, nor could we show cleavage of IgG by collagenase, gelatinase, macrophage metalloelastase or membrane-type (MT)-MMP. This cleavage of IgG, by separating the antigen-binding (Fab′)₂ from the Fc portion, will remove much of the immunoglobulins' functionality, e.g. complement fixation, Fc receptor binding. In the context of a tumour producing matrilysin or stromelysin, this may represent a way in which the tumour protects itself from ADCC. In inflammed or damaged tissues where plasma protein leakage occurs, degradation by MMPs may be a mechanism for clearance of IgG.
Heparin-enhanced zymographic detection of matrilysin and collagenases
2001, Analytical Biochemistry
Unlike the gelatinases (MMP-2 and -9), matrilysin (MMP-7) and collagenases (MMP-1 and -13) are difficult to detect at low levels in conventional casein or gelatin zymography. In this report, heparin was used to enhance the zymographic assays for MMP-7, -1, and -13. With the addition of heparin to the enzyme sample, MMP-7 can be detected at a level of 30 pg in transferrin zymography and MMP-1 and -13 can be detected at a level of 0.2 ng in gelatin zymography. Carboxymethylated transferrin is used instead of casein as a substrate for assaying rat MMP-7. This substrate does not require a prerun step or substrate cross-linking to give uniform staining and clear band formation. It is necessary for heparin to run to the same region of the gel as the enzyme to produce its enhancing effect. For MMP-7 movement of heparin and enzyme is almost equal; for the collagenases it is necessary to add heparin to each well after the electrophoretic run is underway. Possible mechanisms of activity enhancement are discussed.

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Regular ArticleAn Assay for Matrix Metalloproteinases and Other Proteases Acting on Proteoglycans, Casein, or Gelatin

Abstract

Regular Article
An Assay for Matrix Metalloproteinases and Other Proteases Acting on Proteoglycans, Casein, or Gelatin