A High Throughput Fluorogenic Substrate for Interstitial Collagenase (MMP-1) and Gelatinase (MMP-9)

Bickett, D. Mark; Green, M.D.; Berman, Judd; Dezube, Milana; Howe, Anne; Brown, Peter J.; Roth, Jeremy T.; McGeehan, Gerard M.

doi:10.1006/abio.1993.1291

Cited by 106 publications

(61 citation statements)

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“…Stimulation or inhibition of MMP activity can be observed using this technique (Gálvez et al, 2001). Large-scale screening of MMP stimulators or inhibitors can be conducted by adding cell lysates or conditioned medium to biotinconjugated gelatin or to fluorescent dye-conjugated MMP substrates in 96-well plates (Bickett et al, 1993;Menges et al, 1997;Ratnikov et al, 2000). Although these zymogen techniques allow much faster and larger-scale analysis of MMP activity, they do not allow analysis of active vs. inactive MMPs or visualization of their relative levels of expression.…”

Section: Matrix Degradationmentioning

confidence: 99%

In vitro assays of angiogenesis for assessment of angiogenic and anti-angiogenic agents

Goodwin¹

2007

Microvascular Research

251

208

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Blood vessels, either in insufficient numbers or in excess, contribute to the pathogenesis of many diseases. Agents that stimulate angiogenesis can improve blood flow in patients with ischemic diseases, whereas anti-angiogenic agents are used to treat disorders ranging from macular degeneration to cancer. In this review I describe in vitro assays that can be used to assess the activity of agents that affect angiogenesis. Means of quantifying endothelial cell matrix degradation, migration, proliferation, apoptosis and morphogenesis are discussed, as are embryoid body, aortic ring and metatarsal assays of vessel outgrowth. Strengths and limitations of these techniques are also addressed.

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Section: Matrix Degradationmentioning

confidence: 99%

In vitro assays of angiogenesis for assessment of angiogenic and anti-angiogenic agents

Goodwin¹

2007

Microvascular Research

251

208

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“…Barrett et al (1989) assayed a clostridial collagenase using N-(2,4-dinitrophenyl)-ProLeu-Gly-Pro-Trp-Lys substrate. Bickett et al (1993); Gould et al (1999) and Saikumari and Balaram (2008) used similar fluorescently-labeled substrates to achieve high sensitivity in collagenase activity measurements. Variation in these fluorescent techniques include: (a) labeling the products of collagenase activity rather than employing a synthetic substrate, (b) employing a reagent that forms a fluorescent complex with amino acids, (c) adding fluoresamine after incubating the enzyme and (d) using fluorescent substrate (Evans and Ridella, 1984).…”

Section: Synthetic Peptides and Fluorescent Assaymentioning

confidence: 99%

Extraction and Purification of Collagenase Enzymes: A Critical Review

Daboor¹,

Budge²,

Ghaly

et al. 2010

American Journal of Biochemistry and Biotechnology

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Problem statement: Enzymes have vital roles in several industrial processes (foods, cosmetics, nutraceuticals and pharmaceuticals) due to their highly selective nature and high activity at very low concentrations. Recent efforts to identify new sources of useful enzymes have been concentrated on the marine environment because of the potential to make use of processing wastes. About 35-50% of the mass of the fish caught is a waste that is disposed off at sea or in landfills. The extraction of enzymes from fish processing waste can reduce environment problems and improve the economics of the fish industry. Collagenases are a group of enzymes that can be extracted from fish waste. Approach: Comprehensive reviews of the literature on the extraction, purification, characterization and use of collagenases was carried out. Results: Collagenases have different molecular weights based on their types and sources. They have the ability to break down the peptide bonds in collagen at physiological pH. They are classified into two types: serine and metallocollagenase. Collagenolytic activities have been shown at a wide range of temperatures (20-40°C) and pH (6-8). Many activators can be used to achive collagenase activity including 4-Aminophenylmercuric Acetate (APMA), trypsin, potassium or sodium thiocyanate, iodoacetamide and potassium iodide. Dithiothreitol (DTT), mercaptoethanol, ethylendiaminetetracetic acid, ophenanthroline and cysteine inactivate the enzyme. Collagenases enzymes can be extracted with a variety of techniques using different buffering systems (tris-HCl, sodium bicarbonate, calcium chloride and cacodylate). All techniques involve the use of ammonium sulphate fractionation and centrifugation to precipitate the enzyme. Collagenases are normally purified using chromatographic techniques such as gel-filtration, ion-exchange and affinity column chromatography. Collagenase can be assayed with a number of methods, including: colorimetric absorbance, viscometry, radioactivity and fluorescence spectroscopy. Collagenases are partly responsible for toughness in red meats and are used as tenderizers in food industry, have application in the fur and hide tanning to ensure uniform dying of leather, used in medicine to treat burns and ulcers, eliminate scar tissues, transplantation of organs. Conclusion: Understanding of the nature of the enzymes and identifying the most suitable resources and the methods for their extraction and purification will have significant impact on the fish processing, food and medical industries.

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“…After 1 h at 37°C and 5 min of vortexing, the samples were centrifuged (5 min at 10000×g) to remove insoluble material, and were subjected to RP-HPLC to determine the solubility of the substrate. Mca-Pro-Leu-GlyLeu-Dpa-Ala-Arg-NH2 was from Bachem (Bubendorf, Switzerland) and DNP-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys(Nma)-NH2 was a gift from Dr. J. McGeehan [11].…”

Section: Solubility Of Fluorogenic Substratesmentioning

confidence: 99%

“…The former methods are mostly too insensitive for detection of MMP activity in unactivated biological media. Recently, the use of fluorogenic peptides for measuring activity of purified MMPs was described [8][9][10][11]. These substrates consist of a fluorophore and a light-absorbing group (quencher) attached to an amino acid sequence that is recognized by MMPs.…”

Section: Introductionmentioning

confidence: 99%

Convenient fluorometric assay for matrix metalloproteinase activity and its application in biological media

Beekman¹,

Drijfhout

Bloemhoff

et al. 1996

FEBS Letters

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Matrix metalloproteinases (MMPs) are involved in physiological tissue remodeling and pathological conditions like tumour metastasis and joint destruction. Until now, no convenient and sensitive MMP-activity assay in crude media like synodal fluid has been available. Therefore, the highly soluble fluorogenic substrate TNO211 (Dabcyl-Gaba-Pro-Gin-Gly-LeuGIu(EDANS)-AIa-Lys-NH2), containing the MMP cleavable Gly-Leu bond and EDANS/Dabcyl as fluorophore/quencer combination, was synthesized and characterized as an MMP specific substrate. We show that the fluorogenic assay using ] NO211 is sensitive and can detect MMP activity in culture medium from endothelial cells and untreated synovial fluid (SF) from RA and OA patients, and control subjects. MMP activity in SF significantly increased in the order C < OA < RA, thus the frequent use of OA samples as control in studies on RA is debatable.A ey words: Matrix metalloproteinase; Synovial fluid; I luorescence quenching

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A High Throughput Fluorogenic Substrate for Interstitial Collagenase (MMP-1) and Gelatinase (MMP-9)

Cited by 106 publications

References 0 publications

In vitro assays of angiogenesis for assessment of angiogenic and anti-angiogenic agents

In vitro assays of angiogenesis for assessment of angiogenic and anti-angiogenic agents

Extraction and Purification of Collagenase Enzymes: A Critical Review

Convenient fluorometric assay for matrix metalloproteinase activity and its application in biological media

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