An assay for matrix metalloproteinases and other proteases acting on proteoglycans, casein, or gelatin.
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.
Manicourt
D H
DH
Laboratoire de Chimie Physiologique (Connective Tissue Group), University of Louvain, Brussels, Belgium.
Lefebvre
V
V
D000818:Animals; D002364:Caseins / Q000378:metabolism*; D002478:Cells, Cultured; D002623:Chemistry Techniques, Analytical / Q000379:methods; D017077:Culture Media, Conditioned; D010450:Endopeptidases / Q000032:analysis* / Q000378:metabolism; D004800:Enzymes, Immobilized; D005109:Extracellular Matrix / Q000201:enzymology*; D005780:Gelatin / Q000378:metabolism*; D006801:Humans; D008666:Metalloendopeptidases / Q000032:analysis* / Q000378:metabolism; D011509:Proteoglycans / Q000378:metabolism*; D011817:Rabbits; D013379:Substrate Specificity; D013552:Swine; D014357:Trypsin / Q000032:analysis / Q000378:metabolism
D002364:Caseins; D017077:Culture Media, Conditioned; D004800:Enzymes, Immobilized; D011509:Proteoglycans; D005780:Gelatin; D010450:Endopeptidases; D014357:Trypsin; D008666:Metalloendopeptidases