Measurement of activity of collagenolytic MMP and inhibitors of MMPs using radiolabeled collagen substrate.
This protocol describes how to purify and radiolabel collagen for use as a substrate to assay collagenolytic members of the matrix metalloproteinases (MMPs). This assay measures enzymes that specifically cleave native triple helical collagen. After incubation of the MMP enzyme with the collagen substrate at 37 degrees C, undigested collagen is removed by centrifugation. Radiolabeled cleaved fragments remain in the supernatant, which is then counted in a scintillation counter; a linear increase in the release of radiolabeled collagen fragments occurs with enzyme level and time. Methods are included for the activation of the proenzyme forms of these MMPs and the assay can also be adapted to measure inhibitors of the collagenolytic MMPs. This assay can be completed in 18 h.
Cawston
Tim E
TE
Musculoskeletal Research Group, Institute of Cellular Medicine, The Medical School, Newcastle University, Framlington Place, Newcastle Upon Tyne, NE2 4HH, UK. T.E.Cawston@ncl.ac.uk
D000818:Animals; D002417:Cattle; D024042:Collagen Type I / Q000737:chemistry* / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods*; D045726:Metalloproteases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D012867:Skin; D013379:Substrate Specificity
D024042:Collagen Type I; D045726:Metalloproteases