title: Fluorescence polarization assay and SDS-PAGE confirms matrilysin degrades fibronectin and collagen IV whereas gelatinase A degrades collagen IV but not fibronectin.
abstract: Matrilysin and gelatinase A are hypothesized to have significant roles in uterine and ovarian function. However, proteolytic activity assays for these enzymes are limited. We describe the development of simple and rapid assays for the proteolysis of fluorescein-labeled full-length substrates, collagen IV (Col-IV) and fibronectin (FN), and demonstrate the selectivity of matrilysin (MMP-7) compared to gelatinase A (MMP-2) for fibronectin. Changes in fluorescence intensity (FIU) and fluorescence polarization (mP) resulting from the protease activity of matrilysin and gelatinase A were measured. These studies show that the fluorescently labeled substrates, Col-IV and FN, are as reliable and amenable to rapid in vitro assay as peptide substrates. In addition, they are easier to use than previously described, non-fluorescent methods. The results demonstrate that assays using full-length, biological matrix proteins are more sensitive indicators of MMP-specific substrate activity than peptide based assays.
authors.affiliation: The R.W. Johnson Pharmaceutical Research Institute, Raritan, NJ 08869, USA. pkraft@prius.jnj.com
authors.forename: P J
authors.initials: PJ
authors.lastname: Kraft
authors.forename: D E
authors.initials: DE
authors.lastname: Haynes-Johnson
authors.forename: L
authors.initials: L
authors.lastname: Patel
authors.forename: J A
authors.initials: JA
authors.lastname: Lenhart
authors.forename: R A
authors.initials: RA
authors.lastname: Zivin
authors.forename: S S
authors.initials: SS
authors.lastname: Palmer
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D024141:Collagen Type IV / Q000378:metabolism*; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D005353:Fibronectins / Q000378:metabolism*; D005454:Fluorescence Polarization; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D020783:Matrix Metalloproteinase 7 / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D024141:Collagen Type IV; D005353:Fibronectins; D020783:Matrix Metalloproteinase 7; D020778:Matrix Metalloproteinase 2