title: Real-time zymography and reverse zymography: a method for detecting activities of matrix metalloproteinases and their inhibitors using FITC-labeled collagen and casein as substrates.
abstract: Zymography and reverse zymography are widely used techniques for identifying the proteolytic activity of enzymes and the presence of protease inhibitors in polyacrylamide gels. In the current studies, we utilized a fluorescein-isothiocyanate-labeled substrate to develop novel zymographic and reverse zymographic methods for detecting matrix metalloproteinases and tissue inhibitors of the metalloproteinases, respectively. Using a transilluminator, the results can be observed visually without stopping the enzymatic reaction. For this reason, we have named these methods real-time zymography and real-time reverse zymography. These methods have the following advantages compared with conventional protocols: (1) because the reaction can be repeatedly monitored on the polyacrylamide gels, optimization of the incubation time can be achieved without preliminary analyses; (2) higher sensitivity is achieved with a lower amount of substrate than with conventional methods; (3) a semi-quantitative analysis of matrix metalloproteinases is possible. An additional advantage of the real-time reverse zymography is that, because the fluorescence detection is specific for substrate digestion, the inhibitor bands can be easily distinguished from contaminating proteins.
authors.affiliation: Nippi Research Institute of Biomatrix, Tokyo, Adachi-ku, 120-8601, Japan. shuhatt@bekkoame.ne.jp
authors.forename: Shunji
authors.initials: S
authors.lastname: Hattori
authors.forename: Hitomi
authors.initials: H
authors.lastname: Fujisaki
authors.forename: Tomomi
authors.initials: T
authors.lastname: Kiriyama
authors.forename: Tsukao
authors.initials: T
authors.lastname: Yokoyama
authors.forename: Shinkichi
authors.initials: S
authors.lastname: Irie
mesh_terms: D002364:Caseins / Q000737:chemistry / Q000378:metabolism*; D004796:Clinical Enzyme Tests / Q000379:methods*; D003094:Collagen / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004795:Enzyme Stability; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry*; D020782:Matrix Metalloproteinases / Q000032:analysis*; D011489:Protein Denaturation; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism; D013194:Staining and Labeling; D013379:Substrate Specificity; D019714:Tissue Inhibitor of Metalloproteinases / Q000032:analysis*
chemical_list: D002364:Caseins; D019714:Tissue Inhibitor of Metalloproteinases; D012710:Serum Albumin, Bovine; D003094:Collagen; D020782:Matrix Metalloproteinases; D016650:Fluorescein-5-isothiocyanate