Metabolic mapping of proteinase activity with emphasis on in situ zymography of gelatinases: review and protocols.
Proteases are essential for protein catabolism, regulation of a wide range of biological processes, and in the pathogenesis of many diseases. Several techniques are available to localize activity of proteases in tissue sections or cell preparations. For localization of the activity of matrix metalloproteinases, in situ zymography was introduced some decades ago. The procedure is based on zymography using SDS polyacrylamide gels containing gelatin, casein, or fibrin as substrate. For in situ zymography, either a photographic emulsion containing gelatin or a fluorescence-labeled proteinaceous macromolecular substrate is brought into contact with a tissue section or cell preparation. After incubation, enzymatic activity is revealed as white spots in a dark background or as black spots in a fluorescent background. However, this approach does not allow precise localization of proteinase activity because of limited sensitivity. A major improvement in sensitivity was achieved with the introduction of dye-quenched (DQ-)gelatin, which is gelatin that is heavily labeled with FITC molecules so that its fluorescence is quenched. After cleavage of DQ-gelatin by gelatinolytic activity, fluorescent peptides are produced that are visible against a weakly fluorescent background. The incubation with DQ-gelatin can be combined with simultaneous immunohistochemical detection of a protein on the same section. To draw valid conclusions from the findings with in situ zymography, specific inhibitors need to be used and the technique has to be combined with immunohistochemistry and zymography. In that case, in situ zymography provides data that extend our understanding of the role of specific proteinases in various physiological and pathological conditions.
Frederiks
Wilma M
WM
Academic Medical Center, University of Amsterdam, Department of Cell Biology and Histology, Amsterdam, The Netherlands. w.m.frederiks@amc.uva.nl
Mook
Olaf R F
OR
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D010450:Endopeptidases / Q000032:analysis* / Q000378:metabolism; D005456:Fluorescent Dyes; D005780:Gelatin / Q000031:analogs & derivatives; D018093:Gelatinases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism; D007150:Immunohistochemistry; D012680:Sensitivity and Specificity
D005456:Fluorescent Dyes; D005780:Gelatin; D010450:Endopeptidases; D018093:Gelatinases