Metabolic Mapping of Proteinase Activity with Emphasis on In Situ Zymography of Gelatinases

Abstract

S U M M A R Y 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.

Matrix Metalloproteinase-Mediated Disruption of Tight Junction Proteins in Cerebral Vessels is Reversed by Synthetic Matrix Metalloproteinase Inhibitor in Focal Ischemia in Rat

“…Sections were rinsed three times in PBS and mounted in Gel/Mountt (Biomeda Corp. Foster City, CA, USA) for fluorescence microscopy. In each experiment, some sections were preincubated with blocking antibodies against MMP-2 (dilution 1:300) and MMP-9 (dilution 1:300) for 1 h, or 1 mmol/L 1,10-phenanthroline, a potent zinc chelator that inhibits all MMP activities, in the reaction buffer (Rivera et al, 2002;Frederiks and Mook, 2004). Sections incubated without DQ-gelatin were not fluorescent.…”

A Stable Niche Supports Long-Term Maintenance of Human Epidermal Stem Cells in Organotypic Cultures

“…Upon local proteolytic cleavage, quenching is released, and fluorescence signals can be visualized and quantified [24]. Unfixed cryostat sections (6 -8 m) were incubated in a solution containing 40 g/ml DQ-gelatin and 5 g/ml 4,6-diamidino-2-phenylindole for 30 minutes at RT and then coverslipped.…”

MEKK1 controls matrix degradation and tumor cell dissemination during metastasis of polyoma middle-T driven mammary cancer

“…One predicted consequence of the loss of uPA activity in MEKK1-deficient tumors would be reduced gelatinase activity. Tumor-associated gelatinase activity was assessed in situ by gelatin zymography (Frederiks and Mook, 2004), in which fresh tumor sections are overlayed with fluorochrome-labeled gelatin ( Figure 5). In this assay, the fluorescence of labeled gelatin is quenched until the gelatin is cleaved, thus areas of gelatin proteolysis are indicated by fluorescence.…”