Method to analyze collagenase and gelatinase activity by fibroblasts in culture.
The net amount of collagen produced and deposited by fibroblasts in cell culture is determined by the rate of collagen synthesis as well as the rate of collagen degradation. Although collagen synthesis can be analyzed by several techniques, it is more difficult to measure collagen degradation. Breakdown of collagen depends upon the activity of a family of structurally and catalytically related mammalian enzymes termed matrix metalloproteinases (MMPs). Interstitial collagenase (MMP1) initiates the cleavage of fibrillar collagen, whereas gelatinases (MMP2 and MMP9) digest the denatured collagen fragments. A method has been developed to quantitate the activity of collagenase (MMP1) and gelatinase (MMP9) in conditioned medium from fibroblast cell cultures. The assay, which uses the fluorogenic substrate Dnp-Pro-Cha-Gly-Cys(Me)-His-AlaLys(Nma)NH2, is technically simple and amenable to high throughput analysis. Addition of specific inhibitors of the metalloproteinases allows for simultaneous measurement of both collagenase and gelatinase activity.
Gould
L J
LJ
Department of Surgery, Medical College of Virginia Campus of Virginia Commonwealth University, Richmond 23298-0117, USA.
Yager
D R
DR
McGeehan
G M
GM
Diegelmann
R F
RF
D000818:Animals; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D017364:Collagenases / Q000032:analysis* / Q000378:metabolism; D017077:Culture Media, Conditioned; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000201:enzymology*; D005456:Fluorescent Dyes; D018093:Gelatinases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D007700:Kinetics; D011817:Rabbits; D012044:Regression Analysis; D013050:Spectrometry, Fluorescence
D017077:Culture Media, Conditioned; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D003094:Collagen; D017364:Collagenases; D018093:Gelatinases