An improved assay of mammalian collagenase activity, and its use to determine hepatic extracellular matrix susceptibility to degradation.
This rapid, sensitive method of determining collagenase (EC 3.4.24.7) activity incorporates several advantages of previous methods. Soluble [14C]acetylated collagen is prepared as the enzyme substrate and collagen-cleavage products are separated from noncleaved collagen by precipitation with dioxane/methanol. The assay is more reproducible than previous methods and has a lower detection limit, 15 mU of enzyme activity. We used the method in a competitive substrate assay with isolated extracellular hepatic matrix from cirrhotic and normal rat liver. Purified collagenase was consistently bound to normal rat matrix to a greater extent than to cirrhotic matrix, suggesting that in hepatic fibrosis the extracellular matrix is not as susceptible to collagenase degradation as that in normal liver.
D000818:Animals; D003094:Collagen / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008103:Liver Cirrhosis / Q000378:metabolism; D003012:Microbial Collagenase / Q000032:analysis*; D051381:Rats; D013379:Substrate Specificity
D003094:Collagen; D003012:Microbial Collagenase