Authors : Gustavo Hernandez Vidal , Francisco Mora Valdez , Jorge Kawas Garza , Rafael Ramirez Romero , Jose Antonio Salinas Melendez , Luis Edgar Rodriguez Tovar , Ramiro avalos Ramirez , Victor Riojas Valdes , Elisabeth Davies and Leo B Jeffcott
Abstract: The enzymatic activity of the cysteine proteinase cathepsin B (EC 3.4.22.1) was localized in equine normal unfixed cartilage, phenotypically modulated chondrocytes and horse skin fibroblasts, dyschondroplastic chondrocytes and cartilage by immunofluorescence. The final precipitation of the fluorescent reaction of cathepsin B enzymatic activity was monitored during the enzymatic reaction. An intense fluorescence was observed using N-carbobenzoxyl-L-alanyl-L-arginyl-L-arginine-4-methoxy- -naphthyl-amide as specific substrate for cathepsin B and 5-nitrosalicylaldehyde as coupling reagent. The unspecific fluorescence was eliminated by the use of the specific inhibitor for cathepsin B, E-64. Continuous monitoring demonstrated the presence of fluorescent granules after a defined time, with the formation of crystals as the final reaction product. This method shows cathepsin B activity in different types of tissues and cells and can be used as kinetic analysis of the enzymatic activity. The intense enzymatic activity observed in equine chondrocytes and equine skin fibroblasts suggests that cysteine proteinase is not only involved in the degradation of collagen by fibroblasts, but it also plays an important role in the intracellular digestion of collagen within chondrocytes.
Gustavo Hernandez Vidal , Francisco Mora Valdez , Jorge Kawas Garza , Rafael Ramirez Romero , Jose Antonio Salinas Melendez , Luis Edgar Rodriguez Tovar , Ramiro avalos Ramirez , Victor Riojas Valdes , Elisabeth Davies and Leo B Jeffcott , 2008. Localization of Cathepsin B Enzymatic Activity in Equine Articular Cartilage, Skin Fibroblasts and Phenotypically Modulated and Dyschondroplastic Chondrocytes. Journal of Animal and Veterinary Advances, 7: 493-501.