Isolation and characterization of Stromelysin-3(MMP-11) like protein from a case of mammary carcinoma in a dog

Stromelysin-3(MMP-11) is a matrix-metalloproteinase known to be over expressed in invasive tumors of human origin and suppose to have a potential role in metastasis. Dog mammary tumors are usually very invasive. Thus to characterize dog stromelysin-3, mammary tumor tissue (papillary adenocarcinoma) was used to isolate the protein by using affinity chromatography tools including Cibacron-Blue 3GA-CL Agarose, gelatin Sepharose, and heparin Sepharose columns. Western blotting with antihuman stromelysin-3 antibody was done for identification. The result showed a heavier (120kDa) protein than the expected size of stromelysin-3. Western blot analysis of the crude sample did not show any other reactive bands. Under reducing condition, the protein size remained intact. The stromelysin-3 like protein showed mild caseinolytic activity, which increased by activators like APMA and trypsin. Whereas, its activity decreased when inhibitors like EDTA, 1, 10-Phenanthroline and TIMP-1 were used suggesting the requirement of calcium and zinc for enzyme activity just like any other metalloproteinase.