Canine TIMP-2: Purification, characterization and molecular detection

Matrix metalloproteinases (MMPs), which degrade tissues in health and disea se are under the control of the tissue inhibitors of MMPs, the TIMPs. TIMP- 2 is particularly important for control of MMP-2 and both have been implica ted in many pathological processes from arthritis to tumour invasion. This study characterized and detected TIMP-2 from canine cells; including synovi al fibroblasts and thr ee tumour-derived canine cell lines, K1, K6 and DH82 . Gelatin zymography demonstrated that pro-MMP-2 is produced by synovial fi broblasts and die three cells Lines. Reverse zymograms showed that all the cell sources tested secrete both TIMP-1 and TIMP-2. The 22 kDa band was pur ified and n-terminal amino acid sequencing showed it to be highly homologou s to equien and homologous human TIMP-2. Analysis of purified canine MMP-2 and MMP-9 showed that TIMP-2 is associated, and co-purifies with MMP-2. Pol ymerase chain reaction, using consensus primers, was used to detect TIMP-2 mRNA from the cell sour-ces and proved positive in all cases. This work hig hlights the importance of TIMP-2 as the main inhibitor for MMP-2 and, there fore, opens the possibilities of targeting TIMP-2 for therapeutic intervent ion against connective amino acid tissue degradation in a range of diseases . (C) 2000 Harcourt Publishers Ltd.