Isolation of MMP-2 from MMP-2/TIMP-2 complex: characterization of the complex and the free enzyme in pulmonary vascular smooth muscle plasma membrane.

We have previously indicated that bovine pulmonary artery smooth muscle plasma membrane possesses a complex of 72-kDa gelatinase and TIMP-2 (MMP-2/TIMP-2 complex) [Mol. Cell. Biochem. 258 (2004) 73]. In this paper, we described isolation of MMP-2 from the MMP-2/TIMP-2 complex, characterizations of the isolated MMP-2 and also the complex. MMP-2/TIMP-2 complex was purified from bovine pulmonary vascular smooth muscle plasma membrane using a combination of purification steps. Heparin-sepharose (100 mM NaCl eluate)-purified preparation contained the MMP-2/TIMP-2 complex. The MMP-2/TIMP-2 complex, which was electrophoresed under reducing condition on the SDS-PAGE and immunobloted with a mixture of polyclonal MMP-2 and TIMP-2 antibodies, revealed two separate immunoreactive bands at their respective electrophoretic migration. Continuous elution electrophoresis of the complex resulted to MMP-2 free of any detectable TIMP-2. The homogeneity of the isolated MMP-2 and the complex was demonstrated by SDS-PAGE under nonreducing condition and also by nondenaturing native-PAGE. The purified TIMP-2 free enzyme electrophoresed as a single band of 72-kDa, which could be activated rapidly and fully by aminophenylmercuric acetate (APMA) with the formation of 62-kDa and 45-kDa active species like native MMP-2 purified from the same source (bovine pulmonary artery smooth muscle). Identical treatment of the MMP-2/TIMP-2 complex with APMA resulted to significantly slower and partial conversion of the active species. Addition of pure TIMP-2 to the TIMP-2 free MMP-2 formed a complex with the progelatinase and prevented the rapid autolytic conversion induced by APMA. Immunoblot study with polyclonal MMP-2 antibody suggested that the isolated 72-kDa gelatinase is the MMP-2. We have also presented additional data indicating that the isolated preparation of 72-kDa gelatinase exhibited properties that are identical with MMP-2 obtained from different sources.

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