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Identification, purification and characterization of matrix metalloproteinase-2 in bovine pulmonary artery smooth muscle plasma membrane

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Abstract

Bovine pulmonary artery smooth muscle tissue possesses matrix metalloproteinase-2 (72 kDa gelatinase: MMP-2; E.C. 3.4.24.24) as revealed by immunoblot studies of its smooth muscle plasma membrane suspension with polyclonal MMP-2 antibody. In this report, we described the purification and partial characterization of MMP-2 in the plasma membrane fraction of the smooth muscle. MMP-2 has been purified from plasma membrane fraction of bovine pulmonary artery smooth muscle to homogeneity using a combination of purification steps. Heparin sepharose purified preparation of 72 kDa progelatinase is composed of two distinct population of zymogens: a 72 kDa progelatinase tightly complexed with TIMP-2 ∥an ambient tissue inhibitor of metalloprotease in the smooth muscle plasma membrane), and a native 72 kDa progelatinase free of any detectable TIMP-2. The homogeneity of the native 72 kDa progelatinase form is demonstrated by SDS-PAGE under nonreducing condition, nondenaturing native gel electrophoresis. The purified TIMP-2 free proenzyme electrophoresed as a single band of 72 kDa which could be activated by APMA with the formation of 62 and 45 kDa active species. The proenzyme is activated poorly by trypsin but not by plasmin. The purified 72 kDa progelatinase is stable at aqueous solution and does not spontaneously autoactivate. The purified 72 kDa gelatinase exhibited properties that are typical of MMP-2 obtained from other sources. These are: (i) its activity is dependent on the divalent cation, Ca2+, and is inhibited by EDTA, EGTA and 1:10-phenanthroline; (ii) it was inhibited by α2 macroglobulin but not by the inhibitors of serine, cysteine, thiol, aspartic proteinases and calpains; (iii) it was found to be inhibited by TIMP-2, the specific inhibitor of MMP-2; (iv) like MMP-2, obtained from other sources, its major substrates were found to be collagens (type IV and V) and gelatins (type I, IV and V). Additionally, the purified MMP-2 degrades Dnp-Pro-Gln-Gly-Ile-Ala-Gly-Gn-D-Arg-OH (dinitrophenyl labelled peptide), a well known synthetic substrate for the MMP-2.

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Authors and Affiliations

  1. Department of Biochemistry and Biophysics, University of Kalyani, Kalyani, West Bengal, India

    Sudip Das, Malay Mandal, Amritlal Mandal, Tapati Chakraborti & Sajal Chakraborti

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  1. Sudip Das
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Das, S., Mandal, M., Mandal, A. et al. Identification, purification and characterization of matrix metalloproteinase-2 in bovine pulmonary artery smooth muscle plasma membrane. Mol Cell Biochem 258, 73–89 (2004). https://doi.org/10.1023/B:MCBI.0000012838.41792.d2

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