Identification, purification and partial characterization of tissue inhibitor of matrix metalloproteinase-2 in bovine pulmonary artery smooth muscle.
Bovine pulmonary artery smooth muscle possesses the tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) as revealed by Western immunoblot study of its cytosol fraction with bovine polyclonal TIMP-2 antibody. This potent polypeptide inhibitor of matrix metalloproteinases (MMPs) was purified to homogeneity from cytosol fraction of bovine pulmonary artery smooth muscle. This inhibitor was purified by ammonium sulfate precipitation followed by gelatin sepharose and lentil lectin sepharose affinity chromatography and continuous elution electrophoresis by Prep Cell Model 491 (Bio-Rad, USA). SDS-PAGE revealed that the inhibitor has an apparent molecular mass of 21 kDa and was confirmed as TIMP-2 by (i) Western immunoblot assay using bovine polyclonal TIMP-2 antibody; and also by (ii) amino terminal amino acid sequence analysis of the purified inhibitor is found to be identical with TIMP-2 obtained from other sources. The purified 21 kDa inhibitor was found to be active against matrix metalloproteinase-2 (MMP-2, 72 kDa gelatinase) and matrix metalloproteinase-9 (MMP-9, 92 kDa gelatinase), the ambient MMPs in the pulmonary artery smooth muscle. The inhibitor was also found to be sensitive to the activated 72 kDa gelatinase-TIMP-2 complex and also active human interstitial collagenase. By contrast, it was found to be insensitive to the serine proteases: trypsin and plasmin. The inhibitor was heat and acid resistant and it had the sensitivity to trypsin degradation and reduction-alkylation. Treatment of the inhibitor with hydrogen peroxide, superoxide generating system (hypoxanthine plus xanthine oxidase) and peroxynitrite inactivated the inhibitor.
Mandal
Malay
M
Department of Biochemistry and Biophysics, University of Kalyani, Kalyani, West Bengal, India.
Mandal
Amritlal
A
Das
Sudip
S
Chakraborti
Tapati
T
Chakraborti
Sajal
S
D000645:Ammonium Sulfate / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D015153:Blotting, Western; D002417:Cattle; D002847:Chromatography, Agarose; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D037102:Lectins / Q000737:chemistry; D027806:Lens Plant / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D009130:Muscle, Smooth / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011651:Pulmonary Artery / Q000378:metabolism*; D013481:Superoxides; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D014357:Trypsin / Q000494:pharmacology
D000975:Antioxidants; D037102:Lectins; D016877:Oxidants; D013481:Superoxides; D019716:Tissue Inhibitor of Metalloproteinase-2; D030421:Peroxynitrous Acid; D006861:Hydrogen Peroxide; D014357:Trypsin; D005341:Fibrinolysin; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D010100:Oxygen; D000645:Ammonium Sulfate