Identification, purification and partial characterization of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in bovine pulmonary artery smooth muscle.
Bovine pulmonary artery smooth muscle tissue possesses the tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) as revealed by immunoblot studies of the cytosolic fraction with polyclonal TIMP-1 antibody. In this report, we described the purification and partial characterization of the inhibitor from the cytosolic fraction of the smooth muscle. This inhibitor was purified by a series of anion-exchange, gel filtration and affinity chromatographic procedure. The purified inhibitor showed an apparent molecular mass of 30 kDa in SDS-PAGE. Amino terminal sequence analysis for the first 22 amino acids of the purified inhibitor was also found to be identical to bovine TIMP-1. This glycosylated inhibitor was found to be active against matrix metalloproteinase-9 (MMP-9, gelatinase B), the ambient matrix metalloproteinase in the pulmonary smooth muscle. The purified TIMP-1 was also found to be sensitive to pure rabbit and human fibroblast collagenase and type IV collagenase. In contrast, it had minimum inhibitory activity against bacterial collagenase. It was also found to be inactive against the serine proteases trypsin and plasmin. The inhibitor was heat and acid resistant and it had the sensitivity to trypsin degradation and reduction-alkylation.
Mandal
Malay
M
Department of Biochemistry and Biophysics, University of Kalyani, Kalyani, West Bengal, India.
Das
Sudip
S
Chakraborti
Tapati
T
Mandal
Amritlal
A
Chakraborti
Sajal
S
D000595:Amino Acid Sequence; D000645:Ammonium Sulfate / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002482:Cellulose / Q000737:chemistry; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017364:Collagenases / Q000378:metabolism; D003208:Concanavalin A / Q000737:chemistry; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005341:Fibrinolysin / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006493:Heparin / Q000737:chemistry; D006801:Humans; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011651:Pulmonary Artery / Q000378:metabolism*; D011817:Rabbits; D013997:Time Factors; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D014357:Trypsin / Q000378:metabolism
D019715:Tissue Inhibitor of Metalloproteinase-1; D003208:Concanavalin A; D002482:Cellulose; D006493:Heparin; D014357:Trypsin; D005341:Fibrinolysin; D017364:Collagenases; D020780:Matrix Metalloproteinase 9; D000645:Ammonium Sulfate