Purification of Progelatinase A (Matrix Metalloproteinase 2) and a Tissue Inhibitor of Metalloproteinase-2(TIMP-2) from T98G Human Glioblastoma Cells

  • Lee, Ho-Jae (Microbial Chemistry Research Group, Genetic Engineering Research Institute, Korea Institute of Science and Technology) ;
  • Chung, Myung-Chul (Microbial Chemistry Research Group, Genetic Engineering Research Institute, Korea Institute of Science and Technology) ;
  • Lee, Choong-Hwan (Microbial Chemistry Research Group, Genetic Engineering Research Institute, Korea Institute of Science and Technology) ;
  • Chun, Hyo-Kon (Microbial Chemistry Research Group, Genetic Engineering Research Institute, Korea Institute of Science and Technology) ;
  • Kho, Yung-Hee (Microbial Chemistry Research Group, Genetic Engineering Research Institute, Korea Institute of Science and Technology)
  • Received : 0
  • Accepted : 0
  • Published : 0

Abstract

The Gelatinases (typeIV collagenases) are metalloproteinases that may play an important role in tumor invasion and metastasis. Progelatinase A was purified from a conditioned medium of T98G human glioblastoma cells. TIMP-2 complexed progelatinase A and free progelatinase A were separated by heparin affinity HPLC. The final product was homogeneous on SDS-PAGE, with a molecular weight of 64,000 daltons without reduction. TIMP-2 and free progelatinase A were separated from TIMP-2 complexed progelatinase A by reverse-phase HPLC in the presence of trifluoroacetic acid. TIMP-2 complexed progelatinase A was resistant to activation by p-aminophenyl mercuric acetate (APMA), and showed less than 20% of the activity of the TIMP-2 free active enzyme. TIMP-2 free progelatinase A was easily activated to the mature form with a molecular weight of 57,000 daltons by APMA and showed high activity compared to the TIMP-2 complexed enzyme.

Keywords

Matrix metalloproteinases;TIMP;glioblastoma