Purification of progelatinase A (matrix metalloproteinase 2) and a tissue inhibitor of metalloproteinase-2 (TIMP-2) from T98G human glioblastoma cells

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.