Comparison of Extracellular Matrix-Degrading Activities between 64-kDa and 90-kDa Gelatinases Purified in Inhibitor-Free Forms from Human Schwannoma Cells1

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Abstract
Two kinds of gelatinases (or type IV collagenases), 90-kDa and 64-kDa gelatinases, were purified in a tissue inhibitor of metalloproteinases (TIMP)- or TIMP-2-free form from the serum-free conditioned medium of human schwannoma YST-3 cells, and their activities on extracellular matrix proteins were compared. Sequential chromatographies on a gelatin-Sepharose column, an LCA-agarose column, and a gel filtration column in the presence of 5 M urea yielded 600 μg of the 64-kDa enzyme and 45 μg of the 90-kDa enzyme from 2.8 liters of the conditioned medium. The purified enzymes showed high gelatinolytic activities without activation by p-aminophenyl mercuric acetate (APMA), indicating that 5 M urea used in the final chromatography not only dissociated the inhibitors from the progelatinases but also activated the proenzymes. The inhibitor-free gelatinases showed a much higher activity than the APMA-activated inhibitor-bound enzymes. The specific activity of the 90-kDa enzyme was nearly 25 times higher than that of the 64-kDa enzyme. The 90-kDa gelatinase hydrolyzed type I collagen as well as native and pepsin-treated type IV collagens at 30°C, while at 37°C it potently hydrolyzed types I, III, and IV collagens but not fibronectin or laminin. The 64-kDa gelatinase showed a similar substrate specificity to that of the 90-kDa enzyme, except that it did not hydrolyze type I collagen and native type IV collagen at 30°C.

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