Comparison of extracellular matrix-degrading activities between 64-kDa and 90-kDa gelatinases purified in inhibitor-free forms from human schwannoma cells.
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 micrograms of the 64-kDa enzyme and 45 micrograms 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 degrees C, while at 37 degrees 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 degrees C.
Yasumitsu
H
H
Kihara Institute for Biological Research, Yokohama City University, Kanagawa.
Miyazaki
K
K
Umenishi
F
F
Koshikawa
N
N
Umeda
M
M
D003094:Collagen / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007797:Laminin / Q000378:metabolism; D020780:Matrix Metalloproteinase 9; D003012:Microbial Collagenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D009442:Neurilemmoma; D010662:Phenylmercuric Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D007797:Laminin; D011480:Protease Inhibitors; C010024:4-aminophenylmercuriacetate; D003094:Collagen; D003012:Microbial Collagenase; D020780:Matrix Metalloproteinase 9; D010662:Phenylmercuric Acetate