Purification of an Active Gelatinase from Collagen Fiber Fraction of Granulation Tissue in Rats

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Abstract
Gelatinase was extracted at 60°C from the collagen fiber-rich fraction of granulation tissue induced by carrageen in in rats. A large part of the extracted gelatinase was unbound to Zn-chelating Sepharose. The unbound gelatinase gave a single band corresponding to a molecular mass of 57 kDa on SDS-substrate PAGE, but showed a much higher molecular mass (≤ 200 kDa) on Sephadex G-150 gel filtration. In addition, that the unbound fraction contained gelatin fragments was revealed by SDS-PAGE. When the unbound fraction of Zn-chelating Sepharose was incubated at 37°C, the gelatin fragments disappeared and the apparent molecular mass of gelatinase in gel filtration decreased. This gelatin degradation of the unbound fraction was enhanced by treatment with 4-aminophenylmercuric acetate (APMA). The results suggest that the gelatinase is bound to gelatin fragments in the unbound fraction. After the treatment with APMA, the gelatinase was purified to homogeneity; the purified gelatinase gave a single band corresponding to a molecular mass of 57 or 67 kDa on SDS-PAGE under nonreducing or reducing conditions, respectively. The purified gelatinase is a metalloproteinase, and extensively degraded gelatin, but showed no proteolytic activity toward α-casein or types I and IV collagens. The results suggest that the 67-kDa active gelatinase is bound to collagen fibers and plays an important role in a rapid degradation of collagen fibers in granulation tissue.

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