title: Purification of an active gelatinase from collagen fiber fraction of granulation tissue in rats.
abstract: Gelatinase was extracted at 60 degrees C from the collagen fiber-rich fraction of granulation tissue induced by carrageenin 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 (greater than 200 kDa) on Sephadex G-150 gel filtration. In addition, that unbound fraction contained gelatin fragments was revealed by SDS-PAGE. When the unbound fraction of Zn-chelating Sepharose was incubated at 37 degrees 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 a 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 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 alpha-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.
authors.affiliation: Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University.
authors.forename: H
authors.initials: H
authors.lastname: Nakagawa
authors.forename: M
authors.initials: M
authors.lastname: Hirata
authors.forename: K
authors.initials: K
authors.lastname: Hoshino
authors.forename: K
authors.initials: K
authors.lastname: Sakata
authors.forename: S
authors.initials: S
authors.lastname: Hatakeyama
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity; D003094:Collagen / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D018093:Gelatinases; D006097:Granulation Tissue / Q000737:chemistry*; D008297:Male; D010434:Pepsin A / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D003094:Collagen; D010434:Pepsin A; D018093:Gelatinases