Purification and characterization of exudate gelatinases in the chronic-phase of carrageenin-induced inflammation in rats.
Gelatinases have been purified from the exudate in the chronic-phase (day 7) of carrageenin-induced inflammation in rats. The day-7 exudate gelatinases gave two peaks on Sephadex G-150 gel filtration, the initial step of the purification. The molecular weights of the gelatinases corresponding to the two peaks were about 300 kDa (HMW fraction) and about 110 kDa (LMW fraction), respectively. The gelatinase in the HMW fraction has been purified to homogeneity; the purified gelatinase gave a single band corresponding to a molecular weight of 57 kDa on both SDS-polyacrylamide gel electrophoresis (PAGE) and SDS-gelatin PAGE. On the other hand, the gelatinase purified from the LMW fraction was found to consist of three species, with molecular weights of 66, 64, and 57 kDa, as judged on SDS-gelatin PAGE. Granulation tissue-derived fibroblasts in culture mainly produced the 64-kDa species, which was converted to a 57-kDa species on treatment with 4-amino-phenylmercuric acetate, while rat macrophages and polymorphonuclear leukocytes mainly secreted the 96-kDa species. These results suggest that exudate gelatinases are largely produced by fibroblasts in granulation tissue and that they bind to exudate proteins, resulting in the formation of complexes with molecular weights of about 300 kDa and about 110 kDa. The gelatinases purified from the HMW and LMW fractions are metalloproteinases, as judged from the results of inhibitor experiments. Both the gelatinases degraded gelatin, but showed to proteolytic activity toward alpha-casein or type I collagen. Type IV collagen was degraded at 35 degrees C by the gelatinases purified from the LMW fraction but not by that from the HMW fraction.
Sakata
K
K
Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University.
Hoshino
K
K
Nakagawa
H
H
D000818:Animals; D002351:Carrageenan; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D005122:Exudates and Transudates / Q000201:enzymology*; D005347:Fibroblasts; D018093:Gelatinases; D007249:Inflammation / Q000139:chemically induced / Q000201:enzymology*; D008264:Macrophages / Q000201:enzymology; D008297:Male; D010434:Pepsin A / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D002351:Carrageenan; D010434:Pepsin A; D018093:Gelatinases