Gelatin-degrading activity secreted by cultured macrophages from human blood

Gelatin-binding proteins (fibronectin and the 95 000-Mr protein [T. Vartio, T. Hovi & A. Vaheri (1982) J. Biol. Chem. 257, 8862-8866] were isolated by gelatin-agarose from the growth medium of cultured human monocyte/macrophages and the 95 000-Mr protein was further separated from fibronectin under nondenaturing conditions by preparative polyacrylamide gel electrophoresis. In the latter the proteins were eluted from the bottom of the tube gel into fractions which were then tested for ability to degrade native or heat-denatured type I collagen (gelatin). When solutions from fractions containing the 95 000-Mr protein were incubated with gelatin, degradation was revealed by analysis of the reaction mixtures in the sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Native type I collagen as well as native or heat-denatured fibronectin or other plasma proteins were unaffected when tested similarly. The degradation of gelatin was calcium-dependent and was inhibited by serum, sulfhydryl and metal-chelating reagents, but not with serine proteinase inhibitors. Gelatin was degraded optimally at pH 7-9 and at 41 degrees C and 37 degrees C and less effectively at 22 degrees C. Native type I collagen was degraded at 41 degrees C but not at 37 degrees C or 22 degrees C. The results show that cultured human macrophages secrete highly specific gelatin-degrading metal-proteinase activity which is associated with the 95 000-Mr gelatin-binding protein.