Characterization and serum inhibition of neutral collagenase from cultured dog gingival tissue

Abstract

1. 1. Explants of dog gingiva, maintained in culture for 9 days in the absence of serum, released a collagenase (EC 3.4.24.3) into the medium. The yield of active enzyme reached a maximum after 5–8 days with concomitant release of collagen degradation products from the explants. 2. 2. The enzyme attacked undenatured collagen in solution at 25°C resulting in a 58% loss of specific viscosity and producing the two characteristic products TC A ( 3 4 ) and TC B ( 1 4 ) . Electron microscopy of segment-long-spacing crystallites of these reaction products showed the cleavage locus of the collagen molecule at interband 40. 3. 3. Optimal enzyme activity was observed over the pH range 7.5–8.5 and a molecular weight of approximately 35 000 was derived from gel filtration studies. EDTA, 1,10-phenanthroline, cysteine and dithiothreitol all inhibited collagenase activity. Proteoglycan derived from porcine and human cartilage did not inhibit the enzyme. 4. 4. The enzyme was inhibited by the dog serum proteins α 2-macroglobulin and a smaller component of molecular weight approximately 40 000. This smaller component was purified by column chromatography utilising Sephadex G-200, DEAE A-50, and G-100 (superfine grade). Agarose electrophoresis of the purified component showed it to represent a β-serum protein. α 1-Antitrypsin did not inhibit the enzyme. 5. 5. The physiological importance of the natural serum inhibitors and gingival collagenase are discussed in relation to latent enzyme and periodontal disease.