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Collagenase and protease activity in Bacteroides melaninogenicus Gisslow, Mary Therese

Abstract

A rapid sensitive collagenase assay has been developed using ¹⁴C-acetylated collagen as the substrate. Acid-soluble calfskin collagen was labelled with acetic-1-¹⁴C-anhydride at pH 8. The radioactively labelled collagen remained susceptible to Clostridium histolyticum collagenase but resistant to nonspecific proteolysis indicating that denaturation of the collagen had not occurred as a result of the acetylation procedure. The rate of release of ¹⁴C from labelled collagen by C. histolyticum collagenase was 14 proportional to enzyme and substrate concentrations. The results of the ¹⁴C assay correlated with those of other collagenase assays tested. Collagenolytic activity of Bacteroides melaninogenicus was examined both in vitro and the guinea pig model system using known collagenase-producing strains and recent isolates. In contrast to the reported results of previous workers, only two of the thirty B. melaninogenicus strains isolated were found to have collagenase activity. Collagenase was found in washed cells of positive strains of B. melaninogenicus as early as day one. No activity was ever observed in culture supernatants. Studies of material aspirated from guinea pigs infected with pathogenic B. melaninogenicus demonstrated the presence of collagenase in the infectious exudates. The enzyme was associated with the bacterial cells, was dependent on reducing conditions and was stimulated by a factor in filtered exudate. Partial characterization of cell-free protease activity in B. melaninogenicus has shown the protease to be dependent on reducing agents, requiring 5 x 10⁻³ M cysteine for activity. Other reducing agents were also effective in stimulating protease activity. The activity was stable at 4°C, resistant to autodigestion, and sensitive to EDTA at concentrations of 10⁻³ M and above. The pH optimum for activity against Azocoll was 8.0.

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