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Isolation of two trypsin-like proteases associated with the outer membranes of Bacteroides gingivalis Scott, Helen G.


Two proteases, P-1 and P-4, present in the outer membranes of Bacteroides gingivalis ATCC 33277, were isolated and partially purified by SDS-PAGE. The purification procedures resulted in a specific activity 45-fold (P-1) and 40-fold (P-4) greater than that of the crude fractions. Electrophoresis of proteolytically active P-1 produced three bands corresponding to molecular weights of 235, 220 and 200 kD whereas electrophoresis of proteolytically active P-4 produced one major band corresponding to 74 kD. The optimum temperature and pH for activity were determined using N-∂-benzoyl-D-arginine-p-nitroanilide as substrate. The proteases were most active at 37 °C and at pH values between 6.0 and 6.5. Both proteases required a reducing agent for activity and were inhibited by a variety of serine and thiol protease inhibitors. Arginine-containing peptides were readily hydrolyzed whereas the proteases were less active against glycine-containing peptides. The proteases hydrolyzed IgA, IgG, gelatin, azocoll and azoalbumin; acid-soluble collagen was not degraded. The results of this investigation suggest that these are trypsin-like proteases which have a thiol component as part of the active site.

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