Proteolytic profile of Treponema vincentii ATCC 35580 with special reference to collagenolytic and arginine aminopeptidase activity

The proteolytic profile of Treponema vincentii ATCC 35580 was studied using PZ-PLGPA (phenylazobenzyloxycarbonyl-L-prolyl-L-leucylglycyl-L-prolyl-D-argi-nine; a substrate of bacterial collagenases) and amino acid 2-naphthylamides (2NA) as substrates. The cell extracts showed high activity toward PZ-PLGPA, Na -L-arginyl-2NA and N γ-L-glutamyl-2NA. Gel permeation chromatography revealed 2 major endopeptidases (I and II) hydrolyzing PZ-PLGPA, the molecular weights of which were 75,000 and 23,000, respectively. Enzyme I was stable enough for subsequent fast protein liquid chromatography on an anion exchange column. The enzyme had a broad pH optimum of 6.5 to 7.5 with PZ-PLGPA as substrate, hydrolyzed gelatin and was moderately inhibited by metal chelators, but was very sensitive to p -chloromercuribenzoic acid (PCMB). Enzyme II with a pH optimum of 7 to 8 was more labile, quite sensitive to PCMB and moderately inhibited by chelators. A high-molecular weight arginine aminopeptidase (mol. wt. > 200,000) was sensitive to PCMB and showed a value of 0.55 mM for K_m in the hydrolysis of Na -L-arginyl-2NA. The hydrolysis of PZ-PLGPA and gelatin suggests that this organism may contain collagenolytic proteinases. Because the insoluble proteinase substrate Azocoll was not hydrolyzed, these enzymes may be active on soluble collagenous substances only. T. vincentii ATCC 35580 typifies an organism rich in PZ-PLGPA-endopeptidase, arginine aminopeptidase and γ-glutamylpeptidase activity.