Characterization of Extracellular Alkaline Proteases and Collagenase Induction in Vibrio alginolyticus

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Abstract
The number and approximate MW of extracellular alkaline proteases produced by V. alginolyticus were determined by gelatin-PAGE [polyacrylamide gel electrophoresis]. Three major bands of protease activity with apparent MW of .apprx. 28,000, 22,500 and 19,500 (proteases 1, 2 and 3, respectively) and 2 minor bands of protease activity with apparent MW of .apprx. 15,500 and 14,500 (proteases 4 and 5, respectively) were obtained after gelatin-PAGE. The activities of the 5 proteases were inhibited by serine protease inhibitors, but their activities were not affected by inhibitors of trypsin-like enzymes. Histidine, which inhibited V. alginolyticus collagenase, did not inhibit the activities of the alkaline serine proteases. The production of protease 1 was enhanced by histidine. Protease 1 production was also affected by temperature and production was depressed at 37.degree. C. Gelatin-PAGE of a commercial V. alginolyticus collagenase preparation revealed 4 bands of activity which were identified as collagenases with apparent MW of .apprx. 45,000, 38,500, 33,500 and 31,000. The collagenase preparation was contaminated with 2 serine proteases. The release of [3H]proline from collagen matrices produced by smooth muscle cells is a sensitive assay for bacterial collagenases and was used to show that V. alginolyticus produced a basal constitutive level of extracellular collagenase. The constitutive levels of collagenase were affected by aeration.