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Aminopeptidase and caseinolytic activities of Mycoplasma salivarium
Abstract:
Aminopeptidase activity was demonstrated in Mycoplasma salivarium (ATCC 23064) cells disrupted by sonic vibrations and lyophilized (crude enzymes), and weak endopeptidase or carboxypeptidase activity was also suggested. The crude enzymes were suspended in 0.1 M borate buffer, pH 8.0, containing 0.5% (w/v) sodium deoxycholate, and then the suspensions were centrifuged at 100,000 g for 2 h. Thus separated, the supernatants were applied to a column of Sephacryl S-300. As a result, aminopeptidase activity was separated from caseinolytic activity, which had already been demonstrated in this organism. The aminopeptidase activity was inhibited by o-phenanthroline and stimulated by Mn2+, and the enzyme exhibited a strong affinity for leucine and arginine. On the other hand, the caseinolytic activity was inhibited considerably by o-phenanthroline and Ni2+ and slightly by diisopropyl fluorophosphate and Co2+. The caseinolytic activity was therefore believed to be due mainly to metalloproteinases and partly to serine proteinases.
Insights
This study identified aminopeptidase activity in Mycoplasma salivarium, distinct from its previously known caseinolytic enzymes. Further characterization revealed specific inhibitors and activators for these distinct enzymatic activities.
Area of Science:
- Microbiology
- Enzymology
- Biochemistry
Background:
- Mycoplasma salivarium possesses known caseinolytic activity.
- The presence and nature of other enzymatic activities in this organism remain less characterized.
Purpose of the Study:
- To investigate and characterize aminopeptidase, endopeptidase, and carboxypeptidase activities in Mycoplasma salivarium.
- To differentiate these activities from previously identified caseinolytic enzymes.
Main Methods:
- Disruption of Mycoplasma salivarium cells via sonication and lyophilization to obtain crude enzymes.
- Enzyme separation using Sephacryl S-300 chromatography after ultracentrifugation.
- Enzyme activity characterization through inhibition and stimulation assays with specific agents (o-phenanthroline, Mn2+, Ni2+, diisopropyl fluorophosphate, Co2+).
Main Results:
- Aminopeptidase activity was successfully isolated and demonstrated.
- Caseinolytic activity was separated from aminopeptidase activity.
- Aminopeptidase was inhibited by o-phenanthroline and stimulated by Mn2+, showing affinity for leucine and arginine.
- Caseinolytic activity was inhibited by o-phenanthroline, Ni2+, and diisopropyl fluorophosphate, suggesting metalloproteinase and serine proteinase involvement.
Conclusions:
- Mycoplasma salivarium possesses distinct aminopeptidase activity.
- The caseinolytic activity is likely mediated by metalloproteinases and serine proteinases.
- This research clarifies the enzymatic repertoire of Mycoplasma salivarium.