Related Experiment Video
Updated: Aug 12, 2026
Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamAΔ41
Published on: March 25, 2010
Purification and characterization of an arginine-specific carboxypeptidase from Mycoplasma salivarium
1Department of Oral Bacteriology, Nagasaki University School of Dentistry, Japan.
Abstract:
The carboxypeptidase which had been shown to be present exclusively in nonfermentative mycoplasmas was found to be associated with cell membranes of Mycoplasma salivarium. The enzyme was released from the membranes with Triton X-100 and purified by ion-exchange chromatography on DEAE-Sephacel, affinity chromatography on arginine-Sepharose 4B, and chromatofocusing. The purified enzyme had a molecular mass of 218 kilodaltons, as estimated by gel filtration through Sepharose CL-6B, and yielded one band of activity in analytical disc-polyacrylamide gel electrophoresis performed in the presence of 0.5% (wt/vol) Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme treated in the presence or absence of 2-mercaptoethanol revealed one band with a molecular mass of 87 kilodaltons. The enzyme catalyzed selectively the cleavage of the C-terminal arginine residue of peptides such as N-benzoylglycyl-L-arginine, tuftsin, and bradykinin and was inhibited considerably by o-phenanthroline and EDTA but only slightly by NiCl2. The inhibition of the enzyme by EDTA was fully reversed by the addition of ZnCl2, whereas the addition of CoCl2 activated the enzyme.
Insights
A novel carboxypeptidase from Mycoplasma salivarium cell membranes was purified. This enzyme selectively cleaves C-terminal arginine, showing unique properties and potential roles in nonfermentative mycoplasmas.
Area of Science:
- Microbiology
- Enzymology
- Molecular Biology
Background:
- Carboxypeptidases are crucial enzymes involved in protein processing.
- Previous studies indicated carboxypeptidase presence exclusively in nonfermentative mycoplasmas.
- The specific localization and properties of this enzyme in Mycoplasma salivarium were unknown.
Purpose of the Study:
- To investigate the presence and localization of carboxypeptidase in Mycoplasma salivarium.
- To purify and characterize the Mycoplasma salivarium carboxypeptidase.
- To determine the enzyme's substrate specificity and cofactor requirements.
Main Methods:
- Enzyme extraction from cell membranes using Triton X-100.
- Purification via ion-exchange chromatography, affinity chromatography (arginine-Sepharose 4B), and chromatofocusing.
- Molecular mass determination using gel filtration (Sepharose CL-6B) and SDS-PAGE.
- Enzyme activity assays with various peptide substrates.
- Inhibition studies with metal chelators (o-phenanthroline, EDTA) and metal ions (NiCl2, ZnCl2, CoCl2).
Main Results:
- The carboxypeptidase was successfully isolated and purified from Mycoplasma salivarium cell membranes.
- The purified enzyme exhibited a native molecular mass of 218 kDa and a subunit mass of 87 kDa.
- The enzyme selectively cleaved C-terminal arginine from peptides like N-benzoylglycyl-L-arginine, tuftsin, and bradykinin.
- It was significantly inhibited by o-phenanthroline and EDTA, with ZnCl2 reversing EDTA inhibition.
- NiCl2 showed slight inhibition, while CoCl2 activated the enzyme.
Conclusions:
- A novel, membrane-associated carboxypeptidase was purified from Mycoplasma salivarium.
- The enzyme is a metalloenzyme, likely zinc-dependent, with a preference for cleaving C-terminal arginine.
- Its unique characteristics suggest a specific role in the physiology of nonfermentative mycoplasmas.
More Related Videos
08:55Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis
Published on: October 28, 2016
09:47The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes
Published on: May 25, 2018