Purification and characterization of an arginine-specific carboxypeptidase from Mycoplasma salivarium.
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.
Shibata
K
K
Department of Oral Bacteriology, Nagasaki University School of Dentistry, Japan.
Watanabe
T
T
D001120:Arginine / Q000378:metabolism*; D002268:Carboxypeptidases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002855:Chromatography, Thin Layer; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D009174:Mycoplasma / Q000201:enzymology* / Q000648:ultrastructure; D017830:Octoxynol; D011092:Polyethylene Glycols; D013379:Substrate Specificity
D003902:Detergents; D011092:Polyethylene Glycols; D017830:Octoxynol; D001120:Arginine; D002268:Carboxypeptidases