Purification and characterization of an aminopeptidase from Mycoplasma salivarium

Abstract

The aminopeptidase which had been shown to be present in Mycoplasma salivarium was found to be associated with the cell membranes of the organism. The enzyme was solubilized in water by papain digestion of the membranes pretreated with Triton X-100 and purified approximately 130-fold by ion-exchange chromatography on DEAE-Sephadex A-50, affinity chromatography on L-leucylglycine-AH-Sepharose 4B, and gel filtration on Sepharose CL-6B. The purified enzyme had a molecular mass of 397 kilodaltQns, estimated by gel filtration through Sepharose CL-6B, and gave two bands of activiy in analytical disc polyacrylamide gel electrophoresis: a dense, diffuse band and a less dense, narrow one, accounting for 90 and 5% of stained proteins in the gel, respectively. The purified protein revealed two bands with molecular masses of 50 and 46 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme catalyzed selectively the cleavage of the N-terminal arginine and leucine residues of peptides; had a p11 optimum at 8.5; and was inhibited remarkably by bestatin, o-phenanthroline, EDTA, and L-cysteine, but was activated nine-and twofold by MnCI2 and MgC92, respectively. The enzyme pretreated with MnCI2 had much higher maximum velocity (V.,,) for L-leucine-p-nitroanilide than the one not treated. That is, the Michaelis constant (K.) and Vmax values of the pretreated enzyme were 10.5 mM and 12.1 FM/min, respectively, whereas those of the untreated enzyme were 5.8 mM and 1.6 ,uM/min, respectively.Mycoplasma salivarium is a member of the oral microbial flora and preferentially inhabits the gingival sulcus (6, 11). The incidence (6) and number (23) of organisms in oral cavities increase significantly with the advancement of periodontal disease. Furthermore, significantly higher antibody responses occur in patients with periodontal disease than in healthy individuals (11). On the basis of these findings, it is of great interest to know whether M. salivarium plays an etiological role in periodontal disease.Recently, Nakamura and Slots (14) suggested that the aminopeptidase of Capnocytophaga species may be an important virulence factor in Capnocytophaga-associated periodontal disease, because aminopeptidases can be involved in bradykinin formation, degradation of collagen fragments, and other events of the inflammatory process.Aminopeptidase activity has been shown to occur in Ureaplasma urealyticum and some Mycoplasma species (22). M. salivarium also possesses the activity (19,24). The activity has been suggested to be attributable to the action of at least two distinct enzymes, leucine aminopeptidase (EC 3.4.11.1) and aminopeptidase B (EC 3.4.11.6), because the organism cleaves p-nitroanilides of L-arginine, L-leucine, L-lysine, and L-alanine (19, 24). Aminopeptidase B is known to convert kallidin to bradykinin (10), which causes vasodilation, increased permeability, accumulation of leucocytes, and pain. Therefore the aminopeptidase may be a factor in the virulence of M. salivarium.In this study we confirmed that...

Catabolism in Mollicutes

“…M . saliuarium membranes also contain aminopeptidase activity specific for N-terminal arginine and leucine residues (Shibata & Watanabe, 1987).…”

The N-Terminal Lipopeptide of a 44-kDa Membrane-Bound Lipoprotein ofMycoplasma salivariumIs Responsible for the Expression of Intercellular Adhesion Molecule-1 on the Cell Surface of Normal Human Gingival Fibroblasts

“…Cell membranes of M. salivarium were prepared according to the method described previously (16). The lipoproteins were extracted from the cell membranes by using n-octyl-␤-glucopyranoside (OG) according to the method of Mühlradt et al (5) modified slightly.…”

Two new aminopeptidases from Ochrobactrum anthropi active on D-alanyl-p-nitroanilide

“…D-Aminopeptidase assays and kinetic measurements. The enzyme activity on D-Ala-, L-Ala-and Gly-p-nitroanilides was measured in 100 mM Tris-HCl pH 8.0, at 30°C by monitoring the variation of absorbance at 405 nm (Dm= 11,500 M − 1 s − 1 ) [24]. Estimated errors on v 0 values were 9 5%.…”