Purification of a mammalian peptidase selective for N-terminal arginine and lysine residues: Aminopeptidase B

Hopsu, V. K.; Mäkinen, Kauko K.; Glenner, George G.

doi:10.1016/0003-9861(66)90380-8

Cited by 127 publications

(23 citation statements)

References 10 publications

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“…L-Leucylglycine is known as a good substrate for leucine aminopeptidase, but not for aminopeptidase B (7,8). Therefore L-leucylglycine was coupled to AH-Sepharose from the column at 0.4 M NaCl (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In reality, our previous study (20) reveals that the SDS-solubilized enzyme appears at the void volume of a Sephacryl S-300 column, whereas the enzyme obtained by papain digestion of the membranes pretreated with Triton X-100 appears behind the void volume, indicating that the molecular mass of the former is greater than that of the latter. Aminopeptidase B selectively cleaves the N-terminal arginine, but not leucine, residue of peptides, whereas leucine aminopeptidase cleaves the N-terminal leucine, but not arginine (7,8). Thus it seems that an aminopeptidase such as aminopeptidase My, with a strong affinity for two amino acids with quite different characteristics, arginine and leucine, has never been described.…”

Section: Resultsmentioning

confidence: 99%

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Purification and characterization of an aminopeptidase from Mycoplasma salivarium

Shibata¹,

Watanabe²

1987

J Bacteriol

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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...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Purification and characterization of an aminopeptidase from Mycoplasma salivarium

Shibata¹,

Watanabe²

1987

J Bacteriol

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“…We used a modification of the method of Hopus et al 13) to measure aminopeptidase B activity. The incubation mixture consisted of 0.1 ml of 3 MM L-arginine-~-naphthylamide (Sigma Chemical Co.), 0.7 ml of HANKS' balanced salt solution, and 10µl of water, with or without the inhibitor.…”

Section: Assay Of Aminopeptidase B Inhibitionmentioning

confidence: 99%

OF4949, new inhibitors of aminopeptidase B. I Taxonomy, fermentation, isolation and characterization.

et al. 1986

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“…The hydrolysis of AA-NA was measured by the method of Höpsu et al (6) and that of Leu-Nan by the method of Tuppy at al. (7).…”

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confidence: 99%

Purification and partial characterization of Phaseolus vulgaris seed aminopeptidase

Abdala

Takeda

Freitas

et al. 1999

Braz J Med Biol Res

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The aminopeptidase activity of Phaseolus vulgaris seeds was measured using L-Leu-p-nitroanilide and the L-aminoacyl-ß-naphthylamides of Leu, Ala, Arg and Met. A single peak of aminopeptidase activity on Leu-ß-naphthylamide was eluted at 750 µS after gradient elution chromatography on DEAE-cellulose of the supernatant of a crude seed extract. The effluent containing enzyme activity was applied to a Superdex 200 column and only one peak of aminopeptidase activity was obtained. SDS-polyacrylamide gel electrophoresis (10%) presented only one protein band with molecular mass of 31 kDa under reducing and nonreducing conditions. The aminopeptidase has an optimum pH of 7.0 for activity on all substrates tested and the highest Vmax/K M ratio for L-Leu-ß-naphthylamide. The enzyme activity was increased 40% by 0.15 M NaCl, inhibited 94% by 2.0 mM Zn 2+ , inhibited 91% by sodium p-hydroxymercuribenzoate and inhibited 45% by 0.7 mM o-phenanthroline and 30 µM EDTA. Mercaptoethanol (3.3 mM), dithioerythritol (1.7 mM), Ala, Arg, Leu and Met (70 µM), p-nitroaniline (0.25 mM) and ß-naphthylamine (0.53 mM) had no effect on enzyme activity when assayed with 0.56 mM of substrate. Bestatin (20 µM) inhibited 18% the enzyme activity. The aminopeptidase activity in the seeds decayed 50% after two months when stored at 4 o C and room temperature. The enzyme is leucyl aminopeptidase metal-and thiol group-dependent.

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Purification of a mammalian peptidase selective for N-terminal arginine and lysine residues: Aminopeptidase B

Cited by 127 publications

References 10 publications

Purification and characterization of an aminopeptidase from Mycoplasma salivarium

Purification and characterization of an aminopeptidase from Mycoplasma salivarium

OF4949, new inhibitors of aminopeptidase B. I Taxonomy, fermentation, isolation and characterization.

Purification and partial characterization of Phaseolus vulgaris seed aminopeptidase

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