Key words: human mycoplasmas/aminopeptidase activity/mncl2/l-leucine p-nitroanilide

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1 Jpn. J. Oral Biol., 25: , Aminopeptidase activities of human mycoplasmas Ken-ichiro Shibata, Masayoshi Totsuka and Tsuguo Watanabe Department of Oral Bacteriology, Nagasaki University School of Dentistry, 7-1 Sakamoto-machi, Nagasaki 852 (Chief: Prof. Tsuguo Watanabe) (Accepted for publication: September 19, 1983) Key words: human mycoplasmas/aminopeptidase activity/mncl2/l-leucine p-nitroanilide Abstract: Aminopeptidase activity of Mycoplasma salivarium, measured by using L-leucine p-nitroanilide (LeuNA) as a substrate, was enhanced remarkably by 1 mm MnCl2 and slightly by 1 mm MgCl2,b ut not at all by 1 mm CaCl2. In addition, consistency of preincubation time of the crude enzymes (disrupted M. salivarium cells) with MnCl2 was demonstrated to be essential for assaying the activity quantitatively. The crude enzymes hydrolyzed LeuNA, ArgNA, LysNA and AlaNA, in a decreasing order, but not GlyNA and ProNA. Thus, LeuNA was shown to be the best of all tested substrates for activity assay. M. orale ATCC 15539, M. buccale IID 802, M. faucium IID 996, M. hominis IID 801, M. fermentans IID 812, M. pneumoniae IID 815 and IID 817 were also found to possess aminopeptidase activity. M. salivarium ATCC 23064, M. orale ATCC 15539, M. buccale IID 802, M. faucium IID 996, M. hominis IID 801, M. fermentans IID 812, M. pneumonia IID 815 æ Ñ IID 817 ð

3 Fig. 3 Effects of preincubation time of crude enzymes with MnCl2 on aminopeptidase activity. Crude enzymes were incubated with MnCl2 before the addition of LeuNA to the reaction mixtures. Fig. 1 Effects of some metal ions on aminopeptidase activity of M. salivarium. The activity was assayed at 37 Ž and Table 1 The hydrolysis of p-nitroanilide derivatives of various amino acids ph 8.0 in the presence of 1 mm MnCl2 ( ), MgCl2 ( ), CaCl2 ( ) or neither ( œ) of them, and Leu- NA was used as a substrate. Fig. 2 Aminopeptidase activity of M. salivarium. Protein contents of crude enzymes used were 6.0 ( œ), 3.0 ( ), and 1.5mg/m/ ( ). The activity was measured in the presence of 1 mm MnCl2, and LeuNA was used as a substrate.

4 Table 2 Aminopeptidase activities of human mycoplasmas

1) Watanabe, T., Mishima, K. and Horikawa, T.: Proteolytic activities of human mycoplasmas. Jpn. J. Microbiol. 17: 151-153, 1973. 2) Watanabe, T.

5 1) Watanabe, T., Mishima, K. and Horikawa, T.: Proteolytic activities of human mycoplasmas. Jpn. J. Microbiol. 17: , ) Watanabe, T.: Proteolytic activity of Mycoplasma salivarium and Mycoplasma orale 1. Med. Microbiol. Immunol. 161: , ) Pfleiderer, G.: Particle-bound aminopeptidase from pig kidney.in: Methods in Enzymology (G. E. Perlmann and L. Lorand). 19, p , Academic Press, New York, ) Watanabe, T., Totsuka, M. and Shibata, K.: Further studies on proteolytic activities of Mycoplasma salivarium. J. Dent. Res. 61: 600, ) Lowry, 0. H., Rosenbrough, N. J., Farr, A. L. and Randal, R. J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: , ) Himmelhoch, S. R.: Leucine aminopeptidase from swine kidney. In: Methods in Enzymology (G. E. Perlmann and L. Lorand). 19, p , Academic Press, New York, ) Hopsu, V. K., Makinen, K. K. and Glenner, G. G.: Purification of a mammalian peptidase selective for N-terminal arginine and lysine residues Aminopeptidase B. Arch. Biochem. Biophys. 114: , ) Ball, H. J., Neil, S. D. and Reid, L. R.: Use of arginine aminopeptidase activity in characterization of arginine-utilizing mycoplasmas. J. Clin. Microbiol. 15: 28-34, ) Pecht, M., Giberman, E., Keysary, A., Yariv, J. and Katachalski, E..: Hydrolysis of alanine oligopeptides by an enzyme located in the membrane of Mycoplasma laidlawii. Biochim. Biophys. Acta 290: , ) Vinther, O. and Black, F. T.: Aminopeptidase activity of Ureaplasma urealyticum Acta Path. Microbiol. Scand. Sect. B. 82: , 1974.

TRANSPORT OF AMINO ACIDS IN INTACT 3T3 AND SV3T3 CELLS. Binding Activity for Leucine in Membrane Preparations of Ehrlich Ascites Tumor Cells

Journal of Supramolecular Structure 4:441 (401)-447 (407) (1976) TRANSPORT OF AMINO ACIDS IN INTACT 3T3 AND SV3T3 CELLS. Binding Activity for Leucine in Membrane Preparations of Ehrlich Ascites Tumor Cells