AMINOPEPTIDASE ACTIVITY OF UREAPLASMA UREALYTICUM

Acta path. microbiol. scand. Sect. B, 82: 917-918, 1974

AMINOPEPTIDASE ACTIVITY O F UREAPLASMA U R E A L Y T I C U M

0.Vinther and F . T . Black

Proteolytic activity is a well-known property of some mycoplasma species (1, 5, 7, 8). In particular, aminopeptidases have been detected in the membranes of Acholeplasma laidlawii (4, 10) and Mycoplasma fermentans (9). In one case, weak carboxypeptidase activity of A . laidlawii was also noted (4). The human T mycoplasmas, being distinguished from other members of the order Mycoptasmatales primarily by possessing urease activity, have recently been assigned to a separate genus Ureaplasma of the family Mycoplasmataceae (11). Only one species: Ureaplasma urealyticum is presently recognized. In the present study, the action of U. urealyticum cells on a number of synthetic peptidase substrates is reported. By way of comparison, one Acholeplasma species and three Mycoplasma species were included in the investigation.

Material and Methods The following organisms were tested for peptidase activity: Eight strains representing eight serotypes ( 3 ) of U . urealyticum; M . hominis (PG21); M . ferrnentans (PG18); M . pneumoniae (Mac), and A . laidlawii (PG8). U. urealyticum was grown in a medium (S) consisting of Tryptic soy broth (Difco), 2.3 per cent w/v; horse serum, 16.5 per cent v/v; yeast extract corresponding to 1.9 per cent w/v yeast, and sodium penicillin, 1500 i.u./ml. The p H of the final medium was 6.0. The Mycoplasma and Acholeplasma species were cultivated in a modified Hayflick's medium (B) as described previously (6). Determinations of the number of viable organisms in cultures, counted as colony-forming units per ml, were performed on solid S and B media. Peptides and peptide derivatives used as substrates were: Ala,, n = 2-5; Lys-Ala; Ala-Lys; N-acetyl-tri-alanine ; N-acetyl-tetra-alanine; leucine amide; hippuryl-arginine, and hippuryl-phenylalaReceived 9.x.74 Accepted 9 x 7 4 Institute of Medical Microbiology, Bartholin Building, University of Aarhus, DK-8000 Aarhus C, Denmark.

nine, all amino acids being of L configuration. The peptides and derivatives were purchased from Sigma Chemical Company, St. Louis, Mo., U.S.A. Organisms to be tested for peptidase activity were grown in 50 ml aliquots of media and harvested at the end of the log growth phase by centrifugation for one hour at 43500 g in a Sorvall RCZb centrifuge. The cell deposits were washed twice in 10 ml phosphate buffered saline, twice in 10 ml tris buffer, 0.05 M, pH 8.0, and finally resuspended in 50 pl of the latter buffer. Uninoculated S and B media, treated in the same way, served as references. Hydrolysis of substrates was assayed in a mixture containing 4-9 x 108 cells of U. urealyticum or 9 x 107-7 x 108 cells of Mycoplasma and Acholeplasma species, 233 nanomoles peptide or derivative, and 250 nanomoles CaCI,, in a final volume of 70 pl tris buffer, 0.05 M, pH 8.0. After incubation for 2 hours a t 37" C the mixture was centrifuged to remove the cells and 2 pl of the supernatant was spotted on a thin-layer plate for chromatographic analysis of reaction products. Precoated cellulose plates (Merck) or silica plates (Gelman Instrument Company) were used. Separations were achieved by developing in the solvent system n-butanol-acetone-aceticacid-water, 35:35:10:20 v/v/v/v. Leucine and leucine amide were separated as their FLURAMTM derivatives on 5 x 5 cm polyamide sheets (BDH Chemicals Ltd.) by development for 6 min in benzene-acetic acid, 9: 1 v/v. Fluorescence labelling with FLURAMTM was performed according to the manufactures suggestions. ( Hoffman-LaRoche Inc., New Jersey, U.S.A.).

Results and Discussion The results of peptidase tests are summarized in Table 1. All eight serotypes of U. urealyticum released alanine from Ala,. Serotypes V I I and V I I I were chosen for further experiments and were shown to release free amino acids from all the unsubstituted peptides investigated and from leucine amide. No ninhydrin positive spots could be detected after incubation with N-blocked peptides.

917

TABLE I . Release of Amino Acids from Synthetic Peptidase Substrates by Mycoplasma Species

Srr hst rates

Organisms

U . urealyticum A . laidlawii M . pneumoniae M . hominis M . fermentans (

+ )-amino acid

+ + + + +

+

+

+ -

-

t

+

+

-

-

0

0

+

+

0 0 0

TR 0 0

+

+

0

0

+

+

0 0 0

0 0 0

released, ( 0 )-amino acid not released, ( )-not tested, ('I'R) -trace

T h e results clearly indicate that U . urealyticum possesses aminopeptidase activity whereas carboxypeptidase activity is probably not present. Endopeptidase activity against N-acetyl-tri-alanine and N-acetyl-tetra-alanine may also be excluded since such activity would have resulted in release of alanine from liberated Ala, or Ala,,. T h e discovery of aminopeptidase activity in U. urealyticum adds to the limited knowledge of the biochemical activity of these organisms ( 2 ) . I t will also be seen from Table 1 that the three Mycoplasma species all possess aminopeptidase activity. I n addition, weak carhoxypeptidase or endopeptidase activity may he present in M . pneumoniae. Both aminopeptidase and carboxypeptidase activities were demonstrated in A . laidlawii in agreement with results of Choules & Gray ( 4 ) . Pecht et al. ( l o ) , on the other hand, were unable to detect release of free amino acids from a number of N-hlocked di- and tri-peptides not comprising, however, blocked alanine homopeptides. Studies are in progress in this laboratory to evaluate the possible taxonomic significance of the

918

+

demonstrated peptidase activities and to further characterize the peptidases in mycoplasma species.

References: I . Aluotto, B. H., Wittler, K. C . , Williams, C . 0 . & Faber, I . E.: Int. J. Syst. BacBlack, F. 7 . : Ann. teriol. 20: 35 -58, 1970.-2. N.Y. Acad. Sci. 225: 131--143, 1973.---3. Black, F. 7'.: Appl. Microbiol. 25: 528-533, 1973.-.-4. Choules, G . L . & G r a y , W . R . : Biochem. Biophys. Res. Commun. 45: 849--855, 197 I . ~ - 5 .Crekaloruski, J . W . , Hall, D . A . & Woolcock, P . R . : J. Gen. Microbiol. 75; 125--133, 1973.--6. Erna, H . & Stipkovits, I-.: Acta vet. scand. 14: 436-449, 1973.-7. Erno, H . & Stipkooits, L.: Acta vet. Freundt, E . A , : scand. 1 4 : 450-463, 1973-8. Thesis, 1958.--9. Pecht, M.: Israel J. Chem. 8: 187p., 1970.-10. Pecht, M., Giberman, E., K e y sary, A , , YariL', /. & Katchalski, E.: Biochim. BioShepard, M . phys. Acta 290: 267-273, 1972.-11. C . , Lunceford, C . D . , Ford, D . K . , Pucell, R . H . , l'aylor-Robinson, D., Razin, S . & Black, F . T.: Int. J. Syst. Bacteriol. 2 4 : 160-171, 1974.