FEMSMicrobiologyLetters94 (1992)81-84 © 1992Federationof EuropeanMicrobiologicalSocieties0378-1097/92/$05.00 Publishedby Elsevier
FEMSLE 04928
The location of the arginine-specific carboxypeptidase in the membrane of Mycoplasma saliL,arium and its physiological functions Ken-ichiro Shibata and Tsuguo Watanabe
Deintrtmentof OralBacteriology,Hokkaido UnitcruditySchtudof Dentate', Hokkaido.Japan Receivedq March 1992 Revisionreceived31)March 1992 Accepted31 March 1992 Key words: Mycoplasma salit'arium; 2,4,6-Trinitrobenzenesuifonate; Carboxypeptidase 1. SUMMARY A non-penetrating probe, 2,4,6-trinitrobenzenesuifonate, inhibited the activity of the carboxypeptidase purified from the cell membranes of Mycoplasma sali~'arium and the same enzymatic activity of intact Mycoplasma cells as well. Growth of the organism in medium containing benzoylglycyI-L-arginine resulted in a higher pH and higher turbidity than growth in the same medium without this supplement It was concluded that the enzyme existed in the outer surface of the membrane of the cells and probably functioned to supply the organism with arginine as an energy source.
2. INTRODUCTION Mycoplasmas are the smallest known free-living and wail-less microorganisms. On the basis of
Correspondenceto: K. Shibata, Departmentof Oral Bacteriology, HokkaidoUniversitySchoolof Dentistry. Kita 13, Nishi 7, Kita-ku,Hokkaido060,Japan.
acid production from carbohydrate, the genus Mycoplasma can be subdivided into fermentative or non-fermentative species. Non-fermenters are welt known to catabolize L-arginine to ATP by the arginine dihydrolase pathway [1,2]. We have been interested in the arginine metabolism of non-fermentative mycoplasmas. Recently,. arginine-specific aminopeptidase and carboxypeptidase were isolated from the membrane of Mycoplasma salirarium, highly purified and characterized [3,4]. in addition, the carboxypeptidase was found in non-fermentative but not in fermentative mycoplasmas [5], Acholeplasma and Ureaplasma (unpublished data). Recently, we found that the inactivation of vascular permeability-increasing activity of bradykinin by mycoplasmas was due to the action of mycoplasmat arginine-specific carboxypeptidase and aminopeptidase [6]. That is, the carboxypeptidase can function as a kininase. Thus, it is also likely that the enzyme plays some etiological role in mycoplasmal infection. In this study, attempts were made to determine the location of the enzyme in the membrane of M. salit'arium and its physiological roles.
3. MATERIALS AND METHODS
3.I. Chemicals BenzoylglycyI-L-arginine (Bz-Gly-Arg) was purchased from the Protein Research Foundation, Osaka, Japan. 2,4,6-Trinitrobenzenesulfonate (TNBS) was purchased from Wako Pure Chemical Industries, Tokyo, Japan. All other chemicals were obtained from commercial sources and were of analytical or reagent grade. 3.2. Organisms and cultural conditions M. salivarium ATCC 23064 was grown in PPLO broth (Difco) supplemented with 1% (voi/vol) PPLO serum fraction (Difco), 1% (wt/vol) yeast extract (Difco), 1% (wt/vol) L-arginine hydrochloride, 0.002% (wt/vol) phenol red, 0.05% (wt/vol) thallium acetate, and penicillin G (1000 units/mI). Cultures were incubated at 37°C for 48 h, at which point the growth was in the mid-logarithmic phase, and centrifuged at 13000 x g for 15 rain. The cell pellets were washed three times with 0.1 M sodium phosphate buffer (pH 8.0) containing 1% (wt/vol) NaCI (SPS buffer) and resuspended in the same buffer to avoid cell lysis. 3.3. Purification of the carboxypeptidase The carboxypeptidase was purified according to the method described previously [3]. The activity was assayed by the colorimetric ninhydrin method by using Bz-Gly-Arg as a substrate [3].
120~M
"
~-43
3.4. Effects of TNBS on the puttied carboxypeptidase and the enzymatic activity of the bacterial cells A 0.4-ml volume of a solution of the carboxypeptidase (28 #g of protein/mi of SPS) or the organism cell suspension (1.4 mg of protein/ml of SPS) was incubated at 28°C with 2.6 ml of 0.1% (wt/vol) TNBS in SPS buffer. Aliquots (0.1 ml) were withdrawn at timed intervals and assayed for the carboxypeptidase activity.
3.5. Effects of L-arginine and Bz-G!y-Arg on the growth of M. salivarium Stock cultures of M. salivarium, grown in the medium without L-arginine (SF medium), were inoculated to the SF mediun: supplemented with 25 mM each of L-arginine (SF-Arg medium) or Bz-Gly-Arg (SF-Bz-Gly-Arg medium). Aliquots were removed at timed intervals and examined for viable counts (colony-forming units (cfu)/m[). In addition, the stock culture was diluted 1/400 with SF, SF-Arg or SF-Bz-Giy-Arg medium and each dilution was incubated after being transfered into 8 wells of a Linbro E.I.A. microtitration plate (Flow Laboratories, Mclean, VA). The growth in the medium containing phenol red was monitored at timed intervals by optical density at 540 nm (OD540), using a Titertek Multiskan MCC (Flow), which is known to correlate to pH change of the medium as described previously [7]. Then, the optical density obtained was changed to pH
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120.
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®
~
FEMSLE 04928
The location of the arginine-specific carboxypeptidase in the membrane of Mycoplasma saliL,arium and its physiological functions Ken-ichiro Shibata and Tsuguo Watanabe
Deintrtmentof OralBacteriology,Hokkaido UnitcruditySchtudof Dentate', Hokkaido.Japan Receivedq March 1992 Revisionreceived31)March 1992 Accepted31 March 1992 Key words: Mycoplasma salit'arium; 2,4,6-Trinitrobenzenesuifonate; Carboxypeptidase 1. SUMMARY A non-penetrating probe, 2,4,6-trinitrobenzenesuifonate, inhibited the activity of the carboxypeptidase purified from the cell membranes of Mycoplasma sali~'arium and the same enzymatic activity of intact Mycoplasma cells as well. Growth of the organism in medium containing benzoylglycyI-L-arginine resulted in a higher pH and higher turbidity than growth in the same medium without this supplement It was concluded that the enzyme existed in the outer surface of the membrane of the cells and probably functioned to supply the organism with arginine as an energy source.
2. INTRODUCTION Mycoplasmas are the smallest known free-living and wail-less microorganisms. On the basis of
Correspondenceto: K. Shibata, Departmentof Oral Bacteriology, HokkaidoUniversitySchoolof Dentistry. Kita 13, Nishi 7, Kita-ku,Hokkaido060,Japan.
acid production from carbohydrate, the genus Mycoplasma can be subdivided into fermentative or non-fermentative species. Non-fermenters are welt known to catabolize L-arginine to ATP by the arginine dihydrolase pathway [1,2]. We have been interested in the arginine metabolism of non-fermentative mycoplasmas. Recently,. arginine-specific aminopeptidase and carboxypeptidase were isolated from the membrane of Mycoplasma salirarium, highly purified and characterized [3,4]. in addition, the carboxypeptidase was found in non-fermentative but not in fermentative mycoplasmas [5], Acholeplasma and Ureaplasma (unpublished data). Recently, we found that the inactivation of vascular permeability-increasing activity of bradykinin by mycoplasmas was due to the action of mycoplasmat arginine-specific carboxypeptidase and aminopeptidase [6]. That is, the carboxypeptidase can function as a kininase. Thus, it is also likely that the enzyme plays some etiological role in mycoplasmal infection. In this study, attempts were made to determine the location of the enzyme in the membrane of M. salit'arium and its physiological roles.
3. MATERIALS AND METHODS
3.I. Chemicals BenzoylglycyI-L-arginine (Bz-Gly-Arg) was purchased from the Protein Research Foundation, Osaka, Japan. 2,4,6-Trinitrobenzenesulfonate (TNBS) was purchased from Wako Pure Chemical Industries, Tokyo, Japan. All other chemicals were obtained from commercial sources and were of analytical or reagent grade. 3.2. Organisms and cultural conditions M. salivarium ATCC 23064 was grown in PPLO broth (Difco) supplemented with 1% (voi/vol) PPLO serum fraction (Difco), 1% (wt/vol) yeast extract (Difco), 1% (wt/vol) L-arginine hydrochloride, 0.002% (wt/vol) phenol red, 0.05% (wt/vol) thallium acetate, and penicillin G (1000 units/mI). Cultures were incubated at 37°C for 48 h, at which point the growth was in the mid-logarithmic phase, and centrifuged at 13000 x g for 15 rain. The cell pellets were washed three times with 0.1 M sodium phosphate buffer (pH 8.0) containing 1% (wt/vol) NaCI (SPS buffer) and resuspended in the same buffer to avoid cell lysis. 3.3. Purification of the carboxypeptidase The carboxypeptidase was purified according to the method described previously [3]. The activity was assayed by the colorimetric ninhydrin method by using Bz-Gly-Arg as a substrate [3].
120~M
"
~-43
3.4. Effects of TNBS on the puttied carboxypeptidase and the enzymatic activity of the bacterial cells A 0.4-ml volume of a solution of the carboxypeptidase (28 #g of protein/mi of SPS) or the organism cell suspension (1.4 mg of protein/ml of SPS) was incubated at 28°C with 2.6 ml of 0.1% (wt/vol) TNBS in SPS buffer. Aliquots (0.1 ml) were withdrawn at timed intervals and assayed for the carboxypeptidase activity.
3.5. Effects of L-arginine and Bz-G!y-Arg on the growth of M. salivarium Stock cultures of M. salivarium, grown in the medium without L-arginine (SF medium), were inoculated to the SF mediun: supplemented with 25 mM each of L-arginine (SF-Arg medium) or Bz-Gly-Arg (SF-Bz-Gly-Arg medium). Aliquots were removed at timed intervals and examined for viable counts (colony-forming units (cfu)/m[). In addition, the stock culture was diluted 1/400 with SF, SF-Arg or SF-Bz-Giy-Arg medium and each dilution was incubated after being transfered into 8 wells of a Linbro E.I.A. microtitration plate (Flow Laboratories, Mclean, VA). The growth in the medium containing phenol red was monitored at timed intervals by optical density at 540 nm (OD540), using a Titertek Multiskan MCC (Flow), which is known to correlate to pH change of the medium as described previously [7]. Then, the optical density obtained was changed to pH
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120.
E'
®
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![[Significance of Mycoplasma orale and Mycoplasma salivarium in the respiratory tract. 2. Role of Mycoplasma orale and Mycoplasma salivarium in normal individuals and patients with respiratory diseases].](/assets/img/hold.png)
