Mycobacterium, RhodococcusおよびNocardiaによる2, 6-Diamino (G-3H) Pimelic Acid Dihydrochloride取り込みの差異

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Diaminopimelic acid (DPA) is a component of cell wall of Mycobacterium, Rhodococcus, and Nocardia. A comparative study of the uptake of radioactive DPA has revealed that these three genera have different patterns of the uptake of this compound. Test organisms were cultivated on Ogawa egg medium at 37°C for 5 days. The bacteria grown were harvested and washed three times with a 0.9% NaCl solution. The bacteria were weighed asep tically, and 100mg, wet weight, of bacteria were suspended in 4.0ml of a M/15 phosphate buffer solu tion (pH 7.1) containing sodium acetate (2.5μg/ml) and 2, 6-diamino (G-3H) pimelic acid dihydrochloride (25μCi/ml). The radioactive DPA (3H-DPA) was a product of the Radiochemical Center, Amersham, England (specific activity, 2.6Ci/m mol; batch 40). The reaction mixture was incubated at 37°C for 24 hours. The mixture was centrifuged, and supernatant was discarded. The bacteria were washed three times with 5ml of distilled water, and fractionated according to the procedure of Schneider (J. Biol. Chem. 161: 293, 1945). The bacteria were first extracted twice with 2ml of a 10% trichloroacetic acid (TCA) solution (Fraction I), and twice with 2ml of ethanol and then once with 3ml of a boiling ethanol-diethyl ether (1: 1) mixture for 5 minutes (Fraction II), and extracted twice with 2ml of 5% TCA solution at 90°C for 15 minutes (Fraction III). The residue was dissolved in 2ml of a 1% NaOH solution by heating at 100°C for 5 minutes (Fraction IV). A 0.5ml sample of each fraction was added to 5ml (Fractions I to III) or 10ml (Fraction IV) of a scintillator solvent, and the radioactivity was estimated by a liquid scintillation counter (Aloka LSC 651, Nihon Musen Co., Tokyo). The scintillator solvent used was composed of the following: Toluene, 1, 000ml; 2, 5-diphenyloxazole, 4g; 1, 4-bis [2-(5phenyloxazolyl)] -benzene, 100mg (TDP-1-SL). The radioactivity was expressed as disintegrations per minute (dpm). Total radioactivity in a whole fraction was calculated by multiplying the radioactivity estimated by the volume. The radioactivity from 3H-DPA was incorporated first principally into the Fraction II, which contains lipids, and then moved to the Fraction IV, which contains proteins (Table 1). In this study, the radioactivity in various organisms were compared at a stage, at which the majority of the radio activity was present in the Fraction II (Table 2). Comparisons of the radioactivity in Fraction II of various organisms are shown in Table 3. Rapid ly growing mycobacteria showed a large amount of the incorporation (60, 650 to 1, 217, 900dpm), whereas rhodococci and nocardiae showed a small amount of it (2, 648 to 44, 570). An intermediate between rapidly and slowly growing mycobacteria, M. flavescens, showed a radioactivity of in between these above two groups. It is considered that the uptake of the 3H-DPA is influenced by the growth rate, as DPA is a cell wall component. Comparison of the uptake would be meaningful, only when test organisms have the same growth rate. Rapidly growing mycobacteria, rhodococci, and nocardiae grow abundantly after incubation for 3 days, and are considered to have almost the same growth rate. Difference of the uptake of 3H-DPA between the rapidly growing mycobacteria, and the rhodococci and nocardiae would be considered as some difference of cell wall formation of these organisms. In summary, rapidly growing mycobacteria took up more amounts of 2, 6-diamino (G-3H) pimelic acid dihydrochloride than did rhodococci and nocardiae. No marked difference was observed between the latter two.
日本結核病学会の論文

<I>Mycobacterium, Rhodococcus</I>および<I>Nocardia</I>による2, 6-Diamino (G-<SUP>3</SUP>H) Pimelic Acid Dihydrochloride取り込みの差異

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