35S-Sulfate摂取後の薄層クロマトグラフィーによるMycobacterium avium-Mycobacterium intracellulare Complex における2亜群の区別

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Strains of Mycobacterium aviurn-Mycobacterium intracellulare complex could be divided into two subgroups, one showing no detectable spots and another showing two radioactive spots at the Rf values 0.10-0. 14 and 0.32-0.36, by thin-layer chromatography (TLC) of a fraction extracted with acetone after incubation with 35S-sulfate. The strains tested were cultivated on Ogawa egg medium, harvested after incubation at 37 °C for 10 days, and washed twice with a 0.9% NaCl solution. One hundred mg (wet weight) of cells were weighed and added aseptically to the following reaction mixture: 3.0ml of M/15 phosphate buffer solution (pH 7.1) containing 0.1% sodium acetate and 5μCi/ml, 35S-sulfate. The reaction mixture was incubated at 37°C for 24 hours, and the cells were centrifuged. The cells were washed twice with 2.0ml of distilled water and extracted twice with 10% trichloroacetic acid (TCA) solution. The cells were then extracted twice with 2.0ml of petroleum ether, and the petroleumether extracts were discarded. The cells were extracted twice with 2.0ml of acetone, each time for 5 minutes. The acetone extracts were combined and dried by allowing to stand at 37°C. The dried material was dissolved in 0.1ml of acetone, and its 0.02ml sample was placed onto a Silica Gel Hlayer (20 by 20 cm; 0. 25 mm thick) with a micropipette. The sample was subjected to TLC using a solvent: n-propanol-n-butanol-water-ammonia (57: 20: 20: 3, in volume) (ascending system). The chromatogram was then scanned for the radioactivity by an automatic TLC scanner (Nihon-Musen Co., Tokyo) (Scanning speed, 300 mm/hour; chart speed, 150 mm/hour; range, 1, 000 counts perminute (cpm); slit, 30×6 mm; time constant, 30 seconds). The 35S-sulfate was a product of the Radio chemical Centre, Amersham, Buckinghamshire, England (Sodium sulfate (35S) injection, 0. 63 mCi/ml). The results are shown in Table 1 and Fig. 1. The radioactive spot that has appeared at the Rfvalue 0.95 is originally petroleum ether-soluble. The TLC of the concentrate of the petroleumether extracts showed only one spot at this Rf value and the peak was as high as more than 1, 000 cpm. The strains were divided into two subgroups by the absence or presence of some acetone-soluble radioactive compounds. The first subgroup did not show any radioactive spot and the strains showingserotypes 1, 2, 7 and 14 belonged to this group. The second subgroup consisted of the strains which showed the serotypes 9, 16, 18, and 20. Strains which had a M. scrofulaceum serotype 43 (Gause) also entered this group. The former showed no detectable, radioactive spots and the latter showed two radioactive spots at the Rf values 0.10-0.14 and 0.32-0.36.
日本結核病学会の論文

<SUP>35</SUP>S-Sulfate摂取後の薄層クロマトグラフィーによるMycobacterium avium-Mycobacterium intracellulare Complex における2亜群の区別

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