BCG再接種モルモットの肺の細胞顆粒分画からの抗結核菌物質の分離とその性状-1-酸抽出・アルコール沈澱法による調製

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Differential centrifugation of a sucrose homogenate of BCG-revaccinated guinea pig lungs (87.5 g )was conducted to obtain sediments at 1, 000, 3, 900, 8, 400, 15, 000 and 25, 0 00 g fo r 20minutes. Acid phosphatase test indicated that the sediments at 15, 000 g and 25, 000 g were rich in lysosomal granules. Each sediment was resuspended into 20 ml of O.25 M sucrose solution and subjected to extraction with 180 ml of O.2 N H2SO4 at 4°C for 40 minutes. The supernatant of the mixture at 15, 000 g for 20 minutes were added with absolute alcohol to 20% and left to stand at-4°C for 3 hours. The resulting precipitate was collected by centrifugation. The precipitate was dissolved in distilled water and dialysed. The preparation mentioned above was examined for bactericidal activity against M. tuberculosis and B. abortus, incubating 1.8 ml of the diluted sample with O.2 ml of the bacillary suspension of 1 mg per ml at pH 5.0 and 37°C. After 4 hour or 7 hour incubation respectively, serial ten-fold dilutions of the mixture were made, each of which was inoculated onto glycerolegg slants (Ogawa type) for enumeration of viable counts. Table 1 summarizes the yield of each preparat i o n and the bactericidal activity. It is evident from this table that the bactericidal components were present preferentially in so-called "light-mitochondrial fraction" containing lysosomes in higher proportion. Table 2 shows that the preparation was active only in acidic environm ent and at 37°C. It was inactive at neutral pH and 4°C. Besides, the activity was masked or neutralized in the presence of serum. The preparation from 25, 000g sediment (FrIV) was further fractionated by gel-filtration on Sephadex G-150 column with respect to bactericidal activity. There was an indication that the activity was higher in the fraction of larger moleculer weight (Fig.1). Organic solvents were then employed as another procedure for purification (Fig.3 and Table 3). When FrIV was added with acetone to final 90%, the active moiety was separated as acetone-soluble material. After removal of acetone by evaporation, the resulting precipitate and supernatant were separated by centrifugation. The supernatant was highly active at acidic pH, but quite inactive at pH 7.4. The precipitate was extracted with ether, producing an inactive residue. The ether-extracted material was active at both pH 5.0 and 7.4 unlike the starting material. On the other hand, Frill was extracted only with ether and the aquesous phase was subjected to gel-filtration on Sephadex G-50 column (Fig.4 and 5). Two protein peaks were obtained, one of which having the larger molecular weight was of the higher mycobactericidal activity. All the active preparations were stable against heating at 100°C for 5 minutes.
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BCG再接種モルモットの肺の細胞顆粒分画からの抗結核菌物質の分離とその性状-1-酸抽出・アルコール沈澱法による調製

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