家蚕組織に於ける硝酸還元作用に就いて
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The estimation of the nitrate reductase of the silk-worm was carried out as previously reported (Yamafuji, Omura & Sakamoto, Enzymologia, 15, 210, 1952). 5cc of enzyme solution was mixed thoroughly with 5cc of 2x 10^-2M NaNO3 solution in test tube, then was added 1 cc of toluene to cover the surface of reaction mixture. Tubes were allowed to stand in incubator at 40° for 22 hrs. After the reaction had been over the tubes were heated at 100° for 5 min. to stop the reaction, cooled rapidly with flowing water, restored to original volume 10 cc. A few drops of Pb-acetate solution, if water was used as solvent, or 0.2cc of conc. acetic acid in case of phosphate buffer, were added and centrifuged. The clear supernatant liquid obtained was diluted so as to have the final concentration of NO2 formed about 2×10^-5M, added Griess's reagent and the intensity of the red colour developed was compared with that of the standard NO2 solution with concentration of 2×10^-5 M by the Dubosque's colourimeter as usual. As the enzyme solution for this study, the following preparations were tested; 10% (w/v) suspension of whole body of worm which had been prepared by grinding the larvae with small amount of quartz sand and suspended in water or M/10 phosphate buffer, pH 6.4; filtrate of supernatant liquid of the above suspension; and supernatant solution of digestive-canal-free tissue homogenate which was autolyzed at 40° for 20 hrs. under the presence of 2% NaF. It was observed that there are NO2-formations from NO3 by the first but not by the others and by all four enzyme solutions which were heated at 100° for 5 min. before beginning of the reaction as control. Namely, that the enzyme would be associated with tissue fairy closely and that it is impossible to extract by grinding were confirmed. In this work, therefore, the suspension of the ground chyme of tissue was used as enzyme solution. It was peviollsly reported that when ground mass of worm was heated for too long period, the red colour which developed with Griess's reagent disturbed the determination of NO2. If digestive-canal-free tissue was used, however, the intensity of the disturbing colour was negligible after heating even for 20 min. Then the effect of extraction of the tissue on the enzymatic activity was studied. The 10%(w/v) tissue suspension in water or phosphate buffer was prepared as above, agitated thoroughly and centrifuged, The cloudy extract was restored with same solvent to original volume and the residue was also resuspended. The reaction of these enzymes was compared. The amount of NO2 produced by catalytic action of resuspension was about 8～10% of that in original one and of course the extract showed no activity. The nitrate reductase in the silk-worm was existing in the tissue and in the wall of the digestive canal but not in the blood, digestive-juice with high alkalinity, or in the solid in canal which was fine debris of mullberry leaves eaten. The enzymatic activity of the worm homogenate in the phophate buffer as higher than that in water. It seemed that although hydroxylamine feeding brings about the decrease of the reductase activity by whole body, the enzyme in the alimentary-tract-free tissue was activated. The ground mass of worm was repeatedly dehydrated with a large volume of acetone, dried in vaccuo and pulverized. This acetone-dried powder also retained the ability of catalyzing, the conversion of NO3 to NO2 for many months at room temperature and I was unable to extract the enzyme from this powder as living tissue.1.家蚕の硝酸還元酵素は組織並びに消化管に存在し血液, 消化液及び食下された消化管内の桑葉片には検出されなかつた. 2. 酵素は組織にかなり緊密に結合し磨砕によつては水又は燐酸緩衝液で抽出されなかつた. 併し酵素力は減少した. 3. NH2OH添食蚕の全蚕体を酵素剤とした場合は作用力は著しく減少するかの観を示したが組織のみでは却て活性化された. 4. アセトン乾燥粉末は長期間保存し得る酵素剤として有効であり, 又酵素は生体同様抽出されなかつた.
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