The nitrate reductase of maus and ox-liver


Maus-organ (liver, kidney, heart and spleen) and maus-muscle of the hind legs of the same body were repeatedly washed with sterilized water, mixed with small amount of quartz sand, grounded thoroughly in mortar and made up 10 % (w/v) suspension with M/15 Sorensen\u27s phosphate buffer solution of pH 7.4. Ox-liver suspension was prepared with water as in the above procedure and its pH was 6.0. 5 cc of each enzyme solution was mixed with 5 cc of 2x10^-2 M NaNOs solution after careful evacuation in Thunberg\u27s tube, placed the tubes in a water bath at 40°. As control, enzyme solution which had been previously heated at 100° for 5 min. was used. After 1 and 2 hrs. the mixture was heated in the same manner to stop the reaction, added a few drops of conc, acetic acid and centrifuged. The colourimetric estimation of NO2 formed in the clear supernatant liquid was carried out in the usual way. The results were as follows. (snip) It can be seen from the data in the above table, that the detectable quantity of NO2 was produced from NO3 by the catalytic action of these tissue suspension, in other words, the existence of the nitrate reductase in these tissues was confirmed. Then the effect of extraction with water on the enzyme activity was studied as for the silk-worm. The aqueous suspension of ground tissue was defined as original enzyme (O-E). A certain volume of 0-E was centrifuged and the supernatant fluid restored to original volume with water, and it was called extract-enzyme (E-E) and resuspension of residue having the same volume was named residue-enzyme-1 (R-E-1). The suspension of residue which was extracted two times was residue-enzyme-2 (R-E-2) and so on. Each 5 cc of these enzyme solutions contained O.5 g tissue or corresponding amounts of residue or extracted matter. In the next table, the relative activity of the nitrate reductase of these enzyme solutions were presented: (snip) The above table teaches us that the nitrate reductase activity of maus-organ decreased to about half by extracting twice, while that of the muscle of the same animal was extracted about 60% and 40% of that retained in residue and by twice-treating about 35% was involved. To contrast these things, almost whole reductase in ox-liver was entered into extract. The optimum action of the reductase of both maus-organ and ox-liver occurred at pH 6.0~6.2 which is close to the pH of homogenate of these tissues. It has been found to increase their activity by addition of various substances. To the reaction mixture with R-E-2 of maus-organ the same quantity of some acids and sugars as nitrate was added. The relative activities were as follows. (snip) On the other hand, the substances which were five times amounts of nitrate were supplied to reaction mixture with E-E of ox-liver because I doubted if the addition of equal quantity of H-donator cause activation, for E -E contained both enzyme and substrate and the effect of substrate added would be hindered. To contrast to maus-organ and silk-worm the restoration of activity was attained according to the following order : rhamnose, galactose, mannose, glucose, fructose and marmite. The rate of activation, however, were also within 50% accorded with that of maus. Succinic acid and malonic acid which is specific inhibitor of succinodehydrogenase also had high ability of about 2.7 times and 1.9 respectively and by addition of each half amounts of both acids the rate lied between both. Some nitrous compounds such as glycine, alanine and asparagine caused the stronger reactivation. But the most effective one was acetaldehyde having 8-fold rate and the next its reduction product, ethylalcohol, with 4.3 times. On the other hand, citric acid showed the inhibiting action and in case of addition of ascorbic acid NO2 was not detected. However, in pure solution citric acid decreased the rate of coloration of NO2. Therefore, when one thought over above, it is found that the citric acid also have H-donating capacity even small. NO2 solution containing ascorbic acid developed no red colour with Griess\u27s reagent. Now the i n fluences of carbonylic compounds on the enzyme were studied. As seen from data in the table, (snip) though acetaldehyde and α-ketoglutaric acid acted actively and pyruvic acid inhibitory, oximes of these compounds were inhibitingly. I assumed that these oximes would receive hydrogen from H-donators competitively with NO3, therefore, nitrate reductase and oximase, discovered in my laboratory, which catalyze the conversion of oximes to amino compounds, would be the same enzyme.(1) 廿日鼠内臓及び筋肉並びに牛肝臓に硝酸還元酵素作用を確認した. (2) 廿日鼠内臓の硝酸還元酵素力は2回磨砕抽出によつて約1/2に減少し, 一方筋肉では約35%が残存し60%は抽出された. 此の場合にも減少の原因の一つに水素供与体の除去がある事を確かめた. 他方牛肝臓の酵素は大部分抽出された. (3) 水素供与体の効果は廿日鼠内臓に就ては, 枸櫞酸>琥珀酸>林檎酸>乳酸の順に促進作用を示し酒石酸, フマール酸, α-ケトグルタル酸は殆ど影響なく焦性葡萄酸及びマレイン酸は阻害的に作用した. 一方糖類ではガラクトーズ>ラムノーズ>マンノーズ>果糖の順であつた. 又牛肝臓に於ては糖類ではラムノーズ>ガラクトーズ>マンノーズ>葡萄糖>果糖>マンニットの順であり, 琥珀酸, マロン酸の外アスパラギン>グリシンン>アラニンの順に窒素化合物も有効であるが, 最も強力な促進作用を示したのはアセトアルデヒド, 続いてエタノールであつた. (4) 酵素作用の最適pHは廿日鼠内臓6.0~6.2,筋肉6.5,牛肝臓6.0附近と何れも磨砕液のpHに近い点にあつた. (5) 廿日鼠の酵素作用は肝臓, 筋肉, 並びに腎臓に検出され心臓, 脾臓及び肺には見出されなかつた. (6) カルボニール化合物中, アセトアルデヒド及びα-ケトグルタル酸は促進, 焦性葡萄酸は阻害的に作用したが之等のオキシムは何れもその影響を減殺する傾向を示し且つNO3の還元を拮抗的に阻害するように思われた

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