On the nitrate reductase of the fowl liver (IV) : Intracellular distribution of the enzyme

The activity of the nitrate reductase could not be detected in the nuclei which had been isolated from the fowl liver by the citric acid method. Then the liver was fractionated with the isotonic sucrose solution by the differential centrifuge method and the intracellular distribution of the nitrate reductase was surveyed by estimating the activity of the mitochondrial, microsomal and supernatant fractions. It was found that most of the activity was contained in the supernatant, while it was scarcely detected in the former two particles. Since native hydrogen donators were dissolved in cell sap, it was suggested that the nitrate reduction can be performed within the cytoplasm of the liver cell. However, the mitochondria could mediate transfer of hydrogen between some donators and the nitrate reductase. The absorption spectrum of the supernatant fraction was estimated and found to be very similar to that of the purified preparation. The former, however, have two more feeble peaks at 530 and 562 mμ, which were extinguished by dialysis. These observations agreed well with those concerning the livers of mouse and cattle.廿日鼠及び牛肝臓の硝酸還元酵素は可溶性蛋白に属し主として細胞質部分に含まれているが,同時に組織固有の水素供与体その他の補助因子も同じく細胞液に溶存しているため硝酸塩の還元は細胞質に行われている事が推定された. 更にミトコンドリアも水素伝達系として反応に陽与し得る事が示されたがこの粒子の効果は動物或いは組織の種類による顕著な特異性は認められなかつた. 一方従来行つてきた肝臓の硝酸還元酵素に関する研究からホモゼネートでの硝酸還元或いは精製実験に於て多少の差は認められるが本質的には殆ど相違がない事は明らかであつて,その細胞内挙動についても非常に類似している事が推定される

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

On The Efect Of Some Carbonyl Compounds And Their Oximes Upon The Nitrate Reductase

Acetaldehyde interfered with the activity of miee nitrate reductase and did not have any influence upon that of fowl and silkworm tissue. On the other hand, pyruvic acid inhibited the action of reductase of mice and fowl, whereas the activation of worm enzyme was brought about. Against the above, acetone had almost no effect. But their oximes caused the inhibition in all cases, although the degree of the depression of the activity varied with the kinds of oximes and enzymic materials. This inhibition is partly due to the competition of oximes with nitrates for hydrogen atoms, and partly to the direct effect of oximes upon the enzyme itself. In concentrations as far as 10⁻³M acetaldehyde oxime, the inhibition was observed on the mice reductase. Hydroxylamine, too, had about the same degree of influence as that of acetaldehyde oxime

Studies on the reductase of nitrate. Part 2. On the coloration of NO_2-radical

The influences of the phosphate and glycocol buffers on the Griess’s coloration which we employed to estimate NO_2, NH_2OH, acetoxime and pyruvic acid-oxime, and the stabilities of these substances in the buffer solutions at 100°for 10 minutes and at 40° for 24 hours were studied. Some unfavourable effects were observed but found to be avoided by dilution of the buffers. And, the best condition for the estimation of pyruvic acid- and α-ketoglutaric acid-oximes by this colour reaction were determined.1) Griess試薬によるNH2OH, acetoxime及びpyruvic acid-oximeの比色測定はGlycocol-HCl緩衝液によつて阻害されるがNO2は発色遅延するのみである. 然るに緩衝液中40°24時間及び100°10分処理には前3者は安定でありNO2は破壊される. 2) Glycocol-NaOHは呈色へは影響しないが, 稀薄NH20Hは大部分, acetoximeも一部本緩衝液中上記処理で分解される. 3) Phosphateはacetoxime及びpyruvic acid-oximeの呈色を特にアルカリ側に於て阻害する. 稀NH2OHは同様に不安定である. 4) 之等の影響は緩衝液の濃度の減小に従つて緩和されるがNH20Hの安定性のみは酸度の増加に依る. 5) Pyruvicacid-oxime並びにα-ketoglutaric acid-oximeの測定は夫々サンプル10ccにIN-H2SO4 0.2cc添加し4分乃至4分30秒100°に加熱加水分解して生成したNH2OHを呈色比較する. 呈色率は約60%である

Effect of some purines on the nitrate reductase of the cattle liver

From a supplementary experiment with the cattle liver which contains much higher activity of the aldehyde oxidase than that of mouse and fowl livers, it was found that the nitrate reductase of the former was also inhibited similary by some purines as cases of the laters. Furthermore, the rate of inhibition by uric acid was lowered when the activity of the nitrate reductase had been activated by addition of acetaldehyde, increase of the amounts of enzyme employed or selection of liver of high activity. Concerning the nitrate reducing system of the purified preparation of cattle liver enzyme too, the purine inhibition coincided well with those of crude enzyme homogenate, though the former was not accompanied with the inherent hydrogen donators as well as some dehydrogenases which can couple with the nitrate reductase. A variation of ultraviolet absorption spectrum of the enzyme of high purity was brought about by addition of purines which suggest the combination of purines with enzyme protein.動物組織の硝酸還元酵素がキサンチン及びアルデヒド酸化酵素と極めて密接な関係を持つと主張されている事は繰り返し述べてきたが,キサンチン酸化酵素の反応生成物である尿酸は廿日鼠,牛等の哺乳動物ではプリン代謝系の中間体として更に変化を受け尿素を生ずる.之に反して?等鳥類或いは昆虫類では最終生成物として排泄物中の主要成分である点に於て顕著な差異を示し,叉牛肝臓抽出粗酵素液の硝酸塩還元はアセトアルデヒドによつて強く促進されるが廿日鼠及び雛肝酵素の作用は殆ど影響されないか叉は却て多少阻害される傾向を示した. 家蚕組織による硝酸還元阻害が主として尿酸に基く事から更にその前駆物質であるプリン類によつても等しく反応阻害が起る事を見出したが, 当然予測されたように廿日鼠及び?肝臓のプリン代謝系に対しては若干の異なつた影響が観察されたにも拘らず硝酸還元阻害には何等の差異も認められなかつた. 叉他方抽出粗酵素液での硝酸還元作用は動物によつて異なるが精製酵素では大差なく従つて肝臓の硝酸還元酵素に関して供試した3種の動物に於て本質的には差はないと推定される. プリン類の阻害については廿日鼠, ?両肝臓酵素共にプリン核の1,3及び7位の窒素が反応阻害に重要な役割を演じ之等窒素と酵素の活性中心との結合が起るために阻害を惹き起すと推定した. 事実廿日鼠肝臓抽出液の吸収スペクトルは紫外部特に蛋白の吸収に相当する部位に於てプリン類添加によつて多少の変化が起ることを測定したが, 酵素液は多くの不純物を含むために酵素蛋白との結合を示すものと決定的に主張することは出来なかつた. 更に粗酵素液が硝酸還元酵素に水素供与系として共軛し得る種々の脱水素酵素をも含むために,之等の酵素だけがプリン類によつて阻害されても硝酸還元阻害を二次的に惹起するため脱水素酵素系を除いた反応系での試験も必要である. 叉プリン類はキサンチン酸化酵素の基質でもあるため,その同族体は基質との拮抗効果を持つ事も考えられるが,そのためにも他の酵素系例えばアルデヒド酸化酵素系を主とする反応系を用いる事も好都合だと思われる.そこでアルデヒド酸化酵素力が特に強い牛肝臓組織を用い更にその精製酵素標品をも用いて補足実験を行つた

On the nitrate reductase of the fowl liver (I)

The activity of nitrate reductase in the fowl liver homogenate was estimated. It was completely inactivated by heating at 90℃ for 2 minutes and at 80℃ for 5 minutes. However, approximately 15 per cent of the original activity was retained at 70℃ and the slight diminution was observed when the enzyme had been heated at 60℃. The enzyme had a tendency to decrease linearly its activity at the initial stage of heating and then to reach gradually to certain final values according to the temperatures. The optimum pH of the nitrate reductase was about 5.8 coinciding with those of mouse and cattle liver enzymes, though the pH range in which the nitrate reductase is acting was much broader than that of both mammalian reductases. Especially, wide plateau of optimum pH existed between 6 and 7 when the enzyme assays were carried out using phosphate buffer. The enzymatic activity was fallen off considerably by dialysis and restored with the dialysate and boiled extract of liver. Moreover, the activity was also recovered some extent by addition of malate, glutamate, succinate, tartarate, fumarate, lactate, maleiate, malonate, and citrate, since native substances which are acting most predominatingly as hydrogen donator in the liver had been lost by dialysis. However, reactivation with sugars was not observed. The rate of nitrate reduction of the dialysed enzyme was accelerated exceedingly by acetaldehyde which has almost no or slight inhibitory effect on the reductase of the original homogenate. In connection with aldehyde dehydrogenase which was demonstrated so far to be able to reduce nitrate, therefore, some discussions were performed on the active principal reducing nitrate. It was observed that reactivation of dialysed nitrate reductase was attained by the conjunction of raw flavin extract of liver which contains three components FAD, FMN, and ribpflavin. Moreover, it was also suggested that FAD may concern with reduction but both the laters not. From the fact that thiourea, oxine, EDTA, NaN3, KCN, PCMB, monoiod acetic acid, Cu++, Ag+, and hydroxylamine inhibited the reduction of nitrate, the participation of metal and SIi group in the reaction was presumed.(1) ?肝 homogenate に硝酸還元酵素作用を確認した. (2) 酵素力は80℃以上では5分以内で完全に不活性化されるが70℃では20分で約15%残存し60℃でも30分に約20%の減少を来した. 而してその不活性化は初期に於ては加熱時間に比例し爾後徐々に終極値に近づく傾向を示した. (3) 酵素作用の最適pHは5.8附近にあつて哺乳動物のものと大体一致するが作用pH域は更に広かつた. (4) 酵素は透析により活性をかなり低下するが透析外液或は加熱肝臓抽出液により一部恢復した. (5) 酵素作用は種々の水素供与体によつて影響されないが透析すれば林檎酸, Glutamin酸, 琥珀酸, 酒石酸, Fumar酸, 乳酸, Malein酸, Malon酸, 枸櫞酸により促進される様になり, 之等の脱水素酵素が共軛する事を示した. 併し糖類は殆ど影響が認められなかつた. 一方原液に於て多少阻害的に働くか殆ど影響しなかつたAcetaldehydeの促進作用が著しく顕著になつた. (6) 原酵素液に於ける肝臓固有の水素供与体或は透析酵素に於けるAcetaldehydeによるMethyleneblue脱色能とNO3還元力とは必ずしも平行しなかつた. (7) 酵素活性にFADは有効であるが,FMN或はRiboflavinは効果がない事が示唆された. (8) 少くもhomogenateによるNO3還元系に金属並にSH基の関与する事が推定された