Notes |
According to the procedure pr e paring the nitrate reductase of cattle liver, the fowl liver enzyme was partially purified from the homogenate by repeated fractionation with ammonium sulfate and following treatment with Ca-phosphate gel. This preparation was very soluble in water and buffer making a clear pale yellow solution and could be preserved for a considerably long time in cold when it was kept moist after removing water as far as possible by means of an aspirator. However, the enzyme was denatured and lost its solubility as well as activity, if it was further dried in vacuo. As cattle liver enzyme, acetaldehyde was indispensable as a hydrogen donor for the enzymatic reduction of nitrate and the enhancement of the activity was provoked by some ferrous salts. Although this implies the rationality of the aldehyde oxidase hypothesis concerning the principle of the nitrate reductase, a proportionality between both activities of the nitrate reductase and the aldehyde oxidase could not be observed with many preparations which were prepared by the same procedure but from different livers. The final concentration of 2 x 10^-2M of nitrate and acetaldehyde was most suitable for the estimation of the activity. The direct relationship of the activity to the dose of the enzyme and the reaction time was also established. The reductase activity with and without ferrous ion was inhibited by NaN_3, thiourea, 8-hydroxyquinoline, EDTA, KCN, monoiod acetate, PCMB and Cu salts, which might suggest the participation of metal and SH group in the enzymatic nitrate reduction. However, some of the properties of the fowl enzyme were different from those of cattle. There were two peaks in the pH-activity curve, at pH about 4.5 and about 5.8, though the later agreed with the optimum pH of the crude liver homogenate as well as that of the cattle liver enzyme. The reduction was carried out most actively at 60℃ higher than the optimum temperature of the cattle enzyme. Furthermore, the reductase of the fowl liver exhibited a peculiar absorption spectrum which have a plateau from 250 to 280 ma, contrary to that of cattle having a peak at 262 m/1. On the other hand, the spectra of both enzymes in a visible range were approximately similar to each other, presenting a maximum at 405 mμ. In place of acetaldehyde, flavin fraction of liver, raw DPN solution from yeast, and mitochondria of liver could conjugate with the nitrate reductase of fowl liver as a hydrogen donating system. Moreover, dialisable substances of the liver extract could also function as an immediate substrate of the enzyme. Since the substances could be separated into 4 fractions and all these fractions had the ability to bring about the reduction of nitrate by the reductase, there might be at least 4 kinds of the inherent hydrogen donors in the liver tissue. As described above, the nitrate reductase was active in the acid media too. However, the reaction mixture was clarified at pH 5.0 producing a coagulated deposit which became now almost insoluble in buffer of neutrality. Concerning both clear supernatant and suspension of precipitate, the activities of the aldehyde and xanthine oxidases as well as nitrate reductase could be established. The activity of the precipitate was higher than that of the supernatant, while pH-activity curves of both fractions were similar to each other and to that of the original enzyme ; there were two optimum pHs. However, the absorption spectra were quite different. The extinction at 405 m/.c was maintained in the spectrum of the supernatant but lost in that of the precipitate which had been dissolved in weak alkaline solution. On the contrary, the plateau in ultraviolet range remained in the later though it was narrowed, whereas it was lost but a peak at 270 mp was appeared in the former. Similary, the enzyme was divided into two fractions, the clear supernatant and the precipitate, by heating, for example at 60° or 70℃ for 10 min. In this case too, both fractions had the activities of the nitrate reductase and the aldehyde and xanthine oxidases. However, the activities of the supernatant were higher than that of the precipitate, in opposition to the case of acid treatment. Furthermore, it was found that the absorption spectra of these fractions differ from one another. Hence, at any rate, it is supposed that there might be some kinds of enzymes which accompany both activities of the aldehyde and xanthine oxidases as well as nitrate reductase.
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