Study on the Nitrate Reductase (Ⅰ) NAD(P)H-and FMNH-dependent Nitrate Reductase

植物體內硝酸鹽還原酵素活性之研究

黃啓穎(Chi-Ying Huang) ； 藍調(Tao Lon)

《中華農業研究》 19卷4期 (1970/12) Pp. 50-59

https://doi.org/10.29951/JARC.197012.0006

摘要

從前一般學者都認爲於植物體內硝酸鹽還原過程中，祗有一種硝酸鹽還原酵素參與催化作用，又依酵素的特性而言，一種酵素蛋白(Apoenzyme)與一種輔酵素(Coenzyme)構成爲一種完全酵素，所以硝酸鹽還原酵素似乎不可能與兩種輔酵素（NADPH或FADH2）結合。本研究爲針對此問題加以探討，究由一種酵素蛋白可利用兩種輔酵素，或兩種酵素各利用其所需的輔酵素，同時參與硝酸鹽還原作用，效以大豆爲材料經賓驗結果摘要如下：
1.在此實驗我們已獲知：當硝酸鹽被大豆吸收後，在轉變爲銨的過程中有兩種硝酸鹽還原酵素參與作用，則NADPH和FMNH硝酸鹽還原酵素，此兩種酵素在理化性上很近似，但是以活性而言，NADPH硝酸鹽還原酵素大於FMNH硝酸鹽還原酵素。
2. NADPH硝酸鹽還原酵素和FMNH硝酸鹽還原酵素的活性同樣可受P-Chloromercuribenzoate所抑制；可是此種抑制作用，又可被半胱氨酸(Cysteine)所克服，由此可見半胱氨酸對此兩種酵素的活性有促進作用，與對照比較其活性效率可使NADPH硝酸鹽還原酵素的活性約增加43%，而對FMNH硝酸鹽還原酵素也可以增加約20%，所以半胱氨酸對NADPH硝酸鹽還原酵素之作用大於FMNH硝酸鹽還原酵素。
3. FMNH硝酸鹽還原酵素較NADPH硝酸鹽還原酵素耐熱，當溫度大於45℃時，NADPH硝酸鹽還原酵素之活性則急減。
4.依照Km值比較，在NADPH硝酸鹽還原酵素所結台的輔酵素NADPH2(TPNH2)較NADH2(DPNH2)爲佳，而於FMNH硝酸鹽還原酵素則以FADH2比FMMH2爲佳。所以在大豆體內而言，參與硝酸鹽還原作用的酵素，NADPH硝酸鹽還原酵素比FMNH硝酸鹽還原酵素爲佳。

並列摘要

1. There show a little differential activity in NAD (P) H-NR and FMNH-NR. The activity of NAD (P) H-dependent nitrate reductase predominate to that of FMNH-dependent nitrate reductase.
2. NAD (P) H-NR and FMNH-NR are equally susceptible to the inhibition of p-chloromer-curibenzoate, but this inhibitory effort can be reversed by cysteine in the reaction mixture.
3. Presence of cysteine in the extraction medium results in increasing enzyme activity. AND (P) H-NR and FMNH-NR activity arc increased about 43% and 20%, compared to that of the control lacking cysteine, respectively.
4. Activity of NAD (P) H-NR decrease more rapidly that of FMNH-NR at the temperature above 45°C, i.e. NAD (P) H-NR is more heat labile.
5. Relative affinity of cofactors to enzyme are arranged, according to their apparent Km values, in order of
NADPH2＞ NADH2 for NAD (P) H-dependent nitrate reductase.
FADH2＞ FMNH2 for FMNH-dependent nitrate reductase.
Relative affinity of nitrate to enzyme:
NAD (P) H-dependent nitrate reductase＞ FMNH-dopendent nitrate reductase.
By comparison of the characteristics of enzyme described above, the so-called nitrate reductase may be a mixture of NAD (P) H_and FMNH-dependent nitrate reductase. Although these kinds of enzyme are nearly identical in chemical and physical properties.