Pyridine Nucleotide-Nitrate Reductase from Extracts of Higher Plants

Abstract

The purification and properties of an enzyme from soybean leaves is descr. which catalyzes the reduction of nitrate to nitrite by oxidation of TPNH or DPNH. Evidence is presented that this or a similar enzyme is present in the 6 other higher plant spp. examined. Expts. show that the enzyme is a flavoprotein with FAD as the prosthetic group. It is sensitive to heavy metal inhibitors, including potassium cyanide, sodium azide, thiourea and potassium ethylxanthate. Inhibition by p-chloromercuribenzoate was reversed by cysteine hydrochloride, which suggests that a sulfhydryl group is present on the active enzyme surface. No inhibition was obtained by use of CO or NaF. The pH for optimum activity is at 6.0. No stimulation of activity is obtained by additions of ZnSO4, MnSO4, Na2B4O7, Na2MoO4, MgSO4, FeSO4, FeCl3 or CUSO4 at final concentrations of 10-4[image]. Detns. of the TPNH that disappeared in the reaction and the quantities of nitrite that appeared concomitantly, indicate that one mole of nitrate is reduced by one mole of the reduced coenzyme. By use of grana and purified enzyme from soybean leaves, the photochemical reduction of nitrate has been demonstrated. The significance of these results is discussed in relation to previous reports of the effect of light on nitrate assimilation. No measurable quantity of nitrite disappeared in reaction containing purified nitrate reductase fractions. Disappearance, however, was observed in certain homogenates and other preparations. Nitrite disappearance could be partially inhibited by the addition of 10-3 [image] final concn. of NH2OH - HCl. Preliminary evidence indicates that TPNH at least is involved in nitrite as well as nitrate reduction.