Identification of an endogenous NADPH-regenerating system coupled to nitrate reduction in vitro in plant and fungal crude extracts

Abstract

A low molecular weight factor obtained from the crude extract of nit-1 mutant of Neurospora crassa (NEF) was capable of stimulating the activity of reconstituted NADPH specific nitrate reductase (NR, EC 1.6.6.3; Z.Z. Alikulov, N.A. Savidov, H.S. Lips, A low molecular weight regulator of nitrate reductase in higher plants. 4th International Symposium on Inorganic Nitrogen Assimilation and 1st Fohs Biostress Symposium. Seeheim/Darmstadt, Germany, 1995, p. 9). This factor eluted from a G15 column as glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P). Like the hexose-phosphates, NEF allowed rapid regeneration of NADPH from the NADP +, resulting from nitrate reduction. Sequential treatment of the NEF-containing fraction with commercial preparations of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), 6-phosphogluconate dehydrogenase (6PGDH, EC 1.1.1.43) and phosphoglucose isomerase (PGI, EC 5.3.1.9) resulted in the oxidation of NEF and the reduction of NADP +. NEF, therefore, was a substrate of G6PDH and 6PGDH in a reaction which reduced NADP + to NADPH. It was concluded that the low molecular weight factor enhancing NR activity isolated from fungal and plant crude extracts (Z.Z. Alikulov, N.A. Savidov, H.S. Lips, A low molecular weight regulator of nitrate reductase in higher plants. 4th International Symposium on Inorganic Nitrogen Assimilation and 1st Fohs Biostress Symposium. Seeheim/Darmstadt, Germany, 1995, p. 9) may be a mixture of hexose phosphates. G6PDH, 6PGDH and PGI were active in crude extracts of plants and fungi used for NR assays. Oxidation of one mole of G6P led to the production of approximately two moles of NADPH. The activities of PGI, G6PDH and 6PGDH exceeded that of NR in both fungal and plant crude extract. Fungal crude extracts exhibited a considerably higher capacity to regenerate NADPH than plant extracts. The addition of G6P during the in vitro NR assay increased the stability of the reaction allowing detection of very low NR activity levels. The stimulation of NR in vitro by NADPH generating systems points out the physiological importance of these systems in nitrate assimilation and in the nitrate reduction assays in situ and in vivo.