IDENTIFICATION OF AN ENDOGENOUS NADPH-REGENERATING SYSTEM COUPLED TO NITRATE REDUCTION IN-VITRO IN PLANT AND FUNGAL CRUDE EXTRACTS

A low molecular weight factor obtained from the crude extract of nit-1 mutant of Neurospora crassa (NEF) was capable of stimulating the acti vity of reconstituted NADPH specific nitrate reductase (NR, EC 1.6.6.3 ; Z.Z. Alikulov, N.A. Savidov, H.S. Lips, A lour molecular weight regu lator of nitrate reductase in higher plants, 4th International Symposi um on Inorganic Nitrogen Assimilation and 1st Fohs Biostress Symposium . Seeheim/Darmstadt, Germany, 1995, p. 9). This factor eluted from a G 15 column as glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P). Like the hexose-phosphates, NEF allowed rapid regeneration of NADPH f rom the NADP(+), resulting from nitrate reduction. Sequential treatmen t of the NEF-containing fraction with commercial preparations of gluco se-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 molecula r weight factor enhancing NR activity isolated from fungal and plant c rude extracts (Z.Z. Alikulov, N.A. Savidov, H.S. Lips, A low molecular weight regulator of nitrate reductase in higher plants. 4th Internati onal Symposium on Inorganic Nitrogen Assimilation and 1st Fohs Biostre ss symposium. Seeheim/Darmstadt, Germany, 1995, p. 9) may be a mixture of hexose phosphates, G6PDH, 6PGDH ana PGI were active in crude extra cts of plants and fungi used for NR assays. Oxidation of one mole of G 6P led to the production of approximately two moles of NADPH. The acti vities of PGI, G6PDH and 6PGDH exceeded that of NR in both fungal and plant crude extract. Fungal crude extracts exhibited a considerably hi gher capacity to regenerate NADPH than plant extracts. The addition of G6P during the in vitro NR assay increased the stability of the react ion allowing detection of very low NR activity levels. The stimulation of NR in vitro by NADPH generating systems points out the physiologic al importance of these systems in nitrate assimilation and in the nitr ate reduction assays in situ and in vivo. (C) 1998 Elsevier Science Ir eland Ltd. All rights reserved.