Activation of Nitrate Reductase by Extracts from Corn Scutella

Abstract

NADH-nitrate reductase (NR) from the primary leaves and root tips of corn seedlings (var. W64A x W182E) were activated by extracts from corn scutella. The activator extracted in potassium phosphate buffer (pH 7.5) or 80% (v/v) ethanol and fractionated by Dowex I (acetate) and Dowex 50 (H+) resins was recovered in the cationic fraction. The activator was not detected in extracts from shoots, roots, or endosperm of the seedlings. It activated the nitrate-induced cytochrome c reductase of NR complex but had slight inhibitory effects on the activities of FMNH2-NR and reduced methylviologen-NR. In addition the activator inhibited the activities of purified NR-inactivating proteins from corn roots (var. Wf9 x 38-11) and rice cell cultures.NADH-NR4 is considered to be the key enzyme in the nitrate assimilation pathway, and the rapid changes in its activity in response to changes in environmental conditions indicate the presence of effective regulatory systems in plant tissues. The rapid fluctuations could result from changes in relative rates of synthesis or degradation of the enzyme or from its activation or inactivation. For example, upon transfer of Chlorella from ammonium to nitrate medium, Johnson (3) recently showed by density-labeling technique that there was a rapid increase in NADH-NR activity, but only part of this was the consequence of increased de novo synthesis; there was apparently considerable activation of existing enzyme. Many factors can affect the NR level in plant tissues and some of these have recently been reviewed by Hewitt et aL (2).There is increasing evidence relating to the activation or inactivation of NR. For example, NRs isolated from Chlorella (12), spinach leaves (8), and corn scutella (15) are inactivated by NAD(P)H and/or cyanide and the inactivated NRs can be reactivated by ferricyanide. NRs are also inactivated by specific NRinactivating proteins isolated from corn roots (14,15), rice-cell cultures (17, 18), and soybean leaves (4). Two types of uncharacterized protein-like factors have recently been identified in wheat leaves (9, 10). One of these factors reduced the stability, whereas the others seemed to confer stability on NR. There are also reports on the effects of low mol wt components, EDTA, cysteine, FAD, or even N02, on the activation of NR (5,11,13 After 3 days at 26 C, scutella were separated from the seedlings for the isolation of the activator. In some experiments, the seedlings were transferred to liquid medium containing the same nutrients, but without agar, and grown for another 3 days at 26 C with a 12-h photoperiod.Extraction of the Activator. One g of corn scutella was quickly frozen in liquid N2 and then extracted with 10 ml 50 mm Kphosphate buffer (pH 7.5), containing 0.5 mm EDTA and 5 mM L-cysteine. The homogenates were filtered through 3 layers of Miracloth and centrifuged at 30,000g for 20 min. The supernatant was treated at 50 C for 10 min to remove NR activity and recentrifuged. The supernatant solution (crude extract) then was mixed with ethanol ...

Occurrence of three macromolecular inhibitors of nitrate reductase in Chinese cabbage roots

“…The interconversion between active and inactive forms of NR in higher plants might be due to the activity of these endogenous regulators. The activator from corn scutella inhibited the activities of both NR-inactivating proteins isolated from corn roots and rice cells (28).…”

“…The corn protein was a serine-endopeptidase (27,29), whereas inactivation by the proteins of both rice cells (31) and soybean leaves (18) was reversible. Endogenous activating factor of NR has been isolated from corn scutella (28) and from cotton seeds (21), respectively. The interconversion between active and inactive forms of NR in higher plants might be due to the activity of these endogenous regulators.…”