A coupled assay has been worked out to study spinach (Spinacea oleracea L.) nitrate reductase under low, more physiological concentrations of NADH. In this assay the reduction of nitrate is coupled to the oxidation of malate catalyzed by spinach NADmalate dehydrogenase. The use of this coupled system allows the assay of nitrate reductase activity at steady-state concentrations of NADH below micromolar. We have used this coupled assay to study the kinetic parameters of spinach nitrate reductase and to reinvestigate the putative regulatory role of adenine nucleotides, inorganic phosphate, amino acids, and calcium and calmodulin.Beside CO2 assimilation, nitrate assimilation is a major function of a leaf cell. NR3 can be regarded as a key enzyme in this process. In spite of a contradicting report (14), most evidence supports the view that nitrate reductase is located in the cytosolic compartment (2, 21, 32). As sources of redox equivalents for the generation of NADH required for nitrate reduction, the oxidation of glyceraldehyde-3-phosphate via the cytosolic NAD-GAPDH (15) and/or the oxidation of malate catalyzed by the cytosolic MDH (17) have been discussed. In both cases, the redox equivalents could be ultimately provided by the photosynthetic light reaction in the chloroplast, either by a triose phosphate-phosphoglycerate shuttle catalyzed by the phosphate translocator (7) or by a malate-oxaloacetate shuttle mediated by specific malate and oxaloacetate transport (10) across the chloroplast envelope. Alternatively, the cytosolic NAD can also be reduced by NADH generated in the mitochondria by tricarboxylic acid cycle via a malate-oxaloacetate shuttle between the mitochondrial matrix and the cytosol (6,35). This way could make it possible that the redox equivalents required for nitrate reduction might also be provided during darkness.

AbstratUsing a novel coupled enzyme activity assay, with a partially purified preparation of spinach leaf nitrate reductase, the apparent K, for NADH was determined as 1.4 yM. These measurements were carried out in the presence of 0.5 m M NAD, which is within the physiological range found in the cytosol of a leaf cell. The results show that an NADH/NAD ratio of 3 x is sufficient for a half maximal rate of nitrate reductase.

No abstract