Regulation of Corn Leaf Nitrate Reductase : I. Immunochemical Methods for Analysis of the Enzyme's Protein Component

NADH:nitrate reductase was extracted from corn leaves (Zea mays L. W64A x W182E) and purified on blue Sepharose. After the nitrate reductase was further purified by polyacrylamide gel electrophoresis, it was used to immunize mice and a rabbit. Western blots of crude leaf extracts were used to demonstrate monospecificity of the mouse ascitic fluids and the rabbit antiserum. The electrophoretic properties of purified corn and squash NADH:nitrate reductases in both native and denatured states were shown to be similar using western blotting with mouse ascitic fluid. The corn leaf enzyme has a 115,000 polypeptide subunit like that of squash. Western blots could detect 3 to 10 nanograms of nitrate reductase protein. But the detection of proteolytic degradation products using western blotting was inconsistent and remains to be established. An enzyme-linked immunosorbent assay (ELISA) was developed for quantifying nitrate reductase protein in the crude extracts of corn leaves. Using a standard curve based on nitrate reductase activity, the ELISA for corn nitrate reductase could detect 0.5 to 10 nanograms of nitrate reductase protein and was adequately sensitive for quantitative analysis of nitrate reductase in crude extracts of leaves even when activity levels were very low. When the ELISA was used to compare the nitrate reductase protein content of corn roots and leaves, these tissues were estimated to contain 0.24 to 0.5 and 4 to 5 micrograms nitrate reductase protein/gram root and leaf, respectively.