Immunological Comparisons of Nitrate Reductases of Different

Six monoclonal antibodies against different epitopes of maize leaf nitrate reductase were used to compare plant nitrate reductases in enzyme linked immunosorbent assay and enzyme activity inhibition tests. The number of cross-reacting antibodies was shown to vary with species according to phylogenetic classification, ranging from five (sugarcane) to one (dicotyledonous species). Cross-reactions were restricted to higher plant nitrate reductases. Plant nitrate reductase (EC 1.6.6. 1) has been shown to have a dimeric structure with two identical subunits (6, 11) and to contain a heme component, FAD' and a molybdenum cofactor (4). NR has two partial activities in the presence of electron donors or acceptors which shunt the electron flow: NADH-Cyt c reductase and methyl viologen-NR (4). Polyclonal antisera have been obtained against NR of spinach (Spinacia oleracea) (3), barley (Hordeum vulgare) (7), and squash (Cucurbita maxima) (14). Using a rabbit polyclonal antiserum prepared against squash NR in an Ouchterlony test, NRs ofsquash and spinach appeared to be similar, but somewhat different from corn and soybean NRs and very different from Chlorella vulgaris and Neurospora crassa NRs (14). Results obtained by rocket immunoelectrophoresis with a polyclonal antiserum raised against barley NR showed differences between NRs of monocotyledonous and dicotyledonous species (15). Recently, MAbs against plant NR have been obtained (1, 12). MAbs prepared against spinach NR were shown to inhibit NR activity of different monocotyledonous and dicotyledonous species (12). Rabbit polyclonal antiserum and mouse MAbs have been obtained against maize leaf NR. The properties of these MAbs have been previously described (1). Reciprocal competition for binding to the antigenic site showed that the MAbs could be divided into six classes, corresponding to six different epitopes of the enzyme. The aim of this work was to study the reactivity pattern of the MAbs, prepared against maize NR, with NRs of different plant species. MATERIALS AND METHODS Plant Materials and Culture Conditions. Seedlings of maize (Zea mays L. cv INRA 508), soybean (Glycine max L. cv L 28), 'Abbreviations: NR, nitrate reductase; ELISA, enzyme linked immunosorbent assay; FAD, flavine adenine dinucleotide; MAb, monocional antibody; PNPP, 4-nitrophenyl phosphate disodium salt; TGE, transmissible gastroenteritis: control monoclonal antibody. tobacco (Nicotiana tabacum L. cv Xanthi), Nicotiana plumbaginifolia L. cv Viviani, barley (Hordeum vulgare L. cv Sonia), and pearl millet (Pennisetum americanum L.), and shoots of sugarcane (Saccharum officinarum L.) were grown in the greenhouse, watered with a nutrient solution as previously described (2), and harvested as young green plantlets 2 or 3 weeks after sowing. Sunflower (Helianthus annuus L. cv HA 89) was available as adult plants grown as the other species. Asparagus (Asparagus officinalis L.) was grown in the field and harvested as young green sticks. Chlorella pyrenoides was grown according to Lefebvre-Drouet and Calvet (9) and Neurospora crassa as previously described (10). Enzyme Preparation. Leaves (or sticks for asparagus) were frozen and ground in liquid N2 and then placed into extraction buffer (15 g of leaves for 100 ml of buffer), containing 50 mm Hepes (pH 8.2), 0.1 M NaCl, 1 mM Na2EDTA, 1 ,M sodium molybdate, 5 ,M FAD, 1 AM leupeptin, 5 mm cysteine, and 3 g Polyclar AT (water-insoluble PVP), according to T. Moureaux (personal communication). After centrifugation at 16,000g for 45 min, the supernatant was precipitated with (NH4)2SO4 (45% of saturation) for 2 h at 4 to 6°C and centrifuged for 45 min at 16,000g. The pellet was dissolved in a 0.05 M Tris-HCl buffer (pH 7.5) containing 1 mM Na2EDTA, 1 AM sodium molybdate, 1 AM leupeptin, 5 AM FAD, and 5 mM cysteine and desalted on a GF45 column (Pharmacia). The resulting final volume of the extract was 1 ml of Tris-HCI buffer for 15 g of leaves. C. pyrenoides cells were sedimented, resuspended in distilled H20, and broken with the microtip of a Branson B15 sonicator (30% of maximal output). N. crassa nitrate reductase was extracted as previously described (10). Rabbit Polyclonal Antiserum. The rabbit polyclonal antiserum was prepared against maize leaf NR, purified by affinity chromatography (2a). Monoclonal Antibodies. The MAbs were used as ascite fluid (after (NH4)2S04 precipitation) prepared as previously described (1). The concentrations of NR specific immunoglobulins in the different ascites were 0.2 mg/ml for 25(15), 0.3 for 96(9)25, 0.3 for 28(2), 0.3 for 8(23), 0.7 for 15(21), and 0.9 for 42(22). We used also, as control ascite, a MAb against a glycosylated protein of a coronavirus TGE (8). This control antibody did not react with NR. NR Assay and Inhibition of NR Activity. The NR activity determinations were made according to Robin (13), except soybean NR activities which were measured according to Jolly et al. (5). For inhibition experiments 0.2 ml of plant extract were incubated with 0.1 ml ascite fluid (dilution 1/50, 1/150, 1/450, etc.), during 20 min in 0.5 ml K-phosphate, 0.1 M, pH 7.5, before the NR assay. The most active extracts (maize, barley) were diluted in order to have the same activities as the less active ones (asparagus, sunflower) to compare NR inhibition ofthe different plant species. 376 www.plantphysiol. org on May 1, 2019 Published by Downloaded from Copyright © 1986 American Society of Plant Biologists. All rights reserved. IMMUNOLOGICAL COMPARISONS OF NITRATE REDUCTASES Two Sites ELISA Test. Ascite fluids (dilution 1/200 in 0.1 M sodium carbonate buffer, pH 9.6) were distributed into the wells ofELISA plates (Linbro ISFB 96) (100 ,l per well) and incubated 90 mini at 37°C. The plates were then washed 3 times with ELISA buffer (0.01 M K-phosphate (pH 7.5), 0.15 M NaCl, 0.05% (v/v) Tween 20, and 0.02% (w/v) NaN3) and carefully emptied. The plant concentrated extract (100 1A) was added at different concentrations after dilution in the ELISA buffer with 10% (v/v) sheep serum, then incubated 90 min at 37°C and washed as above. The third step was the addition of 100 ul of the rabbit anti-NR polyclonal antiserum (dilution 1/200 in ELISA buffer with sheep serum). After incubation 90 min at 37°C and washing, an anti-rabbit IgG alkaline phosphatase conjugate (Sigma) was added (dilution 1/400 in ELISA buffer with sheep serum). After incubation 90 min at 37°C and washing, the phosphatase assay was performed (with PNPP 2.7 mm in 0.1 M diethanolamine buffer, pH 10.2, 1 mM MgCl2). The OD at 405 nm was measured 15 min later using a Dynatech ELISA autoreader.