NADH-Ferricyanide Reductase of Leaf Plasma Membranes : Partial Purification and Immunological Relation to Potato Tuber Microsomal NADH-Ferricyanide Reductase and Spinach Leaf NADH-Nitrate Reductase

Plasma membranes obtained by two-phase partitioning of microsomal fractions from spinach (Spinacea oleracea L. cv Medania) and sugar beet leaves (Beta vulgaris L.) contained relatively high NADH-ferricyanide reductase and NADH-nitrate reductase (NR; EC 1.6.6.1) activities. Both of these activities were latent. To investigate whether these activities were due to the same enzyme, plasma membrane polypeptides were separated with SDS-PAGE and analyzed with immunoblotting methods. Antibodies raised against microsomal NADH-ferricyanide reductase (tentatively identified as NADH-cytochrome b₅ reductase, EC 1.6.2.2), purified from potato (Solanum tuberosum L. cv Bintje) tuber microsomes, displayed one single band at 43 kilodaltons when reacted with spinach plasma membranes, whereas lgG produced against NR from spinach leaves gave a major band at 110 kilodaltons together with a few fainter bands of lower molecular mass. Immunoblotting analysis using inside-out and right-side-out plasma membrane vesicles strongly indicated that NR was not an integral protein but probably trapped inside the plasma membrane vesicles during homogenization. Proteins from spinach plasma membranes were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio] 1-propane-sulfonate and separated on a Mono Q anion exchange column at pH 5.6 with fast protein liquid chromatography. One major peak of NADH-ferricyanide reductase activity was found after separation. The peak fraction was enriched about 70-fold in this activity compared to the plasma membrane. When the peak fractions were analyzed with SDS-PAGE the NADH-ferricyanide reductase activity strongly correlated with a 43 kilodalton polypeptide which reacted with the antibodies against potato microsomal NADH-ferricyanide reductase. Thus, our data indicate that most, if not all, of the truly membrane-bound NADH-ferricyanide reductase activity of leaf plasma membranes is due to an enzyme very similar to potato tuber microsomal NADH-ferricyanide reductase (NADH-cytochrome b₅ reductase).     

Fluctuations in Spinach Leaf Nitrate Reductase During Light-Dark Transitions

In vivo nitrate reductase (NR) activity declined gradually either in absence or presence of Mg²⁺ In dark grown plants of spinach. The increased sensitivity of the extracted NR from the dark grown plants to Mg²⁺ and ATP is indicative of the post-translational modification as one of the mechanisms to control NR activity. The response of extracted NR was gradual and not instantaneous suggesting a complex interplay of NR regulation, as the dark acclimatized plants when exposed to light caused significant nitrate reduction within 15 min of light exposures even in the presence of Mg²⁺ and ATP.     

Chlorate Reducing Activity of Spinach Nitrate Reductase

The activities of 2-oxoaldehyde-metabolizing enzymes (glyoxalase I, glyoxalase II, methyl- glyoxal reductase, methylglyoxal dehydrogenase and lactaldehyde dehydrogenase) were found to be widely distributed among microorganisms. One of the enzymes, methylglyoxal reductase, which catalyzes the reductive conversion of methylglyoxal into lactaldehyde, was purified from Escherichia coli cells. The enzyme was judged to be homogeneous on polyacrylamide gel electrophoresis and was a monomer with a molecular weight of 43000. The enzyme was most active at pH 6.5 and 45°C. The enzyme utilized both NADPH and NADH for the reduction of 2- oxoaldehydes (glyoxal, methylglyoxal, phenylglyoxal and 4,5-dioxovalerate) and some aldehydes (glycolaldehyde, D,l-glyceraldehyde, propionaldehyde and acetaldehyde). The Km values of the enzyme for methylglyoxal, NADPH and NADH were 4.0 mm, 1.7 fiM and 2.8 /¿m, respectively. The product of methylglyoxal reduction was identified as lactaldehyde. The enzyme from E. coli cells was different from the yeast and goat liver enzymes in both molecular structure and substrate specificity.     

Combining Anti-ERBB3 Antibodies Specific for Domain I and Domain III Enhances the Anti-Tumor Activity over the Individual Monoclonal Antibodies

Background

Inappropriate signaling through the epidermal growth factor receptor family (EGFR1/ERBB1, ERBB2/HER2, ERBB3/HER3, and ERBB4/HER4) of receptor tyrosine kinases leads to unregulated activation of multiple downstream signaling pathways that are linked to cancer formation and progression. In particular, ERBB3 plays a critical role in linking ERBB signaling to the phosphoinositide 3-kinase and Akt signaling pathway and increased levels of ERBB3-dependent signaling is also increasingly recognized as a mechanism for acquired resistance to ERBB-targeted therapies.

Methods

We had previously reported the isolation of a panel of anti-ERBB3 single-chain Fv antibodies through use of phage-display technology. In the current study scFv specific for domain I (F4) and domain III (A5) were converted into human IgG1 formats and analyzed for efficacy.

Results

Treatment of cells with an oligoclonal mixture of the A5/F4 IgGs appeared more effective at blocking both ligand-induced and ligand-independent signaling through ERBB3 than either single IgG alone. This correlated with improved ability to inhibit the cell growth both as a single agent and in combination with other ERBB-targeted therapies. Treatment of NCI-N87 tumor xenografts with the A5/F4 oligoclonal led to a statistically significant decrease in tumor growth rate that was further enhanced in combination with trastuzumab.

Conclusion

These results suggest that an oligoclonal antibody mixture may be a more effective approach to downregulate ERBB3-dependent signaling.     

Preparation of Monoclonal Antibodies against NADH: Nitrate Reductase from the Red Alga Porphyra yezoensis

Twenty-two monoclonal antibodies were raised against the nativeform of nitrate reductase (NR) from Porphyra yezoensis, a marinered alga. All the antibodies were able to bind to NR from P.yezoensis, with resultant inhibition of full (NADH:NR) and/orpartial (NADH:FR, NADH:CR, FMNH₂:NR, and MV:NR) enzymatic activity.Fifteen of the antibodies recognized the denatured form of theenzyme. Size-exclusion gel nitration and immunoblotting of theproducts of the limited proteolysis of NR from P. yezoensisby trypsin or Staphylococcus aureus V8 protease revealed that2 out of the 15 subunit-recognizing antibodies bound to theFAD domain, 6 bound to the heme domain, and 7 bound to the Mo-pterindomain. The products of limited proteolysis of NR from P. yezoensisalso revealed that the sites of proteolytic cleavage that encompassedthe heme domain were inverted as compared to the analogous sitesin NRs of higher plants. Some of the monoclonal antibodies cross-reactedwith NRs from plants belonged to different phyla. From threeto five of the antibodies bound subunits of NR from multicellularred algae, but failed to bind NRs from unicellular red algae.Three or four of the antibodies crossreacted with NRs from higherplants, such as tobacco and spinach. One of the antibodies boundNRs from several types of plant, namely, members of Cryptophyta,Chromophyta, and Chlorophyta. All of the monoclonal antibodiesthat cross-reacted with NRs from plants other than the red algaewere specific for the Mo-pterin domain of NR from P. yezoensis. (Received May 10, 1994; Accepted September 7, 1994)     

Spinach Leaf Sucrose-Phosphate Synthase and Nitrate Reductase Are Phosphorylated/Inactivated by Multiple Protein Kinases in Vitro

The regulation of sucrose-phosphate synthase (SPS) and nitrate reductase (NR) activities from mature spinach (Spinacia oleracea L.) leaves share many similarities in vivo and in vitro. Both enzymes are light/dark modulated by processes that involve, at least in part, reversible protein phosphorylation. Experiments using desalted crude extracts show that the ATP-dependent inactivation of spinach SPS and NR is sensitive to inhibition by glucose-6-phosphate. Also, a synthetic peptide homolog of the spinach SPS phosphorylation site inhibits the ATP-dependent inactivation of both enzymes with a similar concentration dependence. We have addressed the possibility that SPS and NR are regulated by the same protein kinase by partially purifying the protein kinases involved. Three unique kinase activities can be separated by anion-exchange and size-exclusion chromatography. Each peak of activity has a different substrate specificity. By gel filtration, they have apparent molecular masses of approximately 45, 60, and 150 kD. Additionally, the activities of the two smaller kinases are dependent on micromolar concentrations of Ca2+, whereas the 150-kD kinase is not. Finally, the 150-kD kinase has a subunit molecular mass of about 65 kD as determined by renaturing the kinase activity in situ following sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Metabolic Interactions between Spinach Leaf Nitrite Reductase and Ferredoxin-NADP Reductase: Competition for Reduced Ferredoxin

Steady state rates of NADP reduction decline upon commencement of nitrite reduction in reconstituted chloroplast preparations. Similarly, steady state rates of nitrite reduction are lower, but not zero, during concurrent NADP reduction. These results imply that competition for substrate occurs and suggest that nitrite reduction can successfully compete for reduced ferredoxin, even at high rates of NADP reduction.     

Immunological Characterization of Nitrate Reductase in Different Tissues of Spinach Seedlings

In spinach seedlings and roots, NADH-nitrate reductase (NR)activity (per g fresh weight) decreased as the seedlings aged.Experiments using double immunodiffusion analysis and immunotitrationshowed no differences in the immunological properties of NRfrom spinach seedlings at various stages of aging. Comparisonof spinach leaf to the spinach root enzyme using the Ouchterlonydouble diffusion technique revealed a high degree of similaritybetween them. (Received November 6, 1985; Accepted March 4, 1986)     

Partial Purification and Characterization of a Calcium-Dependent Protein Kinase and an Inhibitor Protein Required for Inactivation of Spinach Leaf Nitrate Reductase

Evidence is accumulating that the activity of spinach (Spinacia oleracea L.) leaf NADH:nitrate reductase (NR) is modulated both in vitro and in vivo by protein phosphorylation. From the present study we report the partial purification of the two protein factors needed for NR inactivation. We identified NR-protein kinase (NR-PK) as a calcium-dependent and metabolite-regulated protein kinase and have provided additional evidence that phosphorylation of NR is necessary but not sufficient to inactivate the enzyme. The inhibitor protein required for inactivation of phospho-NR was purified 625-fold by polyethylene glycol fractionation and sequential column chromatography. Using partially purified inhibitor protein and NR-PK, we characterized NR inactivation (increased sensitivity to Mg2+ inhibition) in a reconstituted in vitro system. NR-PK activity was inhibited by a variety of metabolic phosphate esters including di-hydroxyacetone phosphate, glucose-6-phosphate, and fructose-1,6-bisphosphate. Light-to-dark transition experiments with a starchless tobacco (Nicotiana sylvestris) mutant, which accumulates phosphate esters during the photoperiod, indicated that NR inactivation in vivo might, indeed, be down-regulated by metabolites. Additionally, we postulate that cytosolic free calcium could play an important role in the regulation of NR activity in vivo.     

Rapid Modulation of Spinach Leaf Nitrate Reductase by Photosynthesis : II. In Vitro Modulation by ATP and AMP

Assimilatory nitrate reductase activity (NRA) in crude spinach leaf (Spinacia oleracea) extracts undergoes rapid changes following fluctuations in photosynthesis brought about by changes in external CO₂ or by water stress (WM Kaiser, E Brendle-Behnisch [1991] Plant Physiol 96:363-367). A modulation of NRA sharing several characteristics (stability, response to Mg²⁺ or Ca²⁺, kinetic constants) with the in vivo modulation was obtained in vitro by preincubating desalted leaf extracts with physiological concentrations of Mg²⁺ and ATP (deactivating) or AMP (activating). When nitrate reductase (NR) was inactivated in vivo by illuminating leaves at the CO₂ compensation point, it could be reactivated in vitro by incubating leaf extracts with AMP. For the in vitro inactivation, ATP could be replaced by GTP or UTP. Nonhydrolyzable ATP analogs (β, γ-imido ATP, β, γ-methyl-ATP) had no effect on NR, whereas γ-S-ATP caused an irreversible inactivation. This suggests that NR modulation involves ATP hydrolysis. In contrast to NR in crude leaf extracts, partially purified NR did not respond to ATP or AMP. ATP and AMP levels in whole leaf extracts changed in the way predicted by the modulation of NRA when leaves were transferred from photosynthesizing (low ATP/AMP) to photorespiratory (high ATP/AMP) conditions. Adenine nucleotide levels in leaves could be effectively manipulated by feeding mannose through the leaf petiole. NRA followed these changes as expected from the in vitro results. This suggests that cytosolic ATP/AMP levels are indeed the central link between NRA in the cytosol and photosynthesis in the chloroplast. Phosphorylation/dephosphorylation of NR or of NR-regulating protein factors is discussed as a mechanism for a reversible modulation of NR by ATP and AMP.     

Monoclonal Antibodies Specific for Members of the Genus Endotrypanum

Twenty-six monoclonal antibodies were produced against membrane-enriched preparations of Endotrypanum schaudinni or Endotrypanum sp. promastigotes. Fifteen of these monoclonal antibodies (E1-E15) reacted only with the standard strain of E. schaudinni , M6159. Monoclonal antibodies E16-E26 were considered Endotrypanum specific; no cross reactivity was detected with any other genus of the family Trypanosomatidae (Leishmania, Trypanosoma, Leptomonas. Herpetomonas or Crithidia) by dot-blot radioimmune assay. By indirect immunofluorescence assay, the antigens recognized by Endotrypanum specific monoclonal antibodies appear to be associated with the surface of the parasite. Based on Western blot analysis, 4 antigenic molecules ranging in molecular weight from 24 kD to 160 kD were identified by monoclonal antibodies specific for the strain of E. schaudinni , M6159. Monoclonal antibodies specific for the genus Endotrypanum identified an antigen of molecular weight 48 kD as well as a diffuse component migrating with an apparent molecular weight of 64–200 kD.     

Decrease of Nitrate Reductase Activity in Spinach Leaves during a Light-Dark Transition

In leaves of spinach plants (Spinacia oleracea L.) performing CO₂ and NO₃⁻ assimilation, at the time of sudden darkening, which eliminates photosystem I-dependent nitrite reduction, only a minor temporary increase of the leaf nitrite content is observed. Because nitrate reduction does not depend on redox equivalents generated by photosystem I activity, a continuation of nitrate reduction after darkening would result in a large accumulation of nitrite in the leaves within a very short time, which is not observed. Measurements of the extractable nitrate reductase activity from spinach leaves assayed under standard conditions showed that in these leaves the nitrate reductase activity decreased during darkening to 15% of the control value with a half-time of only 2 minutes. Apparently, in these leaves nitrate reductase is very rapidly inactivated at sudden darkness avoiding an accumulation of the toxic nitrite in the cells.     

Immuno-gold Localization of Nitrate Reductase in Spinach (Spinacia oleracea) Leaves

The intracellular location of nitrate reductase in spinach leaveswas examined by applying an immunocytochemical method. Thinsections were first treated with immunopurified anti-nitratereductase monospecific antibodies, followed by incubation withcolloidal gold-labelled goat anti-rabbit immunoglobulin G asa marker. The nitrate reductase was specifically located inthe chloroplast. When anti-nitrate reductase antibodies wereomitted, or when pre-immune serum was used no label was observed. (Received October 30, 1986; Accepted December 25, 1986)     

Control by Glutamine of the Synthesis of Nitrate Reductase in Cultured Spinach Cells

Immunoblotting, using antibodies raised against electrophoreticallypure nitrate reductase, was used to study the regulation ofsynthesis of nitrate reductase in cultured spinach cells. Theextent of the loss of nitrate reductase activity that occurredwhen cultures were transferred to a glutamine-containing mediumwas correlated with the decrease in the level of cross-reactingmaterial (repression). Removal of exogenous glutamine resultedin the appearance of nitrate reductase activity as well as ofimmunoreactive protein (derepression). The activity of nitratereductase in spinach cells under "repressing" or "derepressing"conditions appears to be regulated by changes in the amountof the enzyme protein rather than by inactivation and activationof preexisting protein. (Received August 22, 1991; Accepted May 28, 1992)     

Malate and Dihydroxyacetone Phosphate-dependent Nitrate Reduction in Spinach Leaf Protoplasts

Isolated spinach (Spinacia oleracea L. var. Bloomsdale) leaf protoplasts reduced nitrate at rates of 9 micromoles per milligram chlorophyll per hour in light with a 3- to 4-fold stimulation in the presence of HCO₃⁻. A similar stimulation of nitrate reduction in the absence of CO₂ fixation was obtained by the addition of malate, oxaloacetate (OAA), phospho-3-glyceric acid (PGA), or dihydroxyacetone phosphate (DHAP). Stimulation by malate and DHAP was light-independent, while the PGA and OAA effect was light-dependent. Nitrate reduction was found to be coupled to the cytoplasmic oxidation of DHAP or malate. The PGA/DHAP and OAA/malate shuttle across the chloroplast envelope has been demonstrated to support CO₂ fixation and/or nitrate reduction. The leaf protoplasts readily assimilated nitrate into amino-N in a stoichiometric relationship.     

Establishment of Monoclonal Antibodies Specific for Bacillus subtilis DB9011

Bacillus subtilis DB9011 is a strain with useful functions for agriculture. To establish a method for the discrimination of this strain from others, monoclonal antibodies (MAbs) were prepared. Although two established MAbs (MAb9B6 and MAb14D2) cross-react with some other Bacillus strains in ELISA, only B. subtilis DB9011 vegetative cells are recognized by both MAbs. MAb14D2 recognizes flagellin, a 34-kDa unit protein of flagella. The two MAbs established will provide powerful tools with which detailed analysis of this bacterial strain can be obtained under environmental conditions.     

Magnesium and Calcium Inhibition of Squash Leaf NADH Nitrate Reductase

This paper describes the first experimental evidence that theinhibition of nitrate reductase by Mg²⁺ or Ca²⁺ is related tothe hysteretic properties of the enzyme. The low activity formof nitrate reductase, i.e. the form of nitrate reductase showinghysteretic behaviour, was inhibited 70–90% by 5 mM Ca²⁺or Mg²⁺. However, no inhibition by Ca²⁺ or Mg²⁺ was seen afterthe enzyme was converted to its high activity form by preincubationwith substrates. Addition of thiol compounds or certain aminoacids to the assay mixture also prevented the Mg²⁺ or Ca²⁺ inhibition. (Received June 28, 1993; Accepted August 11, 1993)     

Rapid Modulation of Spinach Leaf Nitrate Reductase Activity by Photosynthesis : I. Modulation in Vivo by CO(2) Availability

It has been shown recently that in spinach leaves (Spinacia oleracea) net photosynthesis and nitrate reduction are closely linked: when net photosynthesis was low because of stomatal closure, rates of nitrate reduction decreased (WM Kaiser, J Förster [1989] Plant Physiol 91: 970-974). Here we present evidence that photosynthesis regulates nitrate reduction by modulating nitrate reductase activity (NRA, EC 1.6.6.1). When spinach leaves were exposed to low CO₂ in the light, extractable NRA declined rapidly with a half-time of 15 minutes. The inhibition was rapidly reversed when leaves were brought back to air. NRA was also inhibited when leaves were wilted in air; this inhibition was due to decreased CO₂ supply as a consequence of stomatal closure. The modulation of NRA was stable in vitro. It was not reversed by gel filtration. In contrast, the in vitro inhibition of nitrate reductase (NR) by classical inhibitors such as cyanide, hydroxylamin, or NADH disappeared after removal of free inhibitors by gel filtration. The negative modulation of NRA in —CO₂-treated leaves became manifest as a decrease in total enzyme activity only in the presence of free Mg²⁺ or Ca²⁺. Mg²⁺ concentrations required for observing half-maximal inhibition were about 1 millimolar. In the presence of EDTA, the enzyme activity was always high and rather independent of the activation status of the enzyme. NRA was also independent of the pH in the range from pH 7 to pH 8, at saturating substrate and Mg²⁺ concentrations. The apparent substrate affinities of NR were hardly affected by the in vivo modulation of NR. Only V_max changed.     

Production and Characterization of Two Monoclonal Antibodies Specific for Plasmopara halstedii

Sunflower downy mildew, caused by the fungus Plasmopara halstedii, is a potentially devastating disease. We produced two monoclonal antibodies (MAbs) (12C9 and 18E2) by immunizing mice with a partially purified extract of P. halstedii race 1. Both MAbs detected in enzyme-linked immunosorbent assay (ELISA) all races of P. halstedii present in France. No cross-reactions were observed with Plasmopara viticola or with other fungi commonly associated with sunflowers. Both MAbs recognized the same three fungal antigens with molecular masses of 68, 140, and 192 kDa. However, the epitopes on the fungal antigens were distinct and repetitive. Seed homogenates from infected plants were incubated in wells coated with MAb 18E2. This resulted in the trapping of P. halstedii antigens that were identified with biotinylated MAb 12C9. No reactions were seen with seed homogenates from healthy plants. Thus, our results suggest that these MAbs might be used to develop a sandwich ELISA detection system for P. halstedii in infected seeds.