Soybean Fractions Research Articles

Isolating antigenotoxic components and cancer cell growth suppressors from agricultural by-products

Commercial processing wastes or by-products of crops were found to be sources of antimutagens and human tumor cell growth suppressors. We developed a microplate method to measure genomic DNA damage in Chinese hamster ovary cells with a modified single cell gel electrophoresis (SCGE) assay. This allowed us to measure the repression of 2-acetoxyacetylaminofluorene (2AAAF)-induced DNA damage by very small amounts of complex mixtures, fractions or individual chemicals isolated from agricultural by-products. We previously demonstrated that PCC, an ethanol extract of a commercial soybean processing by-product, repressed induced genomic DNA damage in mammalian cells. PCC was separated into a series of chemically defined fractions and two fractions (PCC70 and PCC100) repressed mutagen-induced damage. Of the isoflavones isolated from soybean fraction PCC70, daidzein expressed antigenotoxic activity, however, genistin and genistein enhanced DNA damage. An antigenotoxic response also was observed with a fraction isolated from corn distillate solids (CDS40). We developed a microplate assay to measure the suppression of the growth rate of human cancer cells in which the cytostatic/cytotoxic status at each concentration of the test sample was quantitatively determined. Genistein, genistin, daidzein and daidzin isolated from soybean fraction PCC70 expressed a wide range of growth suppression of HT-29 human colon cancer cells. The biological assays were integrated with, and directed, the separation and analytical chemistry component of this project. Compounds were purified from biologically active fractions and the structure of individual chemicals was determined with analytical HPLC and LC-mass spectroscopy (LC-MS). This research may lead to the isolation of novel chemoprotectants from agronomic commercial processing products and by-products.

Partial purification and characterization of pyruvate kinase from the plant fraction of soybean root nodules.

Pyruvate kinase (PK, EC 2.7.1.40) was partially purified from the plant cytosolic fraction of N2-fixing soybean (Glycine max [L.] Merr.) root nodules. The partially purified PK preparation was completely free of contamination by phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), the other major phosphoenolpyruvate (PEP)-utilizing enzyme in legume root nodules. Latency experiments with sonicated nodule extracts showed that Bradyrhizobium japonicum bacteroids do not express either PK or PEPC activity in symbiosis. In contrast, free-living B. japonicum bacteria expressed PK activity, but not PEPC activity. Antibodies specific for the cytosolic isoform of PK from castor bean endosperm cross-reacted with a 52-kDa polypeptide in the partially purified PK preparation. At the optimal assay pH (pH 8.0 for PEPC and pH 6.9 for PK) and in the absence of malate, PEPC activity in crude nodule extracts was 2.6 times the corresponding PK activity. This would tend to favour PEP metabolism by PEPC over PEP metabolism by PK. However, at pH 7.0 in the presence of 5 mM malate, PEPC activity was strongly inhibited, but PK activity was unaffected. Thus, we propose that PK and PEPC activity in legume root nodules may be coordinately regulated by fluctuations in malate concentration in the plant cytosolic fraction of the bacteroid-containing cells. Reduced uptake of malate by the bacteroids, as a result of reduced rates of N2 fixation, may favour PEP metabolism by PK over PEP metabolism by PEPC.

Carbon Dioxide Induced Soybean Protein Precipitation: Protein Fractionation, Particle Aggregation, and Continuous Operation

A novel protein fractionation technique using a volatile electrolyte has been developed. Carbon dioxide was used to isoelectrically precipitate 80% and 95% pure glycinin and beta-conglycinin fractions from soybean isolate. The protein fractions precipitated as primary particles 0.2-0.3 microm in diameter, which under optimum conditions may be recovered as aggregates up to 500 microm in diameter. The dependency of protein fractionation efficiency on aggregate settling rates has been demonstrated. The isoelectric points of the two main soybean fractions, glycinin and beta-conglycinin, were calculated to be pH 5.2 and 4.95, respectively. Solution pH was accurately controlled by pressure in the isoelectric pH range of the different soybean protein fractions, and a pH "overshoot" was eliminated. Volatile electrolyte technology was also applied to a continuous process in order to eliminate the particle recovery concerns associated with batch precipitation and to demonstrate the potential for scale-up. Glycinin was effectively recovered on-line (94% glycinin recovery) with a purity approaching that of the batch process (95%).

Immunodiagnostic methods for detection of 5-enolpyruvylshikimate-3-phosphate synthase in Roundup Ready® soybeans

We have developed immunological-based detection methods to support labeling of protein-containing food fractions derived from Roundup Ready® soybeans. Western blotting and enzyme linked immunosorbent assay (ELISA) procedures were developed to measure the 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) protein derived from the Agrobacterium sp. strain CP4 in the major processed fractions derived from Roundup Ready soybean. Expression of the CP4 EPSPS protein confers tolerance to Roundup® herbicide. The western blotting method utilizes a polyclonal goat anti-CP4 EPSPS antibody which specifically binds to CP4 EPSPS followed by detection of bound goat antibody with biotinylated Protein-G. Detection of this complex is accomplished using horseradish-peroxidase (HRP) labeled NeutrAvidin™ and signal development by enhanced chemiluminesence. Data from western blot analyses of these fractions establish that stable epitopes remain after the seed has been subjected to processing conditions typically employed by the food industry, thereby enabling development of an ELISA method. The ELISA for measurement of CP4 EPSPS is a triple antibody sandwich procedure utilizing a monoclonal capture antibody and a polyclonal detection antibody followed by a third biotin labeled monoclonal anti-rabbit antibody. Sandwich formation is detected using HRP labeled NeutrAvidin™ with color development using TMB substrate. In the sandwich ELISA, the immunological activity of CP4 EPSPS was reduced by the extraction method required to solubilize CP4 EPSPS protein from processed fractions. Sensitivity of the CP4 EPSPS ELISA was sufficient to detect CP4 EPSPS protein in processed soybean fractions that contained 2% Roundup Ready soybean mixed with conventional processed soybean fractions, thereby making the ELISA an acceptable method to assess CP4 EPSPS protein in processed soybean fractions. Data on sensitivity, accuracy, precision and specificity, established that the western blot and ELISA methods are appropriate for compliance with the EC Novel Foods Regulation.

Soy protein, isoflavonoids, and risk of developing coronary heart disease.

The effects of soy protein and isoflavones on blood cholesterol in humans has been variable. Maximal low- density lipoprotein cholesterol lowering appears to be modest and consistently ranges from 5% to 7%. Preliminary evidence suggests a potentially beneficial effect of the isoflavone fraction of soybeans on arterial compliance. The isoflavone fraction has been demonstrated to decrease the in vitro susceptibility of low-density lipoprotein to oxidation; the significance of this finding in vivo is unknown. It is difficult to definitively say at this time whether increased consumption of soy based products will result in a decreased risk of CHD beyond their ability to displace foods high in saturated fat and cholesterol from the diet.

Degradability and digestibility of full-fat soybeans treated with different sugar and heat combinations

Two experiments were conducted to study the effects of different combinations of sugars and heating time and temperature on the ruminal undegradable protein fraction and DM digestibility of full-fat soybeans. In the first experiment, the influence of sugar addition before heating (none, soaked in either a high fructose corn syrup [HFCS] solution, lignosulfonate or molasses), micronization temperatures (100, 110, 120, and 130 °C), and temperature holding times (0, 30, 60, and 120 min) was examined in a 4 × 4 × 4 factorial arrangement of treatments. In the second experiment, the effect of heating in a sugar solution was studied in a 4 × 2 × 4 factorial design with four sugar treatments (none, soaked either in fructose, molasses or a high xylose product), two soaking temperatures (100 and 120 °C), and four soaking times (0, 60, 120, and 240 min). In vitro dry matter digestibility (IVDMD), in vitro ruminal undegradable protein (RUP), and in sacco RUP were measured. In the first experiment, increased temperatures and heating to 120 min resulted in an increased RUP fraction of full-fat soybeans with no effect on DM digestibility, relative to when no sugar or fructose was added. There was a decrease in digestibility with increased temperature when lignosulfonate or molasses was added and with increased heating time when lignosulfonate was added. In the second experiment where soaking in sugar and heating were done simultaneously, lignosulfonate increased the RUP of soybeans without affecting its digestibility but values were numerically lower than when soaking and micronizing were conducted in two different steps as performed in the first experiment. Key words: protein, degradability, digestibility, micronization

Rheological properties of water-soluble soybean polysaccharides extracted under weak acidic condition

We studied the rheological properties of water-soluble soybean polysaccharides (SSPS) directly extracted from the residual by-product of isolated soybean protein for 1.5 h at pH 5.0 and 120°C. The purity was relatively high, and they were composed mostly of saccharides. The molecular weight of the SSPS, as determined by a viscometric method, was approximately 114 000. The SSPS solution was lower in viscosity than that reported by Thompson et al., (Thompson, D.B., Huang, C., & Sieglaff, C. (1987). Rheological behavior of soluble polysaccharides fractions from soybean. Food Hydrocolloids, 1, 333–337) who investigated the polysaccharides extracted by the method of Morita (Morita, M. (1965). Polysaccharides of soybean seeds: polysaccharide constituents of “hot-water-extract” fraction of soybean seed and an arabinogalactan as its major component. Agric. Biol. Chem., 29, 564–573), and the viscosity was higher than polysaccharides extracted by the method of Aspinall et al. (Aspinall, G.O., Begbie, R., Hamilton, A., & Whyte, J.N.C. (1967). Polysaccharides of soybeans: extraction and fractionation of polysaccharides from cotyledon meal. J. Chem. Soc. (C), 1065–1070) The SSPS could be dissolved in water at 30% concentration at 20°C. A 10% solution of SSPS showed Newtonian behavior. This solution did not undergo gelation in the range of 5–80°C. Although this SSPS contained approximately 20% uronic acid, it did not show reactivity with polyvalent cations. The viscosity of the SSPS solution increased slightly with increasing pH, and this increase was reversible. We conclude that SSPS have strong glucosidic bonds that enable them to endure the sterilization at 120°C for 15 min at pH 3–6. Further, the behavior of the viscosities of dilute solutions suggests that galacturonic acids, such as uronic acid, exist closely together in the SSPS structure.

Molecular strategies to improve the nutritional quality of legume proteins.

Legume proteins are relatively deficient in the sulfur amino acids. Among the different strategies to increase the sulfur amino acid contents of legumes, we have chosen to increase the biosynthesis of endogenous, non-abundant sulfur-rich proteins we have identified in soybean seed. We identified and isolated an 8 kDa sulfur-rich protein from the albumin fraction of soybean seed. Subsequently, a cDNA designated Gm2S-1, has been isolated and characterized. The Gm2S-1 cDNA encodes a pre-proprotein that contains a signal peptide and a precursor protein that undergoes post-translational processing to yield the mature 8 kDa protein containing 7.8% methionine and 7.8% cysteine. The Gm2S-1 gene is an ideal candidate for overexpression to improve the nutritional quality of soybean and other legumes.

Biochemical Characterization of Selective Inhibitors for a Glycyrrhizin-binding Lipoxygenase from Soybeans

By GL-affinity column chromatography (HPLC), a GL-binding lipoxygenase (gbLOX) was selectively purified from a partially purified soybean LOX-1 fraction. gbLOX was identified as LOX-3 since the partial N-terminal amino acid sequences of at least four fragments generated from the purified gbLOX (p96 and p94) digested with trypsin were identical to the corresponding sequences of soybean LOX-3. The inhibitory effects of various flavonoids and their modified derivatives, epigallocatechin gallate (EGCG) and a derivative (oGA) of glycyrrhetinic acid on the activities of two LOXs [gbLOX and ngLOX (non-GL-binding LOX)] were examined in vitro. It was found that (i) four compounds [quercetin, EGCG, a derivative (2′, 4′-DDC) of 3, 4-dihydroxycitialcone, and oGA] inhibit gbLOX activity in a dose-dependent manner, but do not affect ngLOX activity; and (ii) quercetin as well as EGCG effectively prevents phosphorylation of gbLOX by casein kinase II (CK-II) in vitro. The results provided here suggest that the potent gbLO...

Effect of Temperature and Genotype on the Crude Glycinin Fraction (11S) of Soybean and Its Analysis by Near-Infrared Reflectance Spectroscopy (Near-IRS)

The crude glycinin fraction (11S) of soybean [Glycine max (L.) Merr.] protein has desirable quality characteristics. The objectives of this soybean study were the following : (1) to develop a near-infrared reflectance spectroscopy (near-IRS) equation for screening of the 11S fraction ; (2) to determine relationships between the 11S fraction, crude protein, and oil concentration ; and (3) to study the effect of growing temperature and genotype on this fraction. Three near-IRS 11S equations were developed, with the best equation having a moderate standard error of calibration (7.7 g/kg) and coefficient of determination (R 2 = 0.84). A positive correlation was observed between the 11S protein subunit and crude protein concentration (r = 0.69, P < 0.01), and a negative correlation was found between the 11S protein subunit and oil concentration (r = -0.67, P < 0.01), based on 319 genotypes. Higher crude protein and 11S protein subunit concentrations were associated with higher growing temperatures, and higher oil concentration was associated with lower growing temperatures, based on 12 genotypes.

Effects of Processing Method on Degradation Characteristics of Protein and Carbohydrate Sources In Situ

Two ruminally cannulated Holstein cows in early lactation were used to determine the effects of heat treatment and particle size on fractional degradation rates and ruminal degradabilities of DM, CP, and total nonstructural carbohydrate of dry shelled corn and soybeans. A randomized complete block design with cows as blocks was used. Carbohydrate sources were cracked corn, chick cracked corn, finely ground corn, and steam-flaked corn. Protein sources were soybeans that were roasted at 144°C, cracked, or ground through a 4-mm screen and raw soybeans that were cracked or ground through a 4-mm screen. Reduction in particle size increased both degradability of CP and total nonstructural carbohydrate in the rumen. Heat treatment decreased degradability of CP and increased degradability of total nonstructural carbohydrate in the rumen for both protein and carbohydrate sources. Roasting of soybeans decreased degradability of all AA except Lys. However, reduction in particle size had a stronger effect than heat treatment on ruminal degradability of CP and AA of soybeans, but had the opposite effect on total nonstructural carbohydrate degradability from corn. Methionine, Leu, and Thr had the lowest ruminal degradability across all soybean sources, and Lys and Val were the most degradable. The AA profile of the RUP fraction of soybeans differed from that of the original feedstuff and was affected by processing method.

Kinetic Properties of NAD-Dependent Glyceraldehyde-3-phosphate Dehydrogenase from the Host Fraction of Soybean Root Nodules

The kinetic mechanism of NAD-dependent glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate:NAD + oxidoreductase (phosphorylating), EC 1.2.1.12] from the host cytosolic fraction of soybean ( Glycine max L. Merr. cv. Williams) root nodules has been investigated by steady-state initial velocity studies and inhibition studies with products and product analogs. The results were consistent with the enzyme having a bi-uni-uni-uni ping-pong mechanism in which NAD + and phosphate interacted sequentially with the enzyme, followed in turn by the release of 1,3-bisphosphoglycerate, the addition of D-glyceraldehyde-3-phosphate, and the release of NADH. At pH 7.2, NAD + bound to the enzyme in a rapid-equilibrium fashion, whereas at pH 8.8 there was rapid-equilibrium addition of both NAD + and phosphate to the enzyme. NADH was a strong competitive inhibitor with respect to NAD + and phosphate. The enzyme was very labile, especially above pH 8, but the rate of loss of activity was considerably reduced in the presence of NAD + and phosphate. The possible role of these properties in the regulation of NAD-dependent glyceraldehyde-3-phosphate dehydrogenase in the host cytosol of soybean nodules is discussed.

Identification of peanut agglutinin and soybean trypsin inhibitor as minor legume allergens.

Peanuts and soybeans are frequent causes of food hypersensitivity reactions in children. Sera from 12 patients with atopic dermatitis and a positive double-blind placebo-controlled food challenge to peanut and sera from 5 patients with atopic dermatitis and a positive double-blind placebo-controlled food challenge to soybean were used to identify and characterize specific legume allergens. Identification of a minor allergen from peanut and a minor allergen from soybean was accomplished using various physicochemical techniques. The peanut fraction, peanut agglutinin, isolated by anion-exchange chromatography and electrolution and confirmed by amino acid sequencing, bound IgE in only 50% of the peanut challenge positive patients. The soybean fraction, soybean trypsin inhibitor, identified by gel filtration and electroelution and confirmed by amino acid sequencing, bound IgE in only 20% of the soy challenge positive patients. The identification of these two known legume proteins as minor allergens should allow further immunologic and structural investigations to compare the major and minor legume allergens.

Composition and Distribution of Adenylates in Soybean (Glycine max L.) Nodule Tissue.

Adenylates (ATP, ADP, and AMP) may play a central role in the regulation of the O2-limited C and N metabolism of soybean nodules. To be able to interpret measurements of adenylate levels in whole nodules and to appreciate the significance of observed changes in adenylates associated with changes in O2-limited metabolism, methods were developed for measuring in vivo levels of adenylate pools in the cortex, plant central zone, and bacteroid fractions of soybean (Glycine max L. Merr cv Maple Arrow x Bradyrhizobium japonicum strain USDA 16) nodules. Intact nodulated roots were either frozen in situ by flushing with prechilled Freon-113(-156[deg]C) or by rapidly (<1 s) uprooting plants and plunging them into liquid N2. The adenylate energy charge (AEC = [ATP + 0.5 x ADP]/[ATP + ADP + AMP]) of whole-nodule tissue (0.65 [plus or minus] 0.01, n = 4) was low compared to that of subtending roots (0.80 [plus or minus] 0.03, n = 4), a finding indicative of hypoxic metabolism in nodules. The cortex and central zone tissues were dissected apart in lyophilized nodules, and AEC values were 0.84 [plus or minus] 0.04 and 0.61 [plus or minus] 0.03, respectively. Although the total adenylate pool in the lyophilized nodules was only 41% of that measured in hydrated tissues, the AEC values were similar, and the lyophilized nodules were assumed to provide useful material for assessing adenylate distribution. The nodule cortex contained 4.4% of whole-nodule adenylates, with 95.6% being located in the central zone. Aqueous fractionation of bacteroids from the plant fraction of whole nodules and the use of marker enzymes or compounds to correct for recovery of bacteroids and cross-contamination of the bacteroid and plant fractions resulted in estimates that 36.2% of the total adenylate pool was in bacteroids, and 59.4% was in the plant fraction of the central zone. These are the first quantitative assessments of adenylate distribution in the plant and bacteroid fractions of legume nodules. These estimates were combined with theoretical calculations of rates of ATP consumption in the cortex (9.5 nmol g-1 fresh weight of nodule s-1), plant central zone (38 nmol g-1 fresh weight of nodule s-1), and bacteroids (62 nmol g-1 fresh weight of nodule s-1) of soybean nodules to estimate the time constants for turnover of the total adenylate pool and the ATP pool within each nodule fraction. The low values for time constant (1.6-5.8 s for total adenylate, 0.9-2.5 s for ATP only) in each fraction reflect the high metabolic activity of soybean nodules and provide a background for further studies of the role of adenylates in O2-limited nodule metabolism.

Identification of Three Phosphoinositide Specific Phospholipase C from Cytosolic Fraction of Soybean (Glycine max L.) Sprouts

Identification of Three Phosphoinositide Specific Phospholipase C from Cytosolic Fraction of Soybean (Glycine max L.) Sprouts

Immunocytochemical localization of NAD‐dependent glyceraldehyde‐3‐phosphate dehydrogenase in soybean nodules

Polyclonal antibodies raised against NAD‐dependent glyceraldehyde‐3‐phosphate dehydrogenase (D‐glyceraldehyde‐3‐phosphate:NAD+ oxidoreductase [phosphorylating], EC 1.2.1.12) from the plant cytosolic fraction of soybean [Glycine max (L.) Merr. cv. Williams] nodules were used to study the subcellular location of the enzyme and its relative distribution between infected, interstitial and cortical cells of soybean (cv. Lincoln) nodules. Post‐embedding immunogold labelling was carried out on nodules harvested 5, 12, 19 and 25 days after the first sign of nodulation. Labelling for glyceraldehyde‐3‐phosphate dehydrogenase was observed over the cytoplasm and nuclei of infected and uninfected cells, as well as over the nucleoid regions of bacteroids. In 5‐day‐old nodules, label also bound adjacent to the peribacteroid membranes. Statistical analysis of the number of gold particles per cell area indicated that in 5‐day‐old nodules, glyceraldehyde‐3‐phosphate dehydrogenase was distributed equally between infected, interstitial and cortical cells, but in older nodules the enzyme was more prominent in the interstitial and cortical cells than in infected cells.

Effect of Sodium Dodecyl Sulphate on 7S Fraction of Soybean

Effect of Sodium Dodecyl Sulphate on 7S Fraction of Soybean

Isoenzymes of Glucose 6-Phosphate Dehydrogenase from the Plant Fraction of Soybean Nodules

Two isoenzymes of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) have been separated from the plant fraction of soybean (Glycine max L. Merr. cv Williams) nodules by a procedure involving (NH(4))(2)SO(4) gradient fractionation, gel chromatography, chromatofocusing, and affinity chromatography. The isoenzymes, which have been termed glucose 6-phosphate dehydrogenases I and II, were specific for NADP(+) and glucose 6-phosphate and had optimum activity at pH 8.5 and pH 8.1, respectively. Both isoenzymes were labile in the absence of NADP(+). The apparent molecular weight of glucose 6-phosphate dehydrogenases I and II at pH 8.3 was estimated by gel chromatography to be approximately 110,000 in the absence of NADP(+) and double this size in the presence of NADP(+). The apparent molecular weight did not increase when glucose 6-phosphate was added with NADP(+) at pH 8.3. Both isoenzymes had very similar kinetic properties, displaying positive cooperativity in their interaction with NADP(+) and negative cooperativity with glucose 6-phosphate. The isoenzymes had half-maximal activity at approximately 10 micromolar NADP(+) and 70 to 100 micromolar glucose 6-phosphate. NADPH was a potent inhibitor of both of the soybean nodule glucose 6-phosphate dehydrogenases.

Effect of Constitutive and Herbivore-induced Extractables from Susceptible and Resistant Soybean Foliage on Nonpest and Pest Noctuid Caterpillars

Soybean foliage from susceptible (‘Bragg’) and resistant (PI 229358 and D75-10169) lines was extracted in several nonpolar and polar organic solvents to assess constitutive (mite-free) and induced (mite-damaged) activity against nonpest and pest noctuid caterpillars. The benzene fraction, incorporated in artificial diet, contained most of the constitutive activity of both the resistant and susceptible lines, as indicated by reduced relative growth rate (RGR) of velvetbean caterpillar, Anticarsia gemmatalis Hubner; corn earworm, Helicoverpa (=Heliothis) zea (Boddie); tobacco budworm, H. vlrescens (F.); and fall armyworm, Spodoptera frugiperda (J. E. Smith) compared with larvae fed the extract- and solvent-free control diet. Induced activity from mite-damaged greenhouse-grown plants and A. gemmatali s- damaged field-grown plants was detected primarily in the petroleum ether fraction. Among the species tested on the induced fractions (A. gemma talis, S. frugiperda , and the cabbage looper, Trichoplusia ni (Hubner)), S. frugiperda was the most sensitive species, as demonstrated by reduced RGR. Overall, among the species (H. zea, H. virescens , and T. nil more tolerant of the soybean foliage fractions, only H. zea is a major soybean foliage pest. Surprisingly, among the two most sensitive species, A. gemmatalis is a legume specialist and major soybean pest, whereas S. frugiperda is a polyphagous species but not a soybean pest. These results suggest that presumed soybean-adapted species may not be more resistant than soybean-naive ∼pecies to the deleterious effects of soybean foliage extracts.

Purification and Immunological Properties of NAD-Dependent Glyceraldehyde-3-Phosphate Dehydrogenase from the Plant Cytosol of Nitrogen Fixing Soybean Nodules

NAD-dependent glyceraldehyde-3-P dehydrogenase (D-glyceraldehyde-3-P: NAD + oxidoreductase phosphorylating, EC 1.2.1.12) has been purified to homogeneity from the plant cytosolic fraction of soybean ( Glycine max L. Merr. cv. Williams) nodules. The enzyme had native and subunit molecular weights of 147 kDa and 37 kDa respectively, indicating a tetrameric structure. Polyclonal antibodies were raised against the purified enzyme and shown to be monospecific by Western blot analysis. The antibodies cross-reacted with glyceraldehyde-3-P dehydrogenase in extracts of soybean roots, stems and leaves, as well as with the enzyme in extracts from a range of legume and non-legume plant species. However, Bradyrhizobium japonicum CB1809 bacteroids contained an immunologically distinct form of the enzyme. The relative intensities of immunoreactive bands in Western blots indicated that NAD-dependent glyceraldehyde-3-P dehydrogenase was expressed to a much greater extent in the plant fraction of soybean nodules than in soybean roots.