Nov 20, 1984 - enzyme-linked immunosorbent assay or by immunodiffusion test. ..... Preimmune serum (P) was also tested against these extracts. locust.
Plant Physiol. (1985) 77, 1004-1009 0032-0889/85/77/1004/06/$0 1.00/0
Intercellular Nodule Localization and Nodule Specificity of Xanthine Dehydrogenase in Soybean' Received for publication July 30, 1984 and in revised form November 20, 1984
ERIC W. TRIPLETT Plant Pathology Department, University of California, Riverside, California 92521 ABSTIRACI The distribution of xanthine dehydrogenase throughout the soybean plant as well as the intercellulr localizaton of xanthine dehydrogenase within soybean nodules was determined. Polyclonal antibodies againt purified xanthine dehydrogenase were prepared and used in an enzymelinked immunosorbent assay to determine whether xanthine dehydrogenase is a nodule-specific protein. This immunological assay showed that xanthine dehydrogenase is present in far greater concentration in the nodule than in any other plant organ. Immunodiffusion tests showed that anti-soybean nodule xanthine dehydrogenase would cross-react with nodule crude extracts from the ureide producers, soybean, cowpea, and lima bean, but would not cross-react with those of the amide producers, alfalfa and lupine. A crude extract from pea nodules cross-reacted slihtly with anti-soybean xanthine dehydrogenase. Anti-soybean xanthine dehydrogenase did not cross-react with buttermilk xanthine oxidase either by enzyme-linked immunosorbent assay or by immunodiffusion test. Fresh nodule sections from the ureide-producers, soybean, cowpea, and lima bean, all stained positively for xanthine dehydrogenase. The substrate-dependent stain was inhibited by aflopurinol and was observed only in the infected nodule cells of these species. Nodules from the amideproducers, alfalfa and white lupine, did not stain for xanthine dehydrogenase.
The ureides, allantoin and allantoic acid, are the predominant forms of nitrogen transported from nitrogen-fixing soybean nodules to other plant parts (15, 23). Synthesis of ureides occurs in the plant cells of nodules via de novo purine synthesis followed by purine oxidation (19). The intracellular and intercellular localizations of the enzymes responsible for ureide synthesis has received significant attention recently (6, 10, 21). Hanks et al. (11) discovered that soybean nodule uricase and allantoinase were localized in the peroxisomes and smooth ER, respectively. Newcomb and Tandon (16) observed that the uninfected cells of soybeans have enlarged peroxisomes and microbodies. Based on that observation, uricase and allantoinase were proposed to be localized in the uninfected cells of soybean nodules (16). Subsequently, Hanks et al. (10) separated uninfected and infected protoplasts from nodules by sucrose density gradient centrifugation and observed that both the total and specific activities of uricase were much higher in the uninfected protoplasts than the infected protoplasts. The specific activity of allantoinase was also much higher in the uninfected protoplasts (10). Bergmann et al. (5) have also shown, by immunohistochemistry, that uricase is
localized in the uninfected cells in the nodule. Shelp et al. (21) also separated uninfected and infected cells. Most of the total uricase activity was associated with infected cells; however, the uricase specific activity was over four times higher in uninfected cells than in infected cells (21). Since the enzymes of de novo purine synthesis and uricase were found in both cell types, Shelp et al. (21) concluded that both cell types were capable of ureide synthesis. However, Shelp et al. (21) proposed that uricase may be active only in the uninfected cells in vivo owing to the 02 requirement for the uricase reaction (30) and the presence of the 02-binding protein, Lb2, in the infected cells (22). XDH, a soluble enzyme which catalyzes the hydroxylations of hypoxanthine to xanthine and xanthine to uric acid in nodules (3, 11, 24), was not assayed in the infected and uninfected cell preparations of either Shelp et al. (21) or Hanks et al. (10). The intercellular localization of this enzyme is unknown. The work described here addresses this problem by describing the histochemical localization of XDH in nodule slices. Elucidation of the intercellular localization of this enzyme will aid in the identification of that intermediate of ureide synthesis which is transported between the infected and uninfected cells of soybean nodules. Bergmann et al. (5) recently identified the nodule-specific protein called nodulin-35 as root nodule uricase. Nodule uricase was found to be distinct from root uricase (5). The distribution of XDH throughout the soybean plant was studied here with antibodies directed against nodule XDH. Also, the cross-reactivities of antibodies to soybean nodule XDH with nodule crude extracts from other leguminous plants and animal xanthine oxidase were examined. Since Lb is known to be nodule-specific (28), the immunochemical experiments were performed with both anti-Lb and anti-XDH. A comparison between the distribution of XDH and Lb was useful in the interpretation of the XDH results. The antiLb data also serve as a positive control for the anti-XDH data. MATERIALS AND METHODS Plant Culture. Nodules used for enzyme purification came from field grown, 7-week-old soybean plants (Glycine max [L]. Meff. cv Pella) inoculated with Rhizobiumjaponicum strain 122 DES. Nodules used for histochemical and immunochemical assays came from greenhouse-grown plants cultured as described by Triplett et al. (26). Peas (Pisum sativum L.), lima beans (Phaseolus lunatus L.), and alfalfa (Medicago sativa L.) were inoculated with R. leguminosarum strain 128C53k, R. sp. 127E1 5, R. meliloti strain YA2, respectively. White lupines (Lupinus albus L.) and cowpeas ( Vigna unguiculata [L.] Walp.) were inoculated with commercial inoculum from Nitragin Co.,
Supported by the United States Department of Agriculture, Science 2Abbreviations: Lb, leghemoglobin; XDH, xanthine dehydrogenase; and Education Administration Competitive Grants Office Grant 83ELISA, enzyme-linked immunosorbent assay. CRCR-1-1287. 1004 I
HISTOCHEMICAL LOCALIZATION OF XANTHINE DEHYDROGENASE
Milwaukee, WI. Rhizobium strains were maintained on yeastextract mannitol medium as described by Triplett et al. (26). Protein Purification. XDH was assayed according to Triplett et al. (25). All spectrophotometric measurements were made with a Beckman DU-7U spectrophotometer. Purification of XDH from field nodules proved to be more difficult than the procedure used for greenhouse nodules (25). All steps were performed at 4°C. The buffer used throughout the purification was 10 mm K-phosphate (pH 7.8) with I mM DTE. Field nodules (200 g) were ground in a Waring Blendor with 500 ml buffer and 50 g insoluble PVP. The extract was then centrifuged at l0,OOOg for 20 min to remove debris, bacteroids, and intact organelles. The supernatant, which is referred to as the crude extract, was brought to 30% saturation with solid (NH4)2SO4. After stirring for 20 min, the sample was centrifuged at 10,OOOg for 20 min. The supernatant was brought to 45% saturation with solid (NH4)2SO4. After stirring and centrifugation, the pellet was resuspended in 30 ml of 20% (NH4)2SO4. The resuspended pellet was then applied to a 50-ml octyl-Sepharose column equilibrated with 20% (NH4)2SO4 in buffer. A 400-ml gradient of 20 to 0% (NH4)2SO4 in buffer was applied to the column. XDH was eluted with 100 ml of the above buffer without (NH4)2SO4. Active fractions were pooled and applied to a 50-ml DEAE-Sephadex column equilibrated with buffer. A 400-ml gradient of 0 to 400 mM KC1 in buffer was applied to the DEAE column. Active XDH fractions were pooled and concentrated on an Amicon YM-5 membrane. The sample was diluted to lower the salt concentration and then applied to a 10-ml reactive blue 2 column equilibrated with buffer. The column was washed with 50 ml buffer. Pure XDH was eluted with 5 mm NAD+ in buffer and concentrated to 1 ml on an Amicon YM-5 membrane. The method of Dilworth (9) was used for the purification of Lbs a and c. Lb a and XDH purified by the methods described above were injected into rabbits for the production of polyclonal antibodies as described below. PAGE. Vertical native and SDS polyacrylamide gels were electrophoresed and stained for protein and XDH activity as described previously (19). Antibody Production and IgG Purification. Antibodies to XDH and Lb a were prepared by multiple intradermal injections into New Zealand white rabbits as described by Vaitukaitis (27). Preimmune and immune IgGs were purified by applying 2 ml crude serum to a 1-ml protein A-agarose column followed by elution of the IgGs with 0.58% acetic acid in 0.15 M NaCl. The IgG fractions were then dialyzed against PBS (pH 7.0) and concentrated to a volume of 2 ml. Enzyme-Linked Immunosorbent Assay (ELISA). The noncompetitive solid phase ELISA described by Weeden et al. (29) was used to measure antigen levels in crude extracts. Purified preimmune and immune IgGs were used in the ELISA measurements and were diluted to 1 ug protein/ml for the XDH ELISAs. The purified IgGs were diluted 1:1000 for the Lb assays. The peroxidase-conjugated goat anti-rabbit IgG was diluted 1:1000 before use. Absorbance values obtained from preimmune samples were subtracted from the immune values in those cases where an endogenous peroxidase activity present in the plant tissue interfered with the assay. The A at 405 nm in each well of the ELISA plates was measured on a Bio-Tek EIA reader model EL 307. Preparation of Crude Extracts for ELISAs. Samples of leaves, stems, roots, pods, and nodules were taken from three 40-d-old soybean plants. Tissue was ground in liquid N2. To the resulting powder was added 3 ml of 50 mM NaHCO3/g fresh weight of tissue followed by filtering through four layers of cheese cloth. Aliquots of the resulting filtrates were then centrifuged for 10 min at 50,000g to remove particulate material. The supernatants were used in the ELISAs. The protein content of the supernatants
1005
was measured according to Bradford (7). Ouchterlony Double Diffusion Plates. The method of Ouchterlony (17) was used for the double diffusion tests. Purified IgGs were used in all double diffusion tests. Sectioning of Nodules. Fresh nodules were sectioned to about 1 mm thickness and fixed in 1% formaldehyde and 0.5 M K-phosphate (pH 7.0) under vacuum for 1 h at 20'C. Sections were then transferred through a series of 0.25, 0.5, 1.0 M sucrose in formaldehyde and buffer. Each step was done for 30 min at 20C. Sections were frozen in Tissue Tek II at -80'C. Nodule slices were cut to 30 Arm thickness at 1 5C with a Cryo-cut model cryomicrotome made by the American Optical Co. Histochemical Staining of XDH. The histochemical staining method of Rieder et al. (20) was modified to stain nodule sections for XDH activity. Sections were allowed to dry at room temperature for 5 min. A drop of the XDH reaction mixture was placed on the slide. The slide was incubated in the dark for 3 h at room temperature. The formaldehyde used in the fixation process slows the XDH reaction rate. This made a 3-h incubation time with the XDH reaction mixture necessary. Photographs of the sections were taken with a Zeiss MC 63 photomicrographic camera mounted onto a Zeiss compound microscope using Polaroid Polachrome 35-mm slide film. The XDH reaction mixture contained 0.55 ml of 1.8 mg/ml nitrobluetetrazolium, 1.15 ml hypoxanthine (3 ml 10 mm hypoxanthine in 0.1 N KOH + 2 ml 0.5 M Tris-HCl [pH 7.1]), 0.05 ml of 10 mg/ml MgC12*6H20, 0.1 ml of 4.5 mg/ml NAD', 2.5 ml of 20 g polyvinylalcohol dissolved in 50 ml of 50 mM Tris-HCl (pH 7.4) at 90C, 0.1 ml of 32 mg/ml NaN3, and 0.1 ml of 1.0 mg/ml phenazine methosulfate. The azide and phenazine methosulfate solutions were added last.
RESULTS Electrophoresis of Purified XDH. Samples of the above XDH preparation (10 ,g/lane) were run on a 7% native polyacrylamide gel. The gel was stained for enzyme activity as well as protein. A single protein band was observed on the gel which coresponds to the activity band on the gel (Fig. 1). No activity bands were observed when the substrate, hypoxanthine, was absent from the
1
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3
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5
6
7
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9
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FIG. 1. Electrophoresis of samples of purified soybean nodule XDH. XDH samples were applied to the second, third, fifth, sixth, eighth, and ninth lanes (from left to right) of a polyacrylamide gel which was stained for XDH and protein. The second and third lanes were exposed to the activity stain without substrate. The fifth and sixth lanes were stained for enzyme activity with the substrate, hypoxanthine. The seventh and eighth lanes were stained for protein. The portion of the gel stained for protein shrunk as it was placed in 10% methanol and 10% acetic acid following
silver staining.
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Table I. Purification of Soybean Nodule XDH Total Total Specific Purfiction Protein Activity Activity mg units units/mga -fold 1. Crude extract 2679 49.9 0.02 1.0 2. 30-45% (NH4)2SO4 806 17.4 0.02 1.2 3. Octyl Sepharose 95.7 3.38 0.10 5.3 4. DEAE-Sephadex 2.34 6.83 0.29 15.7 5. Reactive Blue 2 0.57 1.62 2.86 153.5 'Unit is defined as umol NADH produced/min at 250C.
Plant Physiol. Vol. 77, 1985
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% 100 34.8 18.8 13.7 3.2
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w
015
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FIG. 3. Immunodiffusion test for cross-reactivity between antibodies (I) against soybean nodule XDH and crude nodule extracts (outer wells) from soybean (1), cowpea (2), lima bean (3), pea (4), lupine (5) and alfalfa (6). Preimmune serum (P) was also tested against these extracts.
0 05F
350 3 50
450 550 560 450 WOVELENGTH (nm)
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FIG. 2. Absorption spectrum of purified soybean nodule XDH (1.0
mg/ml) in 10 mm K-phosphate (pH 7.8) with I mM DTE. gel activity stain. Also, a single protein band was observed on a 10% SDS gel corresponding to the mol wt of the XDH subunit. The results of a typical XDH purification are summarized in Table I. Absorption Spectrum of Purified XDH. A visible absorption spectrum of a preparation of I mg/ml purified XDH from soybean nodules was made (Fig. 2). The absorption spectrum of the soybean enzyme is very similar to that of avian livers published by Cleere and Coughlan (8). The spectrum shows a broad peak between 455 and 458 nm. Also, shoulders are observed at 425 and 550 nm. This observation agrees with the iron and flavin content measured in soybean nodule XDH by Triplett et al. (25). This absorption spectrum shows that this preparation has no detectable amounts of contaminating proteins which absorb light in the visible region. Ouchterlony Double Diffusion Tests. Anti-XDH did not crossreact with crude extracts from non-ureide producing nodules of white lupine and alfalfa (Fig. 3). However, nodule crude extracts from the ureide producers soybean, cowpea, and lima bean all cross-reacted with anti-XDH (Fig. 3). A crude extract from pea nodules showed slight cross-reactivity with anti-soybean XDH. No precipitin reaction could be observed between antibodies to soybean nodule XDH and buttermilk xanthine oxidase. In all immunodiffusion tests, the preimmune crude serum or purified IgGs showed no precipitin reaction. Antibodies directed against soybean nodule Lb strongly crossreacted with lima bean and soybean nodule extracts and weakly with cowpea and lupine nodule extracts (Fig. 4). Nodule extracts of pea and alfalfa did not cross-react with antibodies against soybean nodule Lb a (Fig. 4). The lack of cross-reactivity between antibody against soybean Lb a and the Lb of pea and alfalfa was expected since substantial microheterogeneity has been found between Lbs of different genera (1, 9, 12, 13). Jing et al. (13) found that antibodies against the purified alfalfa Lbs did not cross-react with the Lbs of soybean, lupine, jackbean, and black
FIG. 4. Immunodiffusion test for cross-reactivity between antibodies (I) against soybean nodule Lb a and crude extracts (outer wells) from soybean (1), cowpea (2), lima bean (3), pea (4), lupine (5), and alfalfa (6). Preimmune serum (P) was also tested against these extracts.
locust. Hurrell et al. (12) found that antibody against purified lupine Lb did not cross-react with soybean, broad bean, or serradella. Also, antibody against soybean Lb a did not crossreact with lupine Lb (12). ELISA. Anti-XDH does not cross-react with Lb a. Anti-Lb a does not cross react with XDH but does cross-react with Lb c. This observation is consistent with the results of Verma and Bal (28) who found that polyclonal antibodies produced against slow and fast moving components of Lb cross-reacted with each other. XDH and Lb a were measured in extracts of nodules, roots, stems, leaves, and seeds by the ELISA technique. Lb was found to be nodule specific (Fig. 5), confirming the work of Verma and Bal (28). Nodules were found to contain a far greater concentration of XDH than other plant organs (Fig. 6). Other plant parts had detectable but low amounts of XDH. The ELISA method used in this experiment is far more sensitive than the standard assay for enzymic activity and is capable of detecting inactive XDH. Buttermilk xanthine oxidase was not detected with soybean
HISTOCHEMICAL LOCALIZATION OF XANTHINE DEHYDROGENASE
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FIG. 5. An ELISA designed to determine the distribution of Lb throughout the soybean plant. Crude extracts of nodules, stems, roots, leaves, and seeds as well as a mixture of equal weights (in ng) of purified Lbs a and c.
LOG PROTEIN (4g)
was not dependent on the presence of hypoxanthine (data not shown).
/
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PROTEIN
FIG. 6. An ELISA designed to determine the distribution of XDH throughout the soybean plant. Crude extracts of nodules, stems, roots, leaves, pods, and seeds as well as purified XDH were measured.
anti-XDH by an ELISA. Four gg of buttermilk xanthine oxidase could not be detected in this assay which is capable of detecting 5 ng of soybean XDH. Histochemical Staining of XDH. Sections of nodules from six legume species were prepared and stained for XDH activity. Three of the species, soybean, cowpea, and lima bean, produce ureides as the major nitrogenous compounds transported in the xylem (2, 25). The other three species, alfalfa, peas, and white lupine, produce mainly amides in the nodule for subsequent xylem transport (2). The histochemical stain was applied to the nodule slices in the presence or absence of the substrate, hypoxanthine. Photographs of the slices were taken at magnifications of x 10 (Fig. 7, a and b) and x 100 (Fig. 7, c and d). Sections from soybean nodules showed significantly higher staining in the presence of hypoxanthine (Fig. 7, a and c) than in its absence (Fig. 7, b and d). Furthermore, this substratespecific staining was observed only in the infected cells of the central region of the nodule. This is particularly evident at higher magnification (Fig. 7, c and d) where intense substrate-dependent staining was observed in the large infected cells while no such staining was observed between the infected cells in the uninfected cells. Allopurinol inhibited the substrate-specific staining. The low degree of staining observed in the absence of substrate probably is caused by the oxidation of endogenous substrates which may be catalyzed by the many dehydrogenases present in the tissue. Cowpea and lima bean nodule slices showed identical results (data not shown). Sections from white lupine and alfalfa nodules showed no staining of either cell type in the presence or absence of hypoxanthine (data not shown). Pea nodule sections exhibited significant staining ofthe central portion ofthe nodule but this staining
DISCUSSION The experiments described here were done with two objectives in mind. First, ELISA experiments were performed to determine whether XDH was nodule specific. That is, these assays demonstrate the distribution of the enzyme throughout the plant. Second, histochemical staining of XDH on nodule sections was performed to determine the intercellular localization of the enzyme. Auger and Verma (4) first proposed that nodule specific proteins (nodulins) may be present only in the infected cells of nodules. Lb is an example of a nodulin (28) which is present only in the infected cells of the central region of legume nodules (22). Since ureides are only produced by soybeans which are nodulated and actively fixing nitrogen, XDH may be nodule specific and may be present only in the infected cells of soybean nodules. To be more confident of the immunological methods employed in these experiments, anti-Lb a was used as a positive control for the results obtained by using anti-XDH. Lb was chosen since it is a soluble protein, as is XDH, and is a nodulin confined to the infected cells. The nodule specificity of Lb, first demonstrated by Verma and Bal (28), was confirmed by the results presented here (Fig. 5). The results of Verma and Bal (28) were extended in this study, since all plant organs were measured for Lb, not just nodules and roots as had been done by Verma and Bal (28). No Lb was detected in any plant organ other than the nodule. XDH, on the other hand, is not nodule specific. Antigenic XDH was detected in other plant organs, although in much lower concentrations than in nodules (Fig. 6). While this work was in progress, Bergmann et al. (5) reported that nodulin-35 was an isozyme of uricase found only in the uninfected cells of soybean nodules. There appears to be no correlation between nodule specificity of proteins and the intercellular localization of nodule proteins since one nodulin, Lb, is restricted to infected cells, while another nodulin, uricase, is confined to the uninfected cells. According to the histochemical staining, XDH is localized mainly, if not exclusively, in the infected cells of soybean, cowpea, and lima bean nodules. Three controls were used to verify the validity of the stain. First, staining was found to be substratedependent, since the staining done in the absence of substrate was far less intense than in the presence of substrate. Second, allopurinol, a specific inhibitor of XDH (3, 24), inhibited staining of XDH in the presence of the substrate, hypoxanthine. Third, nodules from non-ureide producing legumes did not stain positively for XDH activity. The localization of XDH in the infected cells may have important consequences in the identification of the transport
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Plant Physiol. Vol. 77, 1985
FIG. 7. Photomicrographs of soybean nodule slices stained for XDH activity in the presence (a and c) and absence (b and d) of the substrate hypoxanthine at magnifications of x 10 (a and b) and x 100 (c and d).
form of nitrogen between infected and uninfected cells. Newcomb and Tandon (16) recently proposed that uricase, the enzyme which oxidizes uric acid to allantoin, is present in the enlarged peroxisomes of the uninfected cells of soybean nodules. Hanks et al. (10) have since separated infected and uninfected protoplasts from soybean nodules and have found that uricase is predominantly in the uninfected protoplasts. Bergmann et al. (5) have confirmed the localization of uricase in the uninfected cells by immunohistochemical staining. The results of Shelp et al. (21), however, suggest that uricase is present in the infected as well as the uninfected cells. However, uricase in crude cowpea nodule extracts and in purified form requires 02 and has been shown to have a Km for 02 of 29 and 28 gM, respectively (18, 30). Lucas et al. (14) have recently shown that the Km for 02 of soybean nodule uricase is 31 gM. The free 02 concentration in the infected cells of soybean nodules has been estimated to be about 10 nM (1). With the free 02 concentration in infected cells 1000 times lower than the uricase Km for 02, the uricase reaction may not function appreciably in the infected cells in vivo. Most of the evidence gathered to date supports the notion that the uricase reaction is confined largely to the uninfected cells of nodules. The histochemical evidence presented here suggests that XDH is localized in the infected cells of nodules. Also, the hypoxanthine and xanthine hydroxylation reactions in soybean
nodules will not use 02 as electron acceptor but rather requires NAD' (24). Therefore, XDH is capable of catalyzing the purine oxidation reactions in the microaerophilic environment of the infected cells. The observed vocalizations of XDH and uricase in the infected and uninfected cells, respectively, imply that uric acid is the intermediate of allantoic acid synthesis which is transported between the infected and uninfected cells. The proposed vocalizations of these two enzymes agrees with the distribution of 02 in the nodule and the differences in the electron acceptors which the two enzymes use to catalyze their respective reactions. The results obtained with the P. sativum nodules were ambiguous. Small amounts of ureides have been detected in the xylem sap of modulated plants of Pisum arvense but not P. sativum (2). This faint cross-reactivity observed between anti-soybean XDH and the pea nodule crude extract may be explained in two ways. First, nodules of P. sativum may produce small quantities of ureides and therefore may have only small amounts of those enzymes responsible for ureide synthesis, including XDH. Second, pea nodules may have a significant amount of XDH which is not closely related phylogenetically to soybean nodule XDH. The lack of cross-reactivity observed between antibodies directed against soybean nodule XDH and animal xanthine oxidase is somewhat surprising given the fact that the enzymes from
HISTOCHEMICAL LOCALIZATION OF XANTHINE DEHYDROGENASE the two sources have the same subunit and holoenzyme mol wt (25), similar Mo and Fe content (25), and nearly identical visible absorption spectra (10; Fig. 1). XDH from the three species of ureide-producing legumes appear to be very similar as they all cross-react strongly with soybean anti-XDH. LITERATURE CITED 1. APPLEBY CA 1984 Leghaemoglobin and Rhizobium respiraton. Annu Rev Plant Physiol 35: 443-478 2. ATKINS CA 1982 Ureide metabolism and the significance of ureides in legumes. In NS Subba Rao, ed, Advances in Agricultural Microbiology. Oxford and IBH, New Dehli, pp 25-51 3. ATKINS CA, RM RAINBIRD, JS PATE 1980 Evidence for a purine pathway of ureide synthesis in Nrfixing nodules of cowpea ( Vigna unguiculata L. Walp). Z Pflanzenphysiol 97: 249-260 4. AUGER S, DPS VERMA 1981 Induction and expression of nodule-specific host genes in effective and ineffective root nodules of soybean. Biochemistry 20: 1300-1306 5. BERGMANN H, E PREDDIE, DPS VERMA 1983 Nodulin-35: a subunit of specific uricase (uricase II) induced and localized in the uninfected cells of soybean nodules. EMBO J 2: 2333-2339 6. BOLAND MJ, JF HANKS, PHS REYNOLDS, DG BLEVINs, NE TOLBERT, KR SCHUBERT 1982 Subcellular organization of ureide biogenesis from glycolytic intermediates and ammonium in nitrogen-fixing soybean nodules. Planta 155: 45-51 7. BRADFORD MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254 8. CLEERE WF, MP COUGHLAN 1975 Avian xanthine dehydrogenases-I. Isolation and characterization of the turkey liver enzyme. Comp Biochem Physiol 50B: 311-322 9. DILWORTH MJ 1980 Leghemoglobins. Methods Enzymol 69: 812-823 10. HANKS JF, K SCHUBERT, NE TOLBERT 1983 Isolation and characterization of infected and uninfected cells from soybean nodules. Role of uninfected cells in ureide synthesis. Plant Physiol 71: 869-873 11. HANKS JF, NE TOLBERT, KR SCHUBERT 1981 Localization of enzymes of ureide biosynthesis in peroxisomes and microsomes of nodules. Plant Physiol 68: 65-69 12. HURRELL JG, KR THULBORN, WJ BROUGHTON, MW DILWORTH, SJ LEACH 1977 Leghemoglobins: Immunochemistry and phylogenetic relationships. FEBS Lett 84: 244-246 13. JING Y, AS PAAU, WJ BRILL 1982 Leghemoglobins from alfalfa (Medicago sativa L. Vernal) root nodules. I. Purification and in vitro synthesis of five leghemoglobin components. Plant Sci Lett 25: 119-132 14. LUCAS K, MJ BOLAND, KR SCHUBERT 1983 Uricase from soybean root
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nodules: purification, properties, and comparison with the enzyme from cowpea. Arch Biochem Biophys 226: 190-197
15. MATSUMOTO T, M YATAZAWA, Y YAMAMOTO 1977 Incorporation of '5N into allantoin in nodulated soybean plants supplied with `5N2. Plant Cell Physiol 18: 459-462 16. NEWCOMB EH, SR TANDON 1981 Uninfected cells of soybean root nodules: ultrastructure suggest key role in ureide production. Science 212: 1394-1396 17. OUCHTERLONY 0 1948 In vitro method for testing the toxin producingcapacity of diphtheria bacteria. Acta Pathol Microbiol Scand 25: 186-196 18. RAINBIRD RM, CA ATKINS 1981 Purification and some properties of urate oxidase from nitrogen-fixing nodules of cowpea. Biochim Biophys Acta 659: 132-140 19. REYNOLDS PHS, Mi BOLAND, DG BLEVINS, DD RANDALL, KR SCHUBERT 1982 Ureide biogenesis in leguminous plants. Trends Biochem Sci 7: 366368 20. RIEDER H, HF TEUTSCH, D SASSE 1978 NADP-dependent dehydrogenases in rat liver parenchyma. I. Methodological studies on the qualitative histochemistry of G6PDH, 6PGDH, malic enzyme and ICDH. Histochemistry 56: 283-298 21. SHELP BJ, CA ATKINS, PJ STORER, DT CANVIN 1983 Cellular and subcellular organization of pathways of ammonia assimilation and ureide synthesis in nodules of cowpea ( Vigna unguiculata L. Walp.). Archiv Biochem Biophys 224: 429-441 22. SMITH JD 1949 The concentration and distribution of haemoglobin in the root nodules of leguminous plants. Biochem J 44: 585-591 23. STREETER JG 1979 Allantoin and allantoic acid in tissues and stem exudate from field-grown soybean plants. Plant Physiol 63: 478-480 24. TRIPLEIT EW, DC BLEVINS, DD RANDALL 1980 Allantoic acid synthesis in soybean root nodule cytosol via xanthine dehydrogenase. Plant Physiol 65: 1203-1206 25. TRIPLETr EW, DG BLEVINS, DD RANDALL 1982 Purification and properties of soybean nodule xanthine dehydrogenase. Archiv Biochem Biophys 219: 39-46 26. TRIPLETT EW, JJ HEITHOLT, KB EVENSEN, DG BLEVINS 1981 Increase in internode length of Phaseolus lunatus L. caused by inoculation with a nitrate reductase-deficient strain of Rhizobium sp. Plant Physiol 67: 1-4 27. VAITUKAITIS JL 1981 Production of antisera with small doses of immunogen: multiple intradermal injections. Methods Enzymol 73: 46-52 28. VERMA DPS, AK BAL 1976 Intracellular site of synthesis and localization of leghaemoglobin in soybean root nodules. Proc Natl Acad Sci USA 73: 38433847 29. WEEDEN NF, RC HIGGINS, LD GOTTLIEB 1982 Immunological similarity between a cyanobacterial enzyme and a nuclear DNA-encoded plastidspecific isozyme from spinach. Proc Natl Acad Sci USA 79: 5953-5955 30. Woo KC, CA ATKINS, JS PATE 1981 Ureide synthesis in a cell-free system from cowpea ( Vigna unguiculata L. Walp) nodules. Studies with 02, pH and purine metabolites. Plant Physiol 67: 1156-1160
