Immunoaffinity Purification and Comparison of Allantoinases … · Plant Physiol. (1995) 107: 435-441 Immunoaffinity Purification and Comparison of Allantoinases from Soybean Root

Plant Physiol. (1995) 107: 435-441

Immunoaffinity Purification and Comparison of Allantoinases from Soybean Root Nodules and Cotyledons'

Jennifer A. Bell and Mary Alice Webb*

Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 47907-1 1 55

Allantoinase (allantoin amidohydrolase, EC 3.5.2.5) catalyzes the conversion of allantoin to allantoic acid in the final step of ureide biogenesis. We have purified allantoinase more than 4000-fold by immunoaffinity chromatography from root nodules and cotyledons of soybean (Clycine max [L.] Merr.). We characterized and com- pared properties of the enzyme from the two sources. Seed and nodule allantoinases had 80% identity in the first 24 amino acid residues of the N terminus. Two-dimensional gel electrophoresis of the purified enzymes showed that multiple forms were present in each. Allantoinases from nodules and cotyledons had very low affinity for allantoin with a K,,, for allantoin of 17.3 mM in cotyledons and 24.4 mM in nodules. Both had activity in a broad range of pH values from 6.5 to 7.5. I n addition, purified allantoinase from both sources was very heat stable. Enzyme activity was stable after 1 h at 70"C, decreased gradually with heating to 85"C, and was lost at 90 to 95°C. Although these studies have revealed some differences between allantoinases in seeds and nodules, the differences were not reflected in key enzyme properties. The immunoaffinity approach enabled purification of allantoinase from soybean root nodules and simplified i t s purification from cotyledons, thereby allowing characterization and comparison of the enzyme from the two sources.

Both root nodules and seeds make important contribu- tions to nitrogen nutrition in plants, but they function in different ways and at different stages of plant development. In soybean (Glycine max L.) and some other legumes, ureide metabolism is induced by the onset of rhizobial infection and nodulation (Fujihara et al., 1977; Matsumoto et al., 1977a, 1977b; Tajima et al., 1977), and root nodules assimilate recently fixed nitrogen into the ureides allantoin and allantoic acid for export to the shoot (Pate, 1973; Mat- sumoto et al., 1977a; Herridge et al., 1978). Soybean seedlings also produce ureides in the absence of infection with rhizobia (Matsumoto et al., 1977a; Polayes and Schubert, 1984), and ureide production in the cotyledons may con- tribute to the export of nitrogen reserves to support seed- ling growth (Webb and Lindell, 1993). Ureides arise from de novo purine synthesis in nodules (Atkins et al., 1980; Boland and Schubert, 1982), whereas in cotyledons of germinating seedlings they may arise from both de novo

purine synthesis and purine salvage (Polayes and Schubert, 1984). The presence of ureide biogenesis in nodules and cotyledons provides an opportunity to compare features of the pathway in the two different structures in distinct physiological and developmental contexts.

Studies of nodule-specific proteins, or nodulins, have identified some enzymes of nitrogen assimilation or ureide biogenesis that differ from their counterparts in other plant organs. For example, a nodule-specific isoform of Gln synthetase was identified in Phaseolus vulgaris (Lara et al., 1983); however, no major kinetic differences between the nodule-specific isoform and other isoforms were found (Cullimore et al., 1983). Although recent studies have shown that uricase in soybean root nodules is virtually identical with uricase in seeds (Tajima et al., 1991, 1993; Damsz et al., 1994), it is distinct from the enzymes respon- sible for uricase activity in uninfected roots of soybean (Tajima et al., 1983).

This study focused on allantoinase (allantoin amidohydrolase, EC 3.5.2.5), which catalyzes the hydrolysis of allantoin to allantoic acid in the final step of ureide biogenesis in seeds and nodules. Previous studies of allantoinase in crude preparations (Franke et al., 1965; Thomas et al., 1983; Rao et al., 1988; Reddy et al., 1989) have indicated that there may be tissue-specific isoforms. Webb and Lindell (1993) recently described the purification of allantoinase from soybean seeds. Allantoinase from soybean nodules was previously resistant to purification (Webb, 1988; M.A. Webb and K.R. Schubert, unpublished data). However, in preliminary studies, the ob- servation that antibodies produced against seed allantoinase also reacted with the nodule enzyme suggested that an im- munological approach to purification might be effective.

In this study, our initial objective was to purify allantoinase from nodules and cotyledons of soybean (G. max L., cv Williams 82) by immunoaffinity chromatography. We then sought to compare key properties of cotyledon and nodule allantoinase, as a first step in characterizing and comparing features of the enzyme in the two locations.

MATERIALS AND METHODS

Plant Material

Soybean (Glycine max L., cv Williams 82) seeds were

chlorite and rinsed well with distilled water. For cotyledon extracts, seeds were planted in a 1:l (v/v) mixture of

This work was suPPorted bY grant DCB-9104860 from the surface sterilized in 1% (v/v) commercial sodium hypo- National Science Foundation and funding from Agricultura1 Re- search Programs, I'urdue University. This is Journal I'aper No. 14035 of the Purdue University Agricultural Experiment Station.

* Corresponding author; e-mail [email protected]; fax 1-317-494 -5896. Abbreviation: TBS, Tris-buffered saline.

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436 Bell and Webb Plant Physiol. Vol. 107, 1995

perlite/vermiculite, and cotyledons were harvested by dis- secting them from the seeds 24 h after imbibition. For root nodules, seeds were inoculated with Bvadyrkizobium japoni- cum (Nitragin Co., Milwaukee, WI) and planted in a 1:l (v/v) mixture of perlite/vermiculite. Plants were grown in a greenhouse, watered daily, and fertilized twice weekly with nitrogen-free fertilizer. Soybean nodules were harvested 28 d after inoculation, frozen in liquid nitrogen, and stored at -80°C until use.

Protein Extraction

Cotyledons or nodules were ground with a pestle in a chilled mortar in 4 volumes of TBS (20 mM Tris, 500 mM NaCl), pH 7.5, with 0.1% (v/v) of two protease inhibitor solutions (Ronnet et al., 1984) plus DTT (100 p ~ ) , EDTA (10 mM), and 0.1 % (w/w) polyvinylpolypyrrolidone. The first inhibitor solution contained aprotinin (2 mg/mL), leupep- tin (1 mg/mL), antipain (2 mg/mL), and benzamide (10 mg/mL), and the second solution contained chymostatin (1 mg/mL) and pepstatin (1 mg/mL) dissolved in DMSO (Ronnet et al., 1984). Extracts were filtered through two layers of Miracloth and centrifuged at 30,OOOg for 30 min at 4"C, and the supernatant was collected. The supernatant from cotyledon extract was refiltered through two additional layers of Miracloth to remove any remaining lipid from the sample. Next, the supernatants from cotyledon or nodule extracts were filtered through 0.2-pm Millipore (Bedford, MA) filters to remove particulate matter. Extracts were kept on ice until they were used.

Anti-Allantoinase Antibodies

The polyclonal antibodies used in these studies were made against native allantoinase purified from soybean seeds, as detailed by Webb and Lindell (1993).

lmmunotitrations

The appropriate amount of antibody was added to 50 pL of crude extract from either seeds or nodules in either 0.1 M

Tes, pH 7.5, or 20 mM bis-Tris, pH 6.2, in a microcentrifuge tube. TBS was added as necessary to bring the total volume to 75 pL. Solutions were incubated at room temperature for 1 h and centrifuged at 2000g for 5 min at 4°C. Aliquots of 10 pL were removed from the supernatant for assay.

Affinity Column Preparation and lmmunoaffinity Chromatography

An Affinica Antibody Orientation kit (Schleicher & Schuell) was used to prepare the antibody affinity column according to package directions. Before use the affinity column was equilibrated in TBS. Cotyledon or nodule extracts were passed through the column at room temperature at a flow rate of 0.3 mL/min. Unbound protein was washed through the column with 75 mL of TBS, and bound protein was eluted from the column in 5 mL of 8.0 M urea in 0.05 M Tris (pH 8.0). The column eluate was dialyzed overnight in two changes of 0.05 M Tes (pH 7.5). Protein was precipitated by the addition of 7 volumes of cold

acetone to the eluate and incubated at -20°C overnight. The precipitate was collected by centrifugation at 30,00Og, and the pellet was dissolved in 0.05 M Tes (pH 7.5).

Allantoinase Activity Assay

The assay for allantoinase activity was based on proce- dures modified by Schubert (1981) from Vogels and van der Drift (1970). A11 assays were performed in duplicate, and controls were included to account for nonenzymatic degradation of allantoin and endogenous allantoic acid in the extracts. A unit of activity was defined as the amount of enzyme that catalyzed the formation of 1 pmol of allantoic acid/min at 30°C.

Protein Determination

Protein concentrations were determined by the method of Bradford (1976) or with the Pierce (Rockford, IL) Micro BCA protein assay reagent (Smith et al., 1985).

Cel Electrophoresis and Western Blot Analysis

Protein extracts were separated by SDS-PAGE with a bis:acrylamide ratio of 0.8:30 (w/w) and an acrylamide concentration of 12.5%, according to the method of Laemmli (1970) in a Bio-Rad mini-gel apparatus. Gels were stained with silver or Coomassie brilliant blue (Merril, 1990). For two-dimensional gel electrophoresis, first- dimension tube gels were prepared according to Bio-Rad Mini-Protean 2-D instructions with LKB Ampholines, pH 3.5 to 9.5 and pH 7 to 9. Second-dimension slab gels were prepared according to Laemmli (1970). For western blot analysis, proteins were electroblotted onto nitrocellulose and probed with anti-allantoinase antibodies as described previously (Webb and Lindell, 1993)

100 A a CI

80 8 Y

*. .- > 60 C .- U

ü a 8 40 c O c .- c.

s 20 ã

0 ' " ' . . ' . . I . . ' . . ' O 1 5 30 4 5 6 0 7 5

Antibody Added (pg) Figure 1. lmmunotitration of allantoinase activity in crude extracts of nodules (circles) and seeds (squares). Open symbols indicate preimmune serum; closed symbols indicate anti-allantoinase antibodies. Control activities without antisera were approximately 200 nmol mL-' min-' in nodule and seed extracts.

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Soybean Nodule and Cotyledon Allantoinase 437

Table I. Immunoaffinity purification of allantoinase from soybean root nodulesSample

Crude extractColumn eluate

after dialysis

Total Activity

ILinol min~ '0.781.30

Protein

mg mL~'

6.390.01

Specific Activity

fimol mg" ' min" '0.01

48.3

Purification

-fold1

4830

N-Terminal Sequencing

Affinity-purified allantoinase was subjected to SDS-PAGE according to the method of Yuen et al. (1986) forN-terminal sequence analysis. After electrophoresis, pro-teins were electroblotted onto ProBlott (Foster City, CA)membrane and stained with Coomassie brilliant blue R-250(Matsudaira, 1987). Protein bands corresponding to a mo-lecular mass of 30 kD were excised from the membrane.The N-terminal sequence was determined with an AppliedBiosystems (Foster City, CA) model 477A Pulsed-Liquidsequencer.

Km Determination

The Xm of affinity-purified allantoinase from seeds andnodules was determined with allantoinase assays of bothsamples in concentrations of allantoin from 2.5 to 25 mM.Controls were included as described for each substrateconcentration.

Determination of pH Optima

Samples of purified allantoinase were assayed in 0.1 MMes in the range of pH 5.0 to 6.5 or 0.1 M bis-Tris propanein the range of pH 6.0 to 10.0 at intervals of 0.5 pH unit.Controls were included as described for each pH pointassayed.

Analysis of Heat Stability

Samples of purified allantoinase were heated in a waterbath. The temperature of the sample was monitored con-tinuously, and 50-juL aliquots were removed from the sam-ple at intervals of 5°C between 70 and 95°C. Aliquots wereimmediately added to 1 mL of assay buffer and held atroom temperature until the experiment was completed.Samples were then assayed immediately. During heatingfrom 70 to 95°C, the temperature increased at a rate ofapproximately 0.5°C/min. In a second experiment allanto-inase was rapidly heated to 70°C and held at that temper-ature for 1 h. Samples were removed at 15-min intervals,added to assay buffer, and assayed after completion of theexperiment as above.

RESULTS

To determine whether antibodies against seed allantoin-ase were cross-reactive with the enzyme in nodules, theantibodies were tested for their ability to titrate activity ina crude extract of nodule proteins. Addition of increasingamounts of anti-allantoinase to both crude seed and noduleextracts reduced allantoinase activity, whereas no reduc-

tion in activity was seen with addition of preimmune sera(Fig. 1). Activity was reduced more in seed extracts than innodule extracts.

Because these results suggested that an immunologicalapproach could be used to purify the nodule enzyme,anti-allantoinase antibodies were used to prepare an im-munoaffinity column. Allantoinase from nodules was pu-rified 4830-fold with this column to a final specific activityof 48.3 jumol min"1 mg"1 protein (Table I). Silver-stainedSDS-PAGE gels of affinity-purified protein showed a singlepolypeptide of 30 kD, which was not prominent in crudeextracts (Fig. 2, A and B). Western blot analysis of column-purified protein probed with anti-allantoinase also re-vealed a single immunologically detectable polypeptidecorresponding to 30 kD that reacted weakly (Fig. 2D). The30-kD polypeptide was not detected when crude extracts ofnodule proteins were probed with anti-allantoinase(Fig. 2C).

The immunoaffinity column also enabled purification ofallantoinase from cotyledons for characterization and com-parison with the purified nodule enzyme. Allantoinase waspurified 4160-fold from cotyledons to a specific activity of41.6 /umol min"1 mg"1 protein (Table II). Analysis of col-umn eluate by SDS-PAGE revealed a 30-kD polypeptide insilver-stained gels (Fig. 3A). Western blot analysis of col-umn-purified protein probed with anti-allantoinaseshowed that the 30-kD polypeptide was strongly reactiveand revealed additional reactive bands at 45 and 50 kD(Fig. 3B). The intensity of immunoreaction was much strong-

C D

29.0-

18.4-

14.3-iFigure 2. A and B, SDS-PAGE analysis of nodule proteins, gel stainedwith silver; C and D, immunoblot of duplicate gel, probed withanti-allantoinase antibodies. Lanes A and C were loaded with 100 jagof crude protein extract; lanes B and D were loaded with 10 jj,g ofpurified nodule allantoinase. Numbers refer to molecular massstandards. www.plantphysiol.orgon April 16, 2020 - Published by Downloaded from

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Table II. Immunoaffinity purification of allantoinase from soybean cotyledonsSample

Crude extractColumn eluate

after dialysis

Total Activity

4.691.60

Protein

mg mL~ '

37.80.01

Specific Activity

limol mg~ ' rnirT '

0.0141.6

Purification

-fold1

4160

er with purified seed allantoinase than with an equivalentamount of purified nodule allantoinase (Figs. 2D and 3B).

The purified enzyme preparations were separated bytwo-dimensional gel electrophoresis, blotted onto nitrocel-lulose, and probed with anti-allantoinase. Approximatelysix to eight immunoreactive spots were observed in west-ern blots of cotyledon and nodule enzymes, indicating thatmultiple forms of the 30-kD polypeptide were present inboth (Fig. 4). Similar patterns were observed for cotyledonand nodule allantoinases, with multiple forms beingspaced equidistantly and spanning similar pi values. Inthese blots the immunoreaction with purified nodule allan-toinase (Fig. 4A) was also weaker than that with an equiv-alent amount of seed allantoinase (Fig. 4B).

For sequence comparison, the amino acid sequence ofnodule allantoinase was determined for the first 24 resi-dues of the N terminus of nodule allantoinase. The nodulesequence had 80% identity (Fig. 5) with the N-terminalsequence of seed allantoinase (Webb and Lindell, 1993).Neither sequence shared any significant similarity withother sequences in computer data banks, including se-quences for allantoinase in yeast (Buckholz and Cooper,1991).

Seed and nodule allantoinases had high Xm values forallantoin of 17.3 and 24.4 mM, respectively (Fig. 6). Bothenzymes were active over a broad range of pH values from6.5 to 7.5, with half-maximal activity at pH 5.3 and 8.5 (datanot shown).

Allantoinases purified from seeds and nodules wereremarkably stable to high temperature. The enzymesretained close to the initial activity for up to 1 h ofheating at 70°C (Fig. 7). Activity in both seeds andnodules decreased gradually with heating above 70°Cand was lost above 85°C (Fig. 8). Nodule and seed allan-toinases held at room temperature for 24 h retained fullactivity (data not shown).

DISCUSSION

Immunoaffinity Purification

Immunoaffinity chromatography provided a rapid andeffective means of purifying allantoinase from soybeancotyledons and root nodules. The affinity column approachenabled the first purification of the nodule enzyme, whichhad been resistant to previous attempts at purification(Webb, 1988; MA. Webb and K.R. Schubert, unpublisheddata). Nodule allantoinase showed an increase in totalactivity with purification, suggesting the presence of aninhibitor of enzyme activity in the crude extracts. Immu-noaffinity purification also simplified purification of allan-toinase from soybean seeds over the conventional proce-

dure developed previously (Webb and Lindell, 1993),which was lengthy and required special equipment. Theability to readily purify allantoinase allowed us to charac-terize and compare properties of the enzyme in cotyledonsand nodules.

Properties of Allantoinase

Purified nodule and cotyledon enzymes had the samemolecular mass, identical with that obtained previously forseed allantoinase (Webb and Lindell, 1993) and both exhib-ited similar patterns after two-dimensional gel electro-phoresis (Fig. 4). Analysis of the N-terminal amino acidsequences revealed differences in at least three residuesamong the first 24 amino acids. Since two residues in theseed sequence were undetermined (Fig. 5) and some wereambiguous (Webb and Lindell, 1993), the differences mayextend further. The observed differences in affinity of anti-allantoinase for cotyledon and nodule enzymes in westernblot analysis (Figs. 2B and 3B) indicated that the two pro-teins do not present identical epitopes, suggesting addi-tional sequence disparity beyond the N termini. The inabil-ity of the antibodies to recognize allantoinase in crudenodule extracts may have been due in part to differencesbetween the antigens. However, reactivity was sufficient torecognize the purified nodule allantoinase.

Previous investigators of allantoinase have consistentlyreported that the partially purified enzyme had a high Kmfor allantoin, indicating a low affinity for this substrate.

B

68.0-

43.0-

Figure 3. A, SDS-PACE analysis of purified cotyledon allantoinase,10 jug. Gel stained with silver. B, Immunoblot of duplicate gelprobed with anti-allantoinase antibodies. Numbers refer to molecu-lar mass standards. www.plantphysiol.orgon April 16, 2020 - Published by Downloaded from



Soybean Nodule and Cotyledon Allantoinase 439

A Pi 3.5kD

68-43-

29-

18.4-

p!9.5

B

68-43-

29-

18.4-

Figure 4. Immunoblot analysis of purified nodule allantoinase (A, 5/j.g) and cotyledon allantoinase (B, 5 /j.g) after two-dimensional gelelectrophoresis. Blots were probed with anti-allantoinase antibodies.

Previously reported values for soybeans included 10 ITIM inshoots (Thomas et al., 1983), 6.7 mM in seeds (Franke et al.,1965), and 50 mM in nodules (M.A. Webb and K.R. Schu-bert, unpublished data). In this study, we determined theKm for allantoin of purified allantoinase and found highvalues in both seeds and nodules of 17.3 and 24.4 mM,respectively. Allantoinases purified from other plantsources have generally shown a high Km for allantoin,including 14 mM in germinating castor beans (Ory et al.,1969), 13 mM in nodules of Cajanus cajun (Amarjit andSingh, 1985), and 40 mM in mung bean (Nagai and Funu-hashi, 1961) and french bean (Wells and Lees, 1992). Thelow affinity for allantoin suggests that allantoinase is com-partmented within the cell in proximity to high concentra-tions of substrate.

NODULE: LDPLAYLTYLNTRPPSLEEAAGKQI I I I I I I I I I I I I I I I I I I

SEED: LKPDAYLTYLNTRPPSXEIAAXKQ

Figure 5. N-terminal sequence comparison of the first 24 amino-terminal residues of allantoinase purified from soybean seeds (Webband Lindell, 1993) and root nodules. | denotes amino acid identity; Xindicates undetermined amino acid residues.

0.30

^- 0.25I

| 0.20=LD)E 0.15c

— 0.10

0.05

0.1 0.2 0.3 0.4-0.1

Figure 6. Lineweaver-Burk plot of Km determinations for allantoinasefrom nodules (open circles, solid line) and cotyledons (closedsquares, dashed line). Values are the averages of three replicates.Error bars indicate the SE values and lines were fitted to the data withsimple regression analysis.

Allantoinase from other plant sources had a pH opti-mum between 7.5 and 8 when crude or partially purifiedextracts were assayed (Nagai and Funahashi, 1961; Frankeet al., 1965; Singh et al., 1970; Nirmala and Sastry, 1975;Thomas et al., 1983; Amarjit and Singh, 1985). An exceptionwas allantoinase from Dolichos seedlings, which had bipha-sic optima at pH 4.0 and 7.5 (Mary and Sastry, 1978). In thisstudy, purified allantoinases from soybean seeds and nod-ules had a broad range of optimal activity from pH 6.5 to7.5.

Many previous studies have shown that allantoinase incrude extracts from a variety of plant sources was stable toheating (Franke et al., 1965; Singh et al., 1970; Nirmala andSastry, 1975; Mary and Sastry, 1978; Thomas et al., 1983;Amarjit and Singh, 1985; Webb, 1988; Webb and Lindell,1993). In this study, we have shown that purified allanto-inase from both cotyledons and nodules retained activity

120

5 15Time at 7CTC (min)

60

Figure 7. Effect of continuous heating at 70°C on activity of purifiedallantoinase from cotyledons (open bars) and nodules (stippled bars).Values are the averages of three replicates. Error bars indicate SEvalues. www.plantphysiol.orgon April 16, 2020 - Published by Downloaded from




z L 120 CI C O 0 100

E 80

c O

I

i2 .- 5 60 c

9 a 40 Q)

C

C a

m *ô 20 c

Z o 24 70 75 80 85 90 95

Temperature ('C)

Figure 8. Effect of increasing temperature on activity of purified allantoinase from cotyledons (open bars) and nodules (stippled bars). Values are the averages of three replicates. Error bars indicate SE values.

for up to 1 h at 70°C and retained some activity up to 85°C. This remarkable stability to heat is apparently unique to plant allantoinases, because allantoinases from animal and bacterial sources are not stable to temperatures above 55°C (Vogels et al., 1966).

Thermostability in proteins is not well understood. Pre- vious studies have not found common determinants in different heat-stable proteins. In some cases, stability is intrinsic to the protein; alternatively, it may be conferred by extrinsic factors such as heat-shock proteins (Minton et al., 1982), ions, sugars, or other co-factors (Amelunxen and Murdock, 1978). Our data showing that purified allantoinase retains activity at high temperatures indicate that the enzyme is intrinsically stable. Previous research suggested that features such as hydrogen bonds, salt bridges, and nonpolar bonds are important in stabilizing proteins to withstand high temperatures and that differences between related heat-stable and heat-labile proteins may be slight (Perutz and Raidt, 1975). Heat stability often accompanies stability to other denaturants as well (Amelunxen and Murdock, 1978). A key feature facilitating immunopurifi- cation of allantoinase was the ability to recover activity after elution from the immunoaffinity column with 8 M

urea.

Allantoinase in Relation to Nodulins

The study of nodule-specific proteins, or nodulins, has contributed greatly to an understanding of symbiotic inter- actions (Legocki and Verma, 1980; Verma et al., 1992), and comparisons of nodule enzymes with their counterparts in other tissues have revealed differences potentially related to nodule physiology. Allantoinase is not unique to nodules and, therefore, is not a nodulin, but sequence data and differences in immunoreactivity suggest that a nodule- specific form is present in soybean. Other studies have provided evidence that some enzymes of ureide metabolism are specifically induced during nodule development

(Verma et al., 1986). However, reexamination of uricase, the enzyme preceding allantoinase in the ureide biogenesis pathway, has shown that it is present in seeds and that differences between the seed and nodule enzyme are min- imal (Tajima et al., 1991, 1993; Damsz et al., 1994). Thus, uricase is also not a nodulin, as it was previously consid- ered to be. Additional comparisons of seed and nodule enzymes in this pathway will enhance understanding of features related to nitrogen assimilation in the legume- Rhizobium symbiosis.

ACKNOWLEDGMENTS

We thank Judith Lindell for technical assistance and Drs. Larry Dunkle and Charles Bracker for reading the manuscript. We ac- knowledge the Purdue Laboratory for Macromolecular Structure for protein sequence analysis.

Received June 30, 1994; accepted October 21, 1994. Copyright Clearance Center: 0032-0889/95/ 107/0435/07.

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Immunoaffinity Purification and Comparison of Allantoinases … · Plant Physiol. (1995) 107: 435-441 Immunoaffinity Purification and Comparison of Allantoinases from Soybean Root