Structure and Possible Ureide Degrading Function of the ... - NCBI

Plant Physiol. (1985) 79, 794-800

0032-0889/85/79/0794/07/$01.00/0

Structure and Possible Ureide Degrading Function of the Ubiquitous Urease of Soybean' Received for publication November 15, 1984 and in revised form April 10, 1985

JOSEPH C. POLACCO*, ROGER W. KRUEGER, AND RODNEY G. WINKLER

Biochemistry Department, University ofMissouri, Columbia, Missouri 65212 ABSTRACI

Ubiquitous soybean urease, as opposed to the seed-specific urease, designates the seemingly identical ureolytic activities of suspension cultures and leaves. It also appears to be the basal urease in developing seeds of a variety, Itachi, which lacks the seed-specific urease (Polacco, Winkler 1984 Plant Physiol 74: 8004804). On native polyacrylamide gels the ureolytic activities in crude extracts of these three tissues comigrate as determined by assays of gel slices. At this level of resolution the ubiquitous urease also migrates with or close to the fast (trimeric) form of the seed-specific urease. The ubiquitous urease was purified approximately 100-fold from suspension cultures of two cultivars (Itachi and Prize) as well as from developing seeds of Itachi. These partially purified preparations allowed visualization of native urease on polyacrylamide gels by activity staining and of urease subunits on denaturing lithium dodecyl sulfate gels by electrophoretic transfer to nitrocellulose and immunological detection ("Western Blot"). The ubiquitous urease holoenzyme migrates slightly less rapidly than the fast seed urease in native gels; its subunit migrates slightly less rapidly than the 93.5 kilodaltons subunit of either the fast or slow (hexameric) seed enzyme. The ubiquitous urease elutes from an agarose A-.5 meter column with the fast form of the seed urease species suggesting that the ubiquitous urease, like the fast seed urease, exists as a trimeric holoenzyme. The soybean cultivar, Prize, produces the hexameric seed urease; yet its ubiquitous urease (from leaf and suspension culture) is trimeric. The pH dependence of the ureolytic activity of seed coats of both seed urease-negative (Itachi) and seed urease-positive (Williams) cultivars suggests that this activity is exclusively the ubiquitous urease. Its relatively higher levels in seed coats than in embryos of Itachi suggests that the ubiquitous urease is involved in degradation of urea derived from ureides. Consistent with a ureide origin for urea is the observation that addition of a urease inhibitor, phenylphosphordiamidate, to extracts of developing Itachi seeds (seed coat plus embryo) results in accumulation of urea from allantoic acid.

Soybean produces a ureolytic activity distinct from but related to the seed-specific urease (10, 24). This second urease form has been found in leaves and in suspension cultures as well as in developing seeds of a variety (Itachi) which lacks the seed-specific urease. The ureases from these three sources are virtually indis-

tinguishable with respect to pH dependence. They also appear to be identical in their degree of sensitivity to hydroxyurea and their affinity for seed urease antibodies (24). Here we present evidence for the exclusive presence of this second urease in seed coats. Since this second type of urease activity has been found in all soybean tissues examined we have termed it the ubiquitous urease (24). The ubiquitous urease is distinguishable from the seed-specific urease by pH optimum, degree of inhibition by hydroxyurea (10, 24), degree of binding by seed urease antibodies (22, 24), and migration in native gels (this work). However, both forms are clearly related with respect to heat stability (22), nickel requirement (11, 19, 29), some common antigenic determinants (22, 24), and sensitivity (albeit not necessarily equal) to common inhibitors such as hydroxyurea (10, 24) and PPD2 (10). We report here that similarities extend to subunit size and assembly. There is growing evidence that the ubiquitous urease has a role in nitrogen assimilation. Cell cultures, which require nickel to produce active urease (19, 22, 24), do not assimilate urea in the absence of nickel (19, 20, 22). Leaf urease is also nickel-dependent (11) and Eskew et al. (7) have shown that nickel-deprived soybean plants produce necrotic leaf tips which accumulate urea to 2.5% of their dry weight. A possible source of urea is the ureides, allantoin and allantoic acid, which are transported from nodules that are actively fixing nitrogen (13-15). Consisent with a ureide source of urea is the observation of Eskew et al. (7) that leaf tip necrosis was more severe in plants dependent on fixed nitrogen than in plants utilizing NH4/N03 provided in the nutrient solution. Atkins et al. (1) found ['4C]urea in the phloem of soybean leaflets in which [2-'4Cjallantoin was applied to the upper surface. We report here that inhibition of the ubiquitous urease in extracts of developing Itachi seeds (seed coat plus embryo) results in the accumulation of urea from allantoic acid. The seed coat is a logical tissue for hypothesizing high levels of both an allantoate-degrading activity and ubiquitous urease. Although ureides represent the bulk of fixed nitrogen in soybean xylem sap (14, 15) and about 40% of soluble nitrogen in pod shells (17), only trace amounts of the nitrogen delivered to the soybean embryo by the seed coat is in ureides, the bulk (70%) being in amide amino acids (25). We report here that seed coats are a-rich source of ubiquitous urease and, in the accompanying paper (30), that seed coats are likewise rich in allantoate-degrad-

ing activity.

MATERIALS AND METHODS Plant Material. Three maturity group III soybean cultivars were employed, two seed urease-positive varieties, Prize and Williams, and a seed urease-negative variety, Itachi (P.I.

'Supported by the Missouri Agricultural Experiment Station and by grants from the United States Department of Agriculture, Science and Education Administration Competitive Grants Office, Grant 59-2291-11-672-0 and 84-CRCR-1-1374 and from the National Science Founda2Abbreviations: PPD, phenylphosphordiamidate; TM, Tris maleate; tion, PCM-8219652. This research is a contribution from the Missouri TBS, Tris-buffered saline; LDS, lithium dodecyl sulfate; ,BME, 2-mercapAgricultural Experiment Station, Journal Series 9754. toethanol. 794

UBIQUITOUS UREASE STRUCTURE AND FUNCTION 228.324). Prize produces the hexameric or slow (540,000 mol wt) seed urease, Williams the trimeric or fast (345,000 mol wt) seed urease, and Itachi makes no detectable seed urease antigen (29) or activity (24). All plants were field-grown and nodulated. Suspension cultures were induced and maintained as described previously (24). Mid to late log cells (20 to 30 g fresh weight/350 ml) were exposed to 10 mm potassium citrate/pH 6.0 and 10 Mm NiSO4 for 24 h prior to harvesting. Cells were collected by filtration on Miracloth (VWR Scientific, St. Louis, MO), washed with distilled H20, and immersed in liquid N2. Suspensioncultured cells and developing seeds (mid-fill stage) were stored at -70°C prior to enzyme extraction. Preparation of Crude Extracts. Crude extracts of leaves, suspension culture, and seeds were made as described previously (24). Leaf extracts were enriched for urease activity by acetone precipitation and resuspension in one-tenth the original volume (24). Seed coats were separated from embryos by slitting developing seeds on the edge opposite the axis. Seeds were placed in cold distilled H20 for 20 min, with occasional stirring. The embryo was then extruded through the slit in the seed coat by gentle squeezing. Seed coats were further rinsed for 10 min in cold, distilled H20 and blotted dry on both sides. This procedure yielded seed coats essentially free of contaminating embyro tissue. (If Williams seed coat tissue contained 1% contaminating embryo tissue the ratio of its urease activities at pH 7.0 versus 8.8 would be 1.2 instead of 0.65, a ratio characteristic of the

795

ubiquitous urease [Table I]). Seed coats were ground in a mortar in 5 volumes of TM (0.1 Tris maleate, 1 mM EDTA, 10 mM #ME [pH 7.0]) and further homogenized by four strokes with a loose fitting plunger in a glass homogenizer (Wheaton). Embryo extracts (Table I) were prepared in an identical manner. There was virtually no sedimentation of urease activity upon centrifugation (16,000g x 15 min) which was routinely done to facilitate the pipetting of extracts. Partial Purification of Ubiquitous Urease. All steps, except the heat treatment (60°C) and both column separations (room temperature), were performed at 4°C. Developing Itachi seeds (50 g) or suspension cultured cells (100-300 g, Prize and Itachi) were homogenized for 30 s in a Tek-Mar (Cincinnati, OH) homogenizer in 5 or 3 volumes, respectively, of TM buffer. After removal of insolubles by centrifugation, extracts were heated at 60°C for 30 min. After centrifugation, the supernatant was cooled to 4°C. Acetone (-20°C) was added slowly with stirring to 60% total volume and insolubles were collected by centrifugation, resuspended in one-tenth to one-fifth original volume TM, and dialyzed overnight against TM buffer. Insolubles were removed by centrifugation and the supernatant mixed with 1.22 volumes of 100% saturated (NH4)2SO4 (to a final saturation of 55%). After standing for 0.5 h at 4°C, the precipitate was collected by centrifugation and dialyzed against three 2-L volumes of TM buffer. Insolubles were removed by centrifugation and the supernatant applied to a 1.5 x 27 cm column of hydroxylapatite (HTP, BioRad; Richmond, CA). Without further equilibration, a 180-

FIG. 1. Polyacrylamide gel electrophoretic profiles of ureolytic activity in extracts of developing seed (Prize and Itachi), leaves (Itachi), and cell suspension culture (Itachi). A, Untreated extracts; B, extracts were heated for 30 min at 60C in 5 mM DTT and 50% glycerol. Protein per lane: 2.7 ,gg, developing Prize seed; 0.38 mg, developing Itachi seed; 0.03 mg, Itachi suspension culture; 0.28 mg Itachi leaf.

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4M:Of s. si

FIG. 2. Urease activity stain of a native gel containing partially punfied ubiquitous ureases. Partially purified (59- to 117-fold, "Materials and Methods") ubiquitous urease from developing Itachi seeds (It/Seed, 41 units), and suspension cultures of Itachi (It/SC, 52 units) and Prize (Pr/SC, 74 units) were compared with urease in diluted crude extracts of mature seeds of Williams (Fast, 200 units), Prize (Slow, 150 units) and Itachi (Null, 0.01 units). All mature seed samples contained 200 Ag protein. Active urease species were detected by the activity stain of Fishbein et al. (9). -

ml linear gradient of 10 to 300 mm K-phosphate (pH 7.0, 1 mM EDTA, 10 mm ,3ME, 0.02% NaN3) was applied. Urease-containing fractions were located by mixing 0.1 ml of each fraction with 0.9 ml 0.5 M urea, 10 mm K-phosphate (pH 7.0), 1 ,ug/ml Cresol Red. Urease-positive samples exhibit a urea-dependent pH rise manifested by a color change from yellow to lavender upon standing at 60C for 1 h. These samples also catalyzed the production of NH3 (as determined by Nessler's reagent [18]) from urea and '4CO2 from ["'C]urea (24). Active fractions were pooled, mixed with 1.22 volumes of 100% saturated (NH4)2SO4 and precipitated proteins resuspended in 0.3 ml total volume with TM buffer. This was applied directly to a 2 x 47 cm column of agarose A-0.5 m (200-400 mesh, Bio-Rad). Peak fractions were pooled and subsequently concentrated by (NH4)2SO4 precipitation and dialysis against TM. Enzyme preparations were stored at -70'C. Some seed preparations were homogenized in TM/10 (10 mM Tris maleate, 0.1 mm EDTA, 10 mm ,tiME [pH 7.01). The lower ionic strength of this buffer resulted in greater differential extrac-

Plant Physiol. Vol. 79, 1985

tion of urease from the storage globulins. The specific activities (nmol urea hydolyzed-mg' protein min-') of urease purified from Itachi cell culture (945), Prize cell culture (463), and Itachi seeds (80) represent fold-increases of 117, 125, and 59, respectively, over the crude extract values reported previously (24). Enzyme Assays. Allantoic acid degrading activity was detected by the allantoate-dependent production of glyoxylate as determined by the method of Vogels and van der Drift (28). Developing Itachi beans were ground in a mortar with 10 volumes 0.1 M Tris-HCl, 1 mm EDTA, 5 mM DTT (pH 7.6). After centrifugation, the supernatant was mixed with one-ninth volume 20 mM MnSO4. Aliquots (0.2 ml) of this manganeseactivated extract were assayed in 0.8 ml 10 mm potassium allantoate (Sigma), 0.1 M Tris-HCl, 5 mm DTT (pH 9.0) at 37°C for 2 to 24 h. Reactions were stopped and deproteinized by vortexing with 1 ml of CHC13. To determine urea it was first necessary to separate it from allantoate which liberates urea in the hot acidic conditions employed in the urea determination. A 0.5-ml aliquot of the CHC13treated extract was applied to 3 ml of Dowex AG- 1-X 10 anion exchange resin in a 5-ml plastic syringe. The resin was previously washed successively with 15 ml 1 N NaOH, 10 ml H20, 6 ml 1 N HCOOH, and finally with water until the effluent was neutral. After applying the extract the column was washed with 4.5 ml H20. Urea was determined by mixing 1 ml of pooled effluent with 1 ml 8 M H2SO4, 0.03% (w/v) Fe2(SO4)3, and 3 ml 0.6% (w/v) diacetyl monoxime, 0.03% (w/v) thiosemicarbazide (18). Samples and standards were boiled for 10 min, cooled, and A535 determined. Urea standards were prepared in 10 mm potassium allantoate and treated concurrently with sample aliquots. Ammonia production was measured (26) in separate reactions which were stopped with 1 ml saturated Na borate (pH 11). During the next 5 h diffusing ammonia was trapped by 10 M H2SO4 which coated an etched, rounded end of a glass rod inserted into the stopper of the reaction vessel. The end of the rod was then immersed in 5 ml H20 and NH3 was determined with Nessler's reagent (18). Urease was determined by the release of 14C02 from ['4C]urea (24). Urease was also detected in column fractions by ureadependent release of NH3 (18) or by urea-dependent pH increase as described above. PAGE. Native acrylamide 6.25% gels were run for 4 h at 15 mamp. Gel slices were assayed for 20 h at 37°C in 2 ml TM containing 25 mM ['4C]urea (85 dpm/nmol). Gels were sliced with razor blades regularly spaced (5.4 mm) with washers. A specific urease activity stain (9) was employed for partially purified ubiquitous urease preparations run on 7.5% native polyacrylamide gels at 30 mamp constant current for 5 h. Denaturing gels were run as described by Laemmli (12) except that a 6 to 9% acrylamide gradient was employed, the upper running buffer contained 0.1% (w/v) LDS, and the lower (cathode) buffer had no detergent. Samples were denatured by incubating for 5 min at 65°C in 30 mM DTT, 1% (w/v) LDS, 0.5 x TM prior to electrophoresis for 8 h at 4°C at 8 W constant power. Urease subunits were detected immunologically after the electroblot transfer of gel-resolved proteins to a cellulose nitrate sheet essentially as described by Towbin et al. (27). Proteins were transferred for 2 h at 200 mamp in 20% (v/v) methanol, 19.2 mM Tris-glycine (pH 8.3) (- 20°C at the start of transfer). Blocking solution was 30% (v/v) goat serum (Gibco; Chagrin Falls, OH), 3% (w/v) BSA (Fraction V, Boehringer Mannheim, Indianapolis, IN) in TBS (20 mM Tris-HCl, 500 mM NaCl). Seedurease antiserum (rabbit) was diluted 1:400 in 10% goat serum, 1% BSA in TBS. Goat antirabbit IgG, conjugated to horseradish peroxidase, and peroxidase substrate were used according to the manufacturer's (Bio-Rad) instructions. Bound proteins were ex-

UBIQUITOUS UREASE STRUCTURE AND FUNCTION

SeedSpecif ic -J -J D z

94-I

0 J co

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Co < UL

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%

797

vemew

co H1

Co

N

0

N\

saw,

FIG. 3. Protein blot analysis of LDSacrylamide gels containing seed and ubiquitous urease subunits. Samples equivalent to those of Figure 2 were denatured and subjected to electrophoresis for 8 h in a 6 to 9% gradient acrylamide gel containing 0.1% LDS. After electrophoretic transfer to a nitrocellulose sheet, ureaseantigen was detected with serum against seed urease as described in "Materials and

#

Methods".

-68 'Am

0

43M

,;A,,-..

posed to the first antibody (anti-urease) overnight at 4C and to the second antibody (goat antirabbit) for 1 h at room tempera-

least one standard as well as (NH4)2SO4 (to mark the retention volume) to check the calibration of the column.

ture.

Mol Wt Determination by Gel Filtration on an Agarose A-O.5 Column. A column (2 x 47 cm) of agarose A-0.5 m (200-400 mesh, Bio Rad), equilibrated in TM plus 0.02% NaN3 (w/v), was calibrated with blue dextran (nominal mol wt of 2 x 106), m

apoferritin (2 mg, CalBiochem; LaJolla, CA), catalase, aldolase, ovalbumin, chymotrypsinogen, Cyt c (2 mg each, Boehringer Mannheim), and (NH4)2SO4 (0.4 ml of a 2.5% saturated solution). The flow rate was 15 ml/h at room temperature. The percentage of retardation for each standard (setting those for blue dextran and (NH4)2SO4 as 0 and 100%, respectively) was calculated along with the percentage of retardation ofItachi developing seed urease (24 units), Prize seed urease (approximately 300 units), Williams seed urease (approximately 100 units), Itachi cell suspension urease (50 units), and Prize cell suspension urease (74 units). Each run with a sample of urease also contained at

RESULTS AND DISCUSSION Native Gel Analysis of Ureolytic Activity in Crude Extracts of Various Tissues. We had reported previously that leaves and suspended cell cultures of Prize (seed urease-positive) and Itachi (seed urease-negative) contained the ubiquitous urease (24). The ubiquitous urease was also reported to be the exclusive urease in Itachi seeds (24). Crude extracts of these three tissue types, prepared as described previously (24), were run on native polyacrylamide gels to compare the mobilities of their urease species with that of the predominant urease in Prize seed extracts. Based on its migration in these native gels, Prize seed urease has a mol wt of approximately 480,000 (21). When localized by assays of gel slices the ureases of Itachi developing seed, cell culture, and leaves migrate virtually as a common species, and more rapidly than Prize seed urease (Fig. 1A). Although not shown here, the

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60 50

0 ' Slow Seed-Specific (Prize)

401

-\FastFSeed-Specific (Wilems)

FER ltachi Seed

b

Table II. Glyoxylate Productionfrom Allantoate in Developing Itachi Seed Extracts Extracts of developing urease-negative Itachi seeds were assayed for 5 h at 37C. Glyoxylate Formation Assay Conditions

psmol-g-' fesh wth-' 1.2 Complete, Mn2_-treated extract 1.3 +ImMPPDa 1.3 +10mMurea + 10 mM arginine 1.3 - Allantoate, + I mM PPD 0.0 - Allantoate, + 10 mM arginine 0.0 0.0 Complete, non-Mn2+-treated extract 'PPD, phenylphosphordiamidate, an effective urease inhibitor (10), completely inhibited the urease activity (1.4 umol urea hydrolyzedg-' fresh wt- h-') of these extracts. Mn2' treatment had no effect on urease activity.

i Itochi Cal Cult. q Prize Cell Cult.

301

Plant Physiol. Vol. 79, 1985

U.

CAT

20I ALD E)

10

Table III. Effect of PPD on Levels ofAllantoate Breakdown Products Extracts of urease-negative seeds were assayed for 14 h at 35C. w

i5

Breakdown Product

OA®)

n

Breakdown Products Rate -PPD +PPD (250 MM)

;mol.g' fresh wt-h-'

0

Glyoxylate

3

Urea Ammonia

CHY 2

CYT C I

0

20

aI

I

60 40 PERCENT RETAINED

I

I

80

100

FIG. 4. Analysis of mol wt of ubiquitous and seed-specific urease by agarose (A-0.5 m) gel chromatography. Ubiquitous urease samples (@) were from developing Itachi seed and suspension culture of Itachi and Prize. Fast seed-specific (@) urease was from crude extracts of mature Williams seed. Protein standards were Cyt c (CYT C), chymotrypsinogen (CHY), ovalbumin (OA), aldolase (ALD), catalase (CAT), apoferritin (FER), and slow (Prize) seed-specific urease. The nominal mol wt of the slow urease, 540,000, was that previously determined by agarose gel filtration (25).

Table I. Ureases ofDeveloping Embryos and Seed Coats Seeds picked 25 to 30 d after flowering were separated into embryos and seed coats.

Seed Urease

Phenotype

T

Urease Activitya pH 7.0 pH 8.8 7.0/8.8

jumol urea-g-' fresh wt-h-'

Williams

Williams Itachi Itachi

Positive Positive

Embryo

Negative Negative

Embryo

Seed coat

Seed coat

1005 2.4 0.9 2.3

680 3.7 1.7 3.8

a Values are averages of 2 or 3 separate experiments.

1.48 0.65 0.53 0.61

0.7 0 5.3

0.7 0.7 3.3

ureases of cell cultures and leaves of Prize also migrate with the urease of Itachi seed and not with that of Prize seed. Prize seed urease yields a more rapidly migrating component in buffers of lowered ionic strength (21) or when heated in the presence of glycerol and DTT (22; Fig. 1B). Heat-treated Prize seed urease migrated more rapidly and virtually identically with the ubiquitous species whose migration was not affected by heat treatment (with the possible exception of cell culture urease, Fig. 1B). (The faster species of Prize seed-specific urease has a gelderived mol wt of 280,000 [211). Based on a subunit size of 93.5

kD (21) and the observation that the conversion of slow to fast does not yield a third form (i.e. splitting is equal) indicates that the slow species is hexameric and the fast species trimeric (21). Buttery and Buzzell (4) had earlier concluded, based on migration ratios in native gels of different acrylamide concentrations, that the fast soybean urease had half the mol wt ofthe slow form. The Jack bean urease 480,000 mol wt form is considered to be hexameric based on titration (16) and on electron microscopic (8) studies, while an active species half that size, which can be produced by dissociation in glycols or glycerol (5), is trimeric (8). Native and LDS Gel Analysis of Partialy Purified Ubiquitous Urease. Ureases from developing Itachi seed as well as from cell suspension cultures of Itachi and Prize were purified 50- to 117fold ("Materials and Methods"). The ubiquitous ureases of these tissues could then be compared with the fast and slow seed urease forms by a specific activity stain of native polyacrylamide gels (9). In the gel of Figure 2 the fast and slow forms of the seed urease were provided by crude extracts of mature seeds of the varieties Williams and Prize, respectively. The genetic fast form of Williams seed migrates identically with the heat-induced fast form of Prize (results not shown). It is clear from Figure 2 that the ubiquitous ureases from cell cultures and Itachi seed comigrate. However, they migrate somewhat more slowly than the fast seed urease and thus appear to represent a species distinct from seed urease. Samples ofthe seed-specific and ubiquitous ureases, equivalent to those of Figure 2, were run on an LDS, acrylamide (6-9%)

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UBIQUITOUS UREASE STRUCTURE AND FUNCTION

gradient gel for 8 h (12) to determine their relative subunit sizes. Upon electrophoretic transfer of the gel proteins to a nitrocellulose sheet (27) urease subunits were detected with antiserum raised against the seed (Prize) urease (21). It is apparent (Fig. 3), based on gel migration rates, that ubiquitous and seed-specific ureases have nearly identical subunit sizes, although the ubiquitous urease subunit appears to run slightly slower. The recognition by seed urease antibodies of the ubiquitous urease confirms our earlier observations on the serological relatedness, albeit limited, of the two forms (21, 22, 24). The specificity of the antibody (21) employed here is demonstrated by its lack of recognition of antigen in extracts of mature Itachi seeds (lane 1, Fig. 3). The ubiquitous urease subunit of developing Itachi seeds was detected (lane 4, Fig. 3) because it was first purified 59-fold, because developing Itachi seeds have 20 times the ureolytic activity of mature seeds (24), and because 3.75 times as much developing seed protein (750 ,g) was loaded than mature seed protein (200 ,g for each cultivar in lanes 1-3, Fig. 3). Thus, the fourth lane of Figure 3 has 4500 times (59 x 20 x 3.75) the ureolytic activity of the first lane. Although not shown on this gel, the urease antigenic protein band detected in extracts of mature wild type seeds comigrates with denatured

purified urease.

Agarose Gel Filtration Analysis of the Soybean Ureases. An attempt was made to determine the mol wt of the soybean ureases by gel filtration on an agarose A-0.5 m column equilibrated with TM buffer and calibrated with proteins of known native mol wt. The ubiquitous ureases of Itachi developing seed and of suspension cultures of Prize and Itachi eluted in virtually the same eluant volume (Fig. 4, retardation values of 21.6-21.9%). Surprisingly, in light of its easily distinguished migration rate on native gels (Fig. 2), the fast seed-specific urease of Williams extracts appeared to coelute with the ubiquitous species (Fig. 4, retardation value of 21.4%). The nominal mol wt of 345,000 for the ubiquitous urease (Fig. 4) would indicate that it is either a trimer (280,000) or tetramer (374,000) of 93.5 kD (Fig. 3) subunits. However, its similar mobility to the fast seed urease in native polyacrylamide (Fig. 2) and on an agarose gel sieving column (Fig. 4) suggests that ubiquitous urease, like the fast seedspecific form, is trimeric. We previously reported a gel-derived mol wt of 280,000 for the fast seed urease (21). The slow (hexameric) seed-specific urease has a previously reported mol wt of 540,000, derived by agarose (A- 15 m) gel chromatography (21). Although Prize's seed urease is hexameric, its ubiquitous (cell culture) urease is trimeric (Fig. 4). Seed Coats Contain the Ubiquitous Urease. The nature of the urease, i.e. seed-specifc or ubiquitous, was examined in seed coats. It was shown earlier (10, 24) that the two urease forms differ markedly in pH dependence, which we have used here to identify the urease of seed coats. Seed coats of Williams and Itachi have ureases of similar activity ratios (at pH 7.0 versus pH 8.8, Table I). The activity ratio is much closer to that of the Itachi embryo (seed minus seed coat) than that of the Williams embryo which produces a preponderance of the seed-specific urease (Fig. 2). Thus, seed coats produce predominantly or exclusively the ubiquitous urease. It is also noteworthy that there are comparable urease levels in the seed coats of both varieties

converted to a urea intermediate. Thus, we first sought to identify an allantoate-degrading activity in developing seeds (seed coats plus embryos) and then to identify urea as an intermediate. As can be seen from the results of Table II, developing bean (Itachi) extracts contain a Mn+dependent activity which is essential for the ultimate conversion of allantoic acid to glyoxylate. The production of glyoxylate is not catalyzed by urease since neither PPD, an excellent inhibitor ofthe ubiquitous urease (Table II; 10), nor urea inhibit glyoxylate production. Neither is arginase, an Mn2+-activated enzyme (e.g. 3), involved since arginine did not diminish the allantoatedependent production of glyoxylate nor did it itself serve as a glyoxylate source (Table II). To detect allantoate-derived urea it was necessary to eliminate the considerable seed urease activity. This was accomplished by using developing seeds lacking the abundant seed-specific urease (Table I) and by inhibiting the remaining ubiquitous urease with PPD. Parallel reactions were employed to study the effects of PPD on ammonia production. As in the experiment of Table II, PPD had no effect on glyoxylate accumulation (Table III). However, it resulted in the accumulation of an easily measured equimolar amount of urea. Since the reaction time was long and products could be consumed in other reactions, no valid conclusion can be made of the stoichiometry of the products. However, it is apparent that PPD does not block all ammonia accumulation so that only a portion of the ureide nitrogen of allantoic acid is converted to ammonia via urea. This agrees with our earlier observation that growth of suspension cultures on an allantoin nitrogen source, unlike that on urea-N, is not nickel-dependent (24). Long reaction times were employed (Tables II and III) to ensure the liberation of detectable quantities of urea from allantoate. As shown in the accompaning paper (30) ammonia and CO2 are liberated well before the appearance of glyoxylate. Although glyoxylate and urea are derived from allantoate by action of an Mn2+-stimulated activity, neither glyoxylate nor urea appear to be immediate breakdown products (30). Indeed, their subsequent production may involve nonenzymic breakdown of the actual product. Evidence for an Assimilatory Function for the Ubiquitous Urease. In contrast to the seed-specific urease, whose activity plateaus at maturity (23), the specific activity of the ubiquitous urease in developing Itachi seeds is 20 times higher than in the mature seed (24). This is the developmental pattern expected for an enzyme with a role in pod filling. Two observations strongly suggest that this role is the conversion of ureides to amino acids: the seed coat is a rich source for the ubiquitous urease (Table I) and urea is a breakdown product of allantoic acid (Table III). We report in the accompanying paper (30) that the seed coat has more than 5 times the allantoate-degrading activity of the emI

bryo.

In a nonseed system, namely cell culture, an active (ubiqui-

tous) urease is essential for urea-supported growth (I19, 20). We do not propose that ureide assimilation is the sole function of the ubiquitous urease. Many seeds and storage organs are rich in arginine. Pea, a legume without an abundant seed urease (2), catabolizes large amounts of arginine during germination via arginase and an inducible urease whose activity rises 5-fold over

(Table I). Ureide Degradation in Developing Soybean Seeds. Itachi's seed that found in dry peas (6). coat ureolytic activity is more than twice that of its embryo Acknowledgments-We thank Dale Blevins and Doug Randall for critical read(Table I). Although nitrogen-fixing soybeans transport the bulk ing ofthe manuscript and Peggy Jo Bledsoe for assistance in the gel slice experiment. of their xylem nitrogen as the ureides allantoin and allantoic acid (13-15), most of the nitrogen delivered to the developing LITERATURE ClI TED embryo by the seed coat is in the form of amino acids with only 1. ATKINS JS CA, PATE, A RITCHIE, MB PEOPLES 1982 Metabolism and translotrace amounts as ureides (25). Thus, the seed coat is a candidate cation of allantoin in ureide-producinggrain legumes. Plant Physiol 70: 476-

tissue for the conversion of ureides to amino acids, and its high urease level suggests that at least some of the ureide nitrogen is

482 2. BAILEY CJ, D BOULTER 1971

Urease,a typical seed protein of the Leguminosae.

800 3. 4. 5.

6. 7.

8.

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In JB Harbourne, D Boulter, BL Turner, eds, The Chemotaxonomy of the Leguminosae. Academic Press, New York, pp 485-502 BounIN J-P 1982 Purification, properties and subunit structure of arginase from Iris bulbs. Eur J Biochem 127: 237-243 BUTrERY BR, RI BUZZEL 1971 Properties and inheritance of urease isozymes in soybean seeds. Can J Bot 49: 1101-1105 CONTAXIS CC, FJ REITHEL 1971 Studies on protein multimers. II. A study of the mechanism of urease dissociation in 1,2-propanediol: comparative studies with ethylene glycol and glycerol. J Biol Chem 246: 677-685 DERUITER H, C KOLLOFFEL 1973 Arginine catabolism in the cotyledons of developing and germinating pea seeds. Plant Physiol 73: 525-528 ESKEw DL, RM WELCH, EE CARY 1983 Nickel: an essential micronutrient for legumes and possibly all higher plants. Science 222: 621-623 FISHBEIN WN, WF ENGLER, JL GRIFFIN, W. ScuRzI, GF BAHR 1977 Electron microscopy ofnegatively stainedjackbean urease at three levels of quaternary structure, and comparison with hydrodynamic studies. Eur J Biochem 73:

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66: 720-725 16. NORRIS R, K BROCKLEHURST 1976 A convenient method of preparation of high-activity urease from Canavalia ensiformis by covalent chromatography and an investigation of its thiol groups with 2,2'-dipyridyl disulphide as a thiol titrant and reactivity probe. Biochem J 159: 245-257 17. OHYAMA T 1984 Comparative studies on the distribution of nitrogen in soybean plants supplied with N2 and N037 at the pod filling stage. II. Assimilation and transport of nitrogenous constituents. Soil Sci Plant Nutr 30: 219-229 18. POLACCO JC 1976 Nitrogen metabolism in soybean tissue culture. I. Assimilation of urea. Plant Physiol 58: 350-357 19. POLACCO JC 1977 Nitrogen metabolism in soybean tissue culture. II. Urea utilization and urease synthesis require Ni2 . Plant Physiol 59: 827-830 20. PoLAco JC 1977 Is nickel a universal component of plant ureases? Plant Sci Lett 10: 249-255 21. PoLAcco JC, EA HAVIR 1979 Comparisons of soybean urease isolated from seed and tissue culture. J Biol Chem 254: 1707-1715 22. POLAcco JC, AL THOMAS, PJ BLEDSOE 1982 A soybean seed urease-nuil produces urease in cell culture. Plant Physiol 69: 1233-1240 23. POLACOO JC, RB SPARKS JR 1982 Patterns of urease synthesis in developing soybeans. Plant Physiol 70: 189-194 24. POLACCO JC, RG WINKLER 1984 Soybean leaf urease: a seed enzyme? Plant Physiol 74: 800-803 25. RAINBIRD RM, JH THORNE, RWF HARDY 1984 Role of amides, amino acids, and ureides in the nutrition of developing soybean seeds. Plant Physiol 74: 329-334 26. SELIGSON D, K HIRAHARA 1959 The measurement of ammonia in whole blood, erythrocytes, and plasma. J Lab Clin Med 49: 962-974 27. TowBIN H, T STAEHLIN, J GORDON 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350-4354 28. VOGELS GD, C VAN DER DR[Fr 1970 Differential analyses of glyoxylate derivatives. Anal Biochem 33: 143-157 29. WINKLER RG, JC POLAcco, DL ESKEW, RM WELCH 1983 Nickel is not required for apourease synthesis in soybean seeds. Plant Physiol 72: 262-

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