Plant Physiol. (1985) 79, 794-8000032-0889/85/79/0794/07/$01.00/0

Structure and Possible Ureide Degrading Function of theUbiquitous 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. WINKLERBiochemistry 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 cul-tures and leaves. It also appears to be the basal urease in developingseeds of a variety, Itachi, which lacks the seed-specific urease (Polacco,Winkler 1984 Plant Physiol 74: 8004804). On native polyacrylamidegels the ureolytic activities in crude extracts of these three tissuescomigrate as determined by assays of gel slices. At this level of resolutionthe 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 sus-

pension cultures of two cultivars (Itachi and Prize) as well as fromdeveloping seeds of Itachi. These partially purified preparations allowedvisualization of native urease on polyacrylamide gels by activity stainingand of urease subunits on denaturing lithium dodecyl sulfate gels byelectrophoretic transfer to nitrocellulose and immunological detection("Western Blot"). The ubiquitous urease holoenzyme migrates slightlyless rapidly than the fast seed urease in native gels; its subunit migratesslightly less rapidly than the 93.5 kilodaltons subunit of either the fastor slow (hexameric) seed enzyme. The ubiquitous urease elutes from anagarose A-.5 meter column with the fast form of the seed urease speciessuggesting that the ubiquitous urease, like the fast seed urease, exists asa trimeric holoenzyme. The soybean cultivar, Prize, produces the hex-americ seed urease; yet its ubiquitous urease (from leaf and suspensionculture) is trimeric.The pH dependence of the ureolytic activity of seed coats of both seed

urease-negative (Itachi) and seed urease-positive (Williams) cultivarssuggests that this activity is exclusively the ubiquitous urease. Its rela-tively higher levels in seed coats than in embryos of Itachi suggests thatthe ubiquitous urease is involved in degradation of urea derived fromureides. Consistent with a ureide origin for urea is the observation thataddition of a urease inhibitor, phenylphosphordiamidate, to extracts ofdeveloping Itachi seeds (seed coat plus embryo) results in accumulationof urea from allantoic acid.

Soybean produces a ureolytic activity distinct from but relatedto the seed-specific urease (10, 24). This second urease form hasbeen found in leaves and in suspension cultures as well as indeveloping seeds ofa variety (Itachi) which lacks the seed-specificurease. The ureases from these three sources are virtually indis-

'Supported by the Missouri Agricultural Experiment Station and bygrants from the United States Department of Agriculture, Science andEducation Administration Competitive Grants Office, Grant 59-2291-1-1-672-0 and 84-CRCR-1-1374 and from the National Science Founda-tion, PCM-8219652. This research is a contribution from the MissouriAgricultural Experiment Station, Journal Series 9754.

tinguishable with respect to pH dependence. They also appear tobe identical in their degree of sensitivity to hydroxyurea andtheir affinity for seed urease antibodies (24). Here we presentevidence for the exclusive presence of this second urease in seedcoats. Since this second type of urease activity has been found inall soybean tissues examined we have termed it the ubiquitousurease (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), andmigration in native gels (this work). However, both forms areclearly related with respect to heat stability (22), nickel require-ment (11, 19, 29), some common antigenic determinants (22,24), and sensitivity (albeit not necessarily equal) to commoninhibitors such as hydroxyurea (10, 24) and PPD2 (10). Wereport 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 toproduce active urease (19, 22, 24), do not assimilate urea in theabsence of nickel (19, 20, 22). Leaf urease is also nickel-depend-ent (11) and Eskew et al. (7) have shown that nickel-deprivedsoybean plants produce necrotic leaf tips which accumulate ureato 2.5% of their dry weight. A possible source of urea is theureides, allantoin and allantoic acid, which are transported fromnodules that are actively fixing nitrogen (13-15). Consisent witha ureide source of urea is the observation of Eskew et al. (7) thatleaf tip necrosis was more severe in plants dependent on fixednitrogen than in plants utilizing NH4/N03 provided in the nu-trient solution. Atkins et al. (1) found ['4C]urea in the phloemof soybean leaflets in which [2-'4Cjallantoin was applied to theupper surface. We report here that inhibition of the ubiquitousurease in extracts of developing Itachi seeds (seed coat plusembryo) 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 soybeanxylem sap (14, 15) and about 40% of soluble nitrogen in podshells (17), only trace amounts of the nitrogen delivered to thesoybean embryo by the seed coat is in ureides, the bulk (70%)being in amide amino acids (25). We report here that seed coatsare a-rich source of ubiquitous urease and, in the accompanyingpaper (30), that seed coats are likewise rich in allantoate-degrad-ing activity.

MATERIALS AND METHODSPlant Material. Three maturity group III soybean cultivars

were employed, two seed urease-positive varieties, Prize andWilliams, and a seed urease-negative variety, Itachi (P.I.

2Abbreviations: PPD, phenylphosphordiamidate; TM, Tris maleate;TBS, Tris-buffered saline; LDS, lithium dodecyl sulfate; ,BME, 2-mercap-toethanol.

794

UBIQUITOUS UREASE STRUCTURE AND FUNCTION

228.324). Prize produces the hexameric or slow (540,000 molwt) 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 describedpreviously (24). Mid to late log cells (20 to 30 g fresh weight/350ml) were exposed to 10 mm potassium citrate/pH 6.0 and 10 MmNiSO4 for 24 h prior to harvesting. Cells were collected byfiltration on Miracloth (VWR Scientific, St. Louis, MO), washedwith distilled H20, and immersed in liquid N2. Suspension-cultured cells and developing seeds (mid-fill stage) were stored at-70°C prior to enzyme extraction.

Preparation of Crude Extracts. Crude extracts of leaves, sus-pension culture, and seeds were made as described previously(24). Leaf extracts were enriched for urease activity by acetoneprecipitation and resuspension in one-tenth the original volume(24).

Seed coats were separated from embryos by slitting developingseeds on the edge opposite the axis. Seeds were placed in colddistilled H20 for 20 min, with occasional stirring. The embryowas then extruded through the slit in the seed coat by gentlesqueezing. Seed coats were further rinsed for 10 min in cold,distilled H20 and blotted dry on both sides. This procedureyielded seed coats essentially free of contaminating embyro tis-sue. (If Williams seed coat tissue contained 1% contaminatingembryo tissue the ratio of its urease activities at pH 7.0 versus8.8 would be 1.2 instead of 0.65, a ratio characteristic of the

ubiquitous urease [Table I]). Seed coats were ground in a mortarin 5 volumes of TM (0.1 Tris maleate, 1 mM EDTA, 10 mM#ME [pH 7.0]) and further homogenized by four strokes with aloose fitting plunger in a glass homogenizer (Wheaton). Embryoextracts (Table I) were prepared in an identical manner. Therewas virtually no sedimentation of urease activity upon centrifu-gation (16,000g x 15 min) which was routinely done to facilitatethe pipetting of extracts.

Partial Purification of Ubiquitous Urease. All steps, except theheat treatment (60°C) and both column separations (room tem-perature), were performed at 4°C. Developing Itachi seeds (50 g)or suspension cultured cells (100-300 g, Prize and Itachi) werehomogenized for 30 s in a Tek-Mar (Cincinnati, OH) homoge-nizer in 5 or 3 volumes, respectively, ofTM buffer. After removalof insolubles by centrifugation, extracts were heated at 60°C for30 min. After centrifugation, the supernatant was cooled to 4°C.Acetone (-20°C) was added slowly with stirring to 60% totalvolume and insolubles were collected by centrifugation, resus-pended in one-tenth to one-fifth original volume TM, and di-alyzed overnight against TM buffer. Insolubles were removed bycentrifugation and the supernatant mixed with 1.22 volumes of100% saturated (NH4)2SO4 (to a final saturation of 55%). Afterstanding for 0.5 h at 4°C, the precipitate was collected by cen-trifugation and dialyzed against three 2-L volumes ofTM buffer.

Insolubles were removed by centrifugation and the supernatantapplied 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 cellsuspension 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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Plant Physiol. Vol. 79, 1985

FIG. 2. Urease activity stain of a native gel containing partially pun-fied ubiquitous ureases. Partially purified (59- to 117-fold, "Materialsand 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 ofmature seeds of Williams (Fast, 200 units), Prize (Slow, 150 units) andItachi (Null, - 0.01 units). All mature seed samples contained 200 Agprotein. Active urease species were detected by the activity stain ofFishbein et al. (9).

ml linear gradient of 10 to 300 mm K-phosphate (pH 7.0, 1 mMEDTA, 10 mm ,3ME, 0.02% NaN3) was applied. Urease-contain-ing fractions were located by mixing 0.1 ml of each fraction with0.9 ml 0.5 M urea, 10 mm K-phosphate (pH 7.0), 1 ,ug/ml CresolRed. Urease-positive samples exhibit a urea-dependent pH risemanifested by a color change from yellow to lavender uponstanding at 60C for 1 h. These samples also catalyzed theproduction 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 of100% saturated (NH4)2SO4 and precipitated proteins resus-pended in 0.3 ml total volume with TM buffer. This was applieddirectly to a 2 x 47 cm column of agarose A-0.5 m (200-400mesh, Bio-Rad). Peak fractions were pooled and subsequentlyconcentrated by (NH4)2SO4 precipitation and dialysis againstTM. 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 lowerionic strength of this buffer resulted in greater differential extrac-

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 cellculture (463), and Itachi seeds (80) represent fold-increases of117, 125, and 59, respectively, over the crude extract valuesreported previously (24).Enzyme Assays. Allantoic acid degrading activity was detected

by the allantoate-dependent production of glyoxylate as deter-mined by the method of Vogels and van der Drift (28).

Developing Itachi beans were ground in a mortar with 10volumes 0.1 M Tris-HCl, 1 mm EDTA, 5 mM DTT (pH 7.6).After centrifugation, the supernatant was mixed with one-ninthvolume 20 mM MnSO4. Aliquots (0.2 ml) of this manganese-activated extract were assayed in 0.8 ml 10 mm potassiumallantoate (Sigma), 0.1 M Tris-HCl, 5 mm DTT (pH 9.0) at 37°Cfor 2 to 24 h. Reactions were stopped and deproteinized byvortexing 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 em-ployed in the urea determination. A 0.5-ml aliquot ofthe CHC13-treated extract was applied to 3 ml of Dowex AG-1-X 10 anionexchange resin in a 5-ml plastic syringe. The resin was previouslywashed successively with 15 ml 1 N NaOH, 10 ml H20, 6 ml 1N HCOOH, and finally with water until the effluent was neutral.After applying the extract the column was washed with 4.5 mlH20. Urea was determined by mixing 1 ml of pooled effluentwith 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 A535determined. Urea standards were prepared in 10 mm potassiumallantoate 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 MH2SO4 which coated an etched, rounded end of a glass rodinserted into the stopper of the reaction vessel. The end of therod was then immersed in 5 ml H20 and NH3 was determinedwith Nessler's reagent (18).

Urease was determined by the release of 14C02 from ['4C]urea(24). Urease was also detected in column fractions by urea-dependent release ofNH3 (18) or by urea-dependent pH increaseas 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 TMcontaining 25 mM ['4C]urea (85 dpm/nmol). Gels were slicedwith razor blades regularly spaced (5.4 mm) with washers. Aspecific urease activity stain (9) was employed for partially puri-fied ubiquitous urease preparations run on 7.5% native poly-acrylamide gels at 30 mamp constant current for 5 h. Denaturinggels were run as described by Laemmli (12) except that a 6 to9% acrylamide gradient was employed, the upper running buffercontained 0.1% (w/v) LDS, and the lower (cathode) buffer hadno detergent. Samples were denatured by incubating for 5 minat 65°C in 30 mM DTT, 1% (w/v) LDS, 0.5 x TM prior toelectrophoresis for 8 h at 4°C at 8 W constant power.

Urease subunits were detected immunologically after the elec-troblot transfer ofgel-resolved proteins to a cellulose nitrate sheetessentially as described by Towbin et al. (27). Proteins weretransferred for 2 h at 200 mamp in 20% (v/v) methanol, 19.2mM Tris-glycine (pH 8.3) (- 20°C at the start of transfer).Blocking solution was 30% (v/v) goat serum (Gibco; ChagrinFalls, OH), 3% (w/v) BSA (Fraction V, Boehringer Mannheim,Indianapolis, IN) in TBS (20 mM Tris-HCl, 500 mM NaCl). Seed-urease antiserum (rabbit) was diluted 1:400 in 10% goat serum,1% BSA in TBS. Goat antirabbit IgG, conjugated to horseradishperoxidase, and peroxidase substrate were used according to themanufacturer's (Bio-Rad) instructions. Bound proteins were ex-

796 POLACCO ET AL.

4M:Of sis.

UBIQUITOUS UREASE STRUCTURE AND FUNCTION

Seed-

Specific

-J-J 0 CoD J <z co UL

U biquitous

coNH1 Co

N\ 0

FIG. 3. Protein blot analysis of LDS-acrylamide gels containing seed and ubiq-uitous urease subunits. Samples equiva-lent to those of Figure 2 were denaturedand subjected to electrophoresis for 8 hin a 6 to 9% gradient acrylamide gel con-taining 0.1% LDS. After electrophoretictransfer to a nitrocellulose sheet, urease-antigen was detected with serum againstseed urease as described in "Materials andMethods".

posed to the first antibody (anti-urease) overnight at 4C and tothe second antibody (goat antirabbit) for 1 h at room tempera-ture.Mol Wt Determination by Gel Filtration on an Agarose A-O.5

m Column. A column (2 x 47 cm) of agarose A-0.5 m (200-400mesh, Bio Rad), equilibrated in TM plus 0.02% NaN3 (w/v), wascalibrated with blue dextran (nominal mol wt of 2 x 106),apoferritin (2 mg, CalBiochem; LaJolla, CA), catalase, aldolase,ovalbumin, chymotrypsinogen, Cyt c (2 mg each, BoehringerMannheim), and (NH4)2SO4 (0.4 ml of a 2.5% saturated solu-tion). The flow rate was 15 ml/h at room temperature. Thepercentage ofretardation for each standard (setting those for bluedextran and (NH4)2SO4 as 0 and 100%, respectively) was calcu-lated along with the percentage ofretardation ofItachi developingseed urease (24 units), Prize seed urease (approximately 300units), Williams seed urease (approximately 100 units), Itachicell suspension urease (50 units), and Prize cell suspension urease(74 units). Each run with a sample of urease also contained at

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

RESULTS AND DISCUSSIONNative Gel Analysis of Ureolytic Activity in Crude Extracts of

Various Tissues. We had reported previously that leaves andsuspended cell cultures of Prize (seed urease-positive) and Itachi(seed urease-negative) contained the ubiquitous urease (24). Theubiquitous urease was also reported to be the exclusive urease inItachi seeds (24). Crude extracts of these three tissue types,prepared as described previously (24), were run on native poly-acrylamide gels to compare the mobilities of their urease specieswith that ofthe predominant urease in Prize seed extracts. Basedon its migration in these native gels, Prize seed urease has a molwt of approximately 480,000 (21). When localized by assays ofgel slices the ureases of Itachi developing seed, cell culture, andleaves migrate virtually as a common species, and more rapidlythan Prize seed urease (Fig. 1A). Although not shown here, the

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Plant Physiol. Vol. 79, 1985

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FIG. 4. Analysis of mol wt of ubiquitous and seed-specific urease byagarose (A-0.5 m) gel chromatography. Ubiquitous urease samples (@)were from developing Itachi seed and suspension culture of Itachi andPrize. Fast seed-specific (@) urease was from crude extracts of matureWilliams 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 theslow urease, 540,000, was that previously determined by agarose gelfiltration (25).

Table I. Ureases ofDeveloping Embryos and Seed CoatsSeeds picked 25 to 30 d after flowering were separated into embryos

and seed coats.

Seed Urease T Urease ActivityaPhenotype pH 7.0 pH 8.8 7.0/8.8

jumol urea-g-' freshwt-h-'

Williams Positive Embryo 1005 680 1.48Williams Positive Seed coat 2.4 3.7 0.65Itachi Negative Embryo 0.9 1.7 0.53Itachi Negative Seed coat 2.3 3.8 0.61a Values are averages of 2 or 3 separate experiments.

Table II. Glyoxylate Productionfrom Allantoate in Developing ItachiSeed Extracts

Extracts of developing urease-negative Itachi seeds were assayed for 5h at 37C.

Assay Conditions Glyoxylate Formation

psmol-g-' fesh wth-'Complete, Mn2_-treated extract 1.2+ImMPPDa 1.3+10mMurea 1.3+ 10 mM arginine 1.3- Allantoate, + I mM PPD 0.0- Allantoate, + 10 mM arginine 0.0Complete, non-Mn2+-treated extract 0.0

'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 ureaseactivity.

Table III. Effect ofPPD on Levels ofAllantoate Breakdown ProductsExtracts of urease-negative seeds were assayed for 14 h at 35C.

Breakdown Breakdown Products RateProduct -PPD +PPD (250 MM)

;mol.g' fresh wt-h-'Glyoxylate 0.7 0.7Urea 0 0.7Ammonia 5.3 3.3

ureases of cell cultures and leaves of Prize also migrate with theurease of Itachi seed and not with that of Prize seed.

Prize seed urease yields a more rapidly migrating componentin buffers of lowered ionic strength (21) or when heated in thepresence of glycerol and DTT (22; Fig. 1B). Heat-treated Prizeseed urease migrated more rapidly and virtually identically withthe ubiquitous species whose migration was not affected by heattreatment (with the possible exception of cell culture urease, Fig.1B). (The faster species of Prize seed-specific urease has a gel-derived mol wt of 280,000 [211). Based on a subunit size of 93.5kD (21) and the observation that the conversion of slow to fastdoes not yield a third form (i.e. splitting is equal) indicates thatthe slow species is hexameric and the fast species trimeric (21).Buttery and Buzzell (4) had earlier concluded, based on migra-tion ratios in native gels of different acrylamide concentrations,that the fast soybean urease had halfthe mol wt ofthe slow form.The Jack bean urease 480,000 mol wt form is considered to behexameric based on titration (16) and on electron microscopic(8) studies, while an active species half that size, which can beproduced by dissociation in glycols or glycerol (5), is trimeric (8).

Native and LDS Gel Analysis of Partialy Purified UbiquitousUrease. Ureases from developing Itachi seed as well as from cellsuspension cultures of Itachi and Prize were purified 50- to 117-fold ("Materials and Methods"). The ubiquitous ureases ofthesetissues could then be compared with the fast and slow seed ureaseforms by a specific activity stain of native polyacrylamide gels(9). In the gel of Figure 2 the fast and slow forms of the seedurease were provided by crude extracts of mature seeds of thevarieties Williams and Prize, respectively. The genetic fast formof Williams seed migrates identically with the heat-induced fastform of Prize (results not shown).

It is clear from Figure 2 that the ubiquitous ureases from cellcultures and Itachi seed comigrate. However, they migrate some-what more slowly than the fast seed urease and thus appear torepresent 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%)

0 ' Slow Seed-Specific (Prize)FER-\FastFSeed-Specific (Wilems)

b ltachi Seedi Itochi Cal Cult.q Prize Cell Cult.U.

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798 POLACCO ET AL.

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 nitrocellu-lose sheet (27) urease subunits were detected with antiserumraised against the seed (Prize) urease (21). It is apparent (Fig. 3),based on gel migration rates, that ubiquitous and seed-specificureases have nearly identical subunit sizes, although the ubiqui-tous urease subunit appears to run slightly slower. The recogni-tion by seed urease antibodies of the ubiquitous urease confirmsour earlier observations on the serological relatedness, albeitlimited, of the two forms (21, 22, 24).The specificity of the antibody (21) employed here is demon-

strated by its lack of recognition of antigen in extracts of matureItachi seeds (lane 1, Fig. 3). The ubiquitous urease subunit ofdeveloping Itachi seeds was detected (lane 4, Fig. 3) because itwas first purified 59-fold, because developing Itachi seeds have20 times the ureolytic activity of mature seeds (24), and because3.75 times as much developing seed protein (750 ,g) was loadedthan 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 x20 x 3.75) the ureolytic activity of the first lane. Although notshown on this gel, the urease antigenic protein band detected inextracts of mature wild type seeds comigrates with denaturedpurified urease.Agarose Gel Filtration Analysis of the Soybean Ureases. An

attempt was made to determine the mol wt ofthe soybean ureasesby gel filtration on an agarose A-0.5 m column equilibrated withTM buffer and calibrated with proteins ofknown native mol wt.The ubiquitous ureases of Itachi developing seed and of suspen-sion cultures of Prize and Itachi eluted in virtually the sameeluant volume (Fig. 4, retardation values of 21.6-21.9%). Sur-prisingly, in light of its easily distinguished migration rate onnative gels (Fig. 2), the fast seed-specific urease of Williamsextracts appeared to coelute with the ubiquitous species (Fig. 4,retardation value of 21.4%). The nominal mol wt of 345,000 forthe ubiquitous urease (Fig. 4) would indicate that it is either atrimer (280,000) or tetramer (374,000) of 93.5 kD (Fig. 3)subunits. However, its similar mobility to the fast seed urease innative polyacrylamide (Fig. 2) and on an agarose gel sievingcolumn (Fig. 4) suggests that ubiquitous urease, like the fast seed-specific form, is trimeric. We previously reported a gel-derivedmol 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) gelchromatography (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 seedcoats. It was shown earlier (10, 24) that the two urease formsdiffer markedly in pH dependence, which we have used here toidentify the urease of seed coats. Seed coats of Williams andItachi have ureases of similar activity ratios (at pH 7.0 versus pH8.8, Table I). The activity ratio is much closer to that of theItachi embryo (seed minus seed coat) than that of the Williamsembryo which produces a preponderance of the seed-specificurease (Fig. 2). Thus, seed coats produce predominantly orexclusively the ubiquitous urease. It is also noteworthy that thereare comparable urease levels in the seed coats of both varieties(Table I).

Ureide Degradation in Developing Soybean Seeds. Itachi's seedcoat ureolytic activity is more than twice that of its embryo(Table I). Although nitrogen-fixing soybeans transport the bulkof their xylem nitrogen as the ureides allantoin and allantoicacid (13-15), most of the nitrogen delivered to the developingembryo by the seed coat is in the form of amino acids with onlytrace amounts as ureides (25). Thus, the seed coat is a candidatetissue for the conversion of ureides to amino acids, and its highurease level suggests that at least some of the ureide nitrogen is

converted to a urea intermediate.Thus, we first sought to identify an allantoate-degrading activ-

ity in developing seeds (seed coats plus embryos) and then toidentify urea as an intermediate. As can be seen from the resultsof Table II, developing bean (Itachi) extracts contain a Mn+-dependent activity which is essential for the ultimate conversionof allantoic acid to glyoxylate. The production of glyoxylate isnot catalyzed by urease since neither PPD, an excellent inhibitorofthe ubiquitous urease (Table II; 10), nor urea inhibit glyoxylateproduction. Neither is arginase, an Mn2+-activated enzyme (e.g.3), involved since arginine did not diminish the allantoate-dependent production of glyoxylate nor did it itself serve as aglyoxylate source (Table II).To detect allantoate-derived urea it was necessary to eliminate

the considerable seed urease activity. This was accomplished byusing developing seeds lacking the abundant seed-specific urease(Table I) and by inhibiting the remaining ubiquitous urease withPPD. Parallel reactions were employed to study the effects ofPPD on ammonia production.As in the experiment of Table II, PPD had no effect on

glyoxylate accumulation (Table III). However, it resulted in theaccumulation of an easily measured equimolar amount of urea.Since the reaction time was long and products could be con-sumed in other reactions, no valid conclusion can be made ofthe stoichiometry of the products. However, it is apparent thatPPD does not block all ammonia accumulation so that only aportion of the ureide nitrogen of allantoic acid is converted toammonia via urea. This agrees with our earlier observation thatgrowth 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 allan-toate. As shown in the accompaning paper (30) ammonia andCO2 are liberated well before the appearance of glyoxylate.Although glyoxylate and urea are derived from allantoate byaction of an Mn2+-stimulated activity, neither glyoxylate norurea appear to be immediate breakdown products (30). Indeed,their subsequent production may involve nonenzymic break-down of the actual product. I

Evidence for an Assimilatory Function for the UbiquitousUrease. In contrast to the seed-specific urease, whose activityplateaus at maturity (23), the specific activity of the ubiquitousurease in developing Itachi seeds is 20 times higher than in themature seed (24). This is the developmental pattern expected foran enzyme with a role in pod filling. Two observations stronglysuggest 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 hasmore than 5 times the allantoate-degrading activity of the em-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

ofthe ubiquitous urease. Many seeds and storage organs are richin arginine. Pea, a legume without an abundant seed urease (2),catabolizes large amounts of arginine during germination viaarginase and an inducible urease whose activity rises 5-fold overthat found in dry peas (6).

Acknowledgments-We thank Dale Blevins and Doug Randall for critical read-ing ofthe manuscript and Peggy Jo Bledsoe for assistance in the gel slice experiment.

LITERATURE ClITED

1. ATKINS CA, JS PATE, A RITCHIE, MB PEOPLES 1982 Metabolism and translo-cation ofallantoin in ureide-producinggrain legumes. Plant Physiol 70:476-482

2. BAILEY CJ, D BOULTER 1971 Urease,a typical seed protein ofthe Leguminosae.

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