ARCHIVES

Vol.

287,

OF BIOCHEMISTRY

No.

1, May

AND

15, pp.

BIOPHYSICS

151-159,

1991

Ureidoglycolate Amidohydrolase from Developing French Bean Fruits (Phaseok vulgaris [L.].)’ Xanthe

E. Wells

Department

Received

and Edith

of Agricultural

September

12, 1990,

M. Lees’

Chemistry,

and

in revised

University

form

January

of Sydney, New South

12, 1991

Ureidoglycolate is an intermediate of allantoin catabolism in ureide-transporting legumes. This report describes the first purification of ureidoglycolate degrading activity (UGDA) from plant tissue in which the enzyme has been separated from urease. The enzyme from developing fruits of Phaseolus vulgaris has been purified 4%fold to give a preparation free of allantoinase and urease activity. UGDA was inhibited by EDTA while the V mex was increased in the presence of Mn”. The K,,, values for ureidoglycolate in the presence and the absence of Mn” were 2.0 and 5.4 mM, respectively. In the absence of Mn2- UGDA was heat labile at 4O”C, but in the presence of Mn2+ the activity was stable up to temperatures of 60°C. The M, of UGDA was determined to be 300,000 by gel filtration chromatography and the pH optimum ranged from pH 7.0 to 8.5. Ammonia was determined to be the nitrogen-containing product of UGDA by a microdiffusion assay. This enzyme should therefore be described as ureidoglycolate amidohydrolase. The activity was shown to be associated with peroxisomes by fractionation of a crude extract on a sucrose density gradient. The products of ureidoglycolate degradation are glyoxylate, ammonia, and presumably carbon dioxide, which can be readily utilized by pathways of metabolism that are known to be present in this organelle. c8 1991 Academic Press,

Inc.

Allantoin and allantoate are the major transport forms of nitrogen in many legumes such as soybeans, cowpeas, and french beans. They are formed in the nodules of these plants by de nouo pathways of purine synthesis and oxidation and are transported from the nodules to the sites of metabolism throughout the plant (l-4). Ureidoglycolate is an intermediate in the degradation of allantoin, via allantoate, irrespective of whether allantoate amidinohydrolase (EC 3.5.3.4) or allantoate amidohydrolase (EC 3.5.3.9) catalyzes the initial reaction of ’ Supported ’ To whom

hy a Commonwealth correspondence should

OOW9861/91 $3.00 Copyright %, 1991 by Academic Press. All rights of reproduction in any form

Postgraduate he addressed.

Research

Wales 2006, Australia

Award

allantoate breakdown. Ureidoglycolate may be degraded by ureidoglycolate amidinohydrolase (ureidoglycolase, UGL,” EC 4.3.2.3) to release urea and glyoxylate or ureidoglycolate amidohydrolase (UGAH) to produce NH:, , C02, and glyoxylate. There is no clear evidence for the activity of UGL in plants. Studies using labeled allantoin (5) showed that all C-N bonds were broken during allantoin degradation. Although these results could indicate a metabolic pathway using UGL, labeled urea could not be detected. Another paper (6) reports the production of very low levels of labeled urea in plants supplied with labeled allantoin. The presence of increased levels of urease in nodulated plants is interpreted as support for the UGL pathway. Other reports (7-9) have shown that the complete metabolism of allantoin proceeds without the release of urea. The release of glyoxylate and carbon dioxide from allantoate (9) by a partially purified extract of soybean seedcoat proceeds under conditions in which urease in the preparation has been completely inhibited. Ureidoglycolate dehydrogenase (EC 1.1.1.154) which catalyzes the NAD(P)’ -dependent conversion of ureidoglycolate to oxaluric acid (10, 11) has not been reported in plants. Partial purification and characterization of ureidoglycolate-degrading activity (UGDA) from bacteria, fungi, plants, and animals has been reported. The ureidoglycolate-degrading enzyme purified from Candida utilis (12) is the only preparation which has been shown to release both glyoxylate and urea from ureidoglycolate and can therefore be unequivocally described as UGL. The presence of this enzyme is indicated in Bacillus fastidiosus, as when extracts of a urease-deficient strain were incubat,ed with ureidoglycolate glyoxylate was produced but release of NHZ1 was dependent on the addition of jack bean urease ” Abbreviations used: LJGI,, ureidoglycolase; 1 J(:AH, ureidoglycolate amidohydrolase; LJGL)A, ureidoglycolate-degrading activity; Taps, N[tris(hydroxymethyl)rnet hyl]-~-aminopropanesulfonic acid; TEA, t riethanolamine; DTT, dithiothreitol; RSA, bovine senm altmmin; Mes, 2-[N-morpholino]ethanesulfonic acid; Tes, N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid: Ches, 2.(N-c~clohexylamino). ethanesulfonic acid. 151

Inc. reserved.

152

WELLS

AND

(13). Ureidoglycolate-degrading activity (UGDA) has been purified from other microorganisms including Streptococcus allantoicus (14, 15), Pseudomonas aeruginosa (16), and Candida tropicalis (17), but only glyoxylate production was assayed. Similarly, the nitrogenous product of the enzymes purified from frog liver and kidney (18) and sardine liver (19) was not determined. There has been no substantial purification of UGDA from a plant source, and no direct identification of the nitrogenous product of ureidoglycolate degradation. A major problem in the determination of this product of ureide breakdown in in vivo experiments and using partially purified cell-free extracts is the presence of urease activity in the tissues. Although urease inhibitors have been used to allow determination of the pathway of allantoate degradation (9) the detailed mechanism of the pathway cannot be understood without separation of the enzymes concerned from urease. Known urease inhibitors may affect enzymes involved in ureide metabolism (7). This paper describes the partial purification and characterization of UGDA from whole developing fruits of Phaseolus vulgaris. UGDA has been separated from urease, which has allowed the determination of the nitrogen-containing product of the reaction and demonstrated that this enzyme is ureidoglycolate amidohydrolase. The role of manganese in the activity of the enzyme has been examined. MATERIALS Developing whole french bean fruits were purchased from fruit and vegetable retail outlets in the Sydney metropolitan area. Allantoin, ureidoglycolic acid (sodium salt), and jack bean urease, Type III, were obtained from Sigma. Sepharose CL-GB, DEAE-Sephacel, Sephadex G200, Sephacryl S-200, blue dextran, and molecular weight standards were from Pharmacia. All other chemicals were the best grade available for the purpose.

of chilled buffer A, B, or C was added to the assays which were placed in ice for 5 min. Immediately after transfer to room temperature, 1.0 ml 32% (w/v) HCl was added followed by 0.2 ml 1.6% (w/v) potassium ferricyanide (prepared fresh daily). After standing at room temperature for 10 min the absorbance of the red 1,5-diphenylformazan was read at 535 nm. In assays of crude extracts in which protein precipitated in the assay procedure interfered with the absorbance readings the formazan was extracted into 4 ml of chloroform/isoamyl alcohol (3/2 (v/v)) and the absorbance read at 520 nm (21). Standard curves for glyoxylate were constructed in assay buffers A, B, C, and D and the effect of any addition to the reaction mixtures was checked for interference with the assays. Blank reactions containing all components except enzyme (replaced by buffer A, B, C, or D) were processed whenever UGDA assay was performed to monitor the effect that components may have on glyoxylate determination and the nonenzymatic degradation of ureidoglycolate.

Allantoinase Samples were incubated at 25°C for periods of up to 30 min in 25 potassium phosphate, pH 7.5, containing 10 mM allantoin in a total volume of 1.0 ml. Assays were initiated by the addition of allantoin. Allantoate produced was determined as glyoxylate, following acid hydrolysis of the allantoate, essentially as described by Vogels and Van Der Drift (20). Reactions were stopped by the addition of 0.1 ml of 11% (w/v) HCl, stoppered, placed in a boiling water bath for 2 min, and then on ice for 5 min. The tubes were then transferred to room temperature, and 1.0 ml 0.3% (w/v) phenylhydrazine-HCl (prepared within 3 h of use) was added. After 10 min 1.0 ml 24% (w/v) HCl was added followed by 1.0 ml 1.6% (w/v) potassium ferricyanide (prepared daily). The red formazan was determined as described for the UGDA assay. mM

Urease Urease activity was determined by incubating samples at 25°C for periods of up to 30 min in 25 mM potassium phosphate, pH 7.5, containing 10 mM urea. Ammonia released was determined by the phenate-hypochlorite method (22).

Protein Protein measuring

Purification

METHODS The following buffers were used throughout this Buffer A: 50 mM TapssNaOH, 25 mM potassium containing 0.2 mM DTT (dithiothreitol), and 10% Buffer B: 50 mM triethanolamine-HCl (TEA-HCl), 0.2 mM DTT and 10% (v/v) glycerol. Buffer C: 25 mM potassium phosphate, pH 7.5, DTT and 10% (v/v) glycerol. Buffer D: 50 mM TEA-HCl, pH 7.5, containing (v/v) glycerol, and 2 mM MnC12.

Enzyme

LEES

study. phosphate, pH 7.8, (v/v) glycerol. pH 7.6, containing containing 0.2

mM

0.2 DTT,

was determined by Coomassie absorbance at 280 nm.

of UGDA

from French

blue dye binding

(23)

or by

Beans

UGDA was partially purified from french beans in buffers B, C, and D. All operations were carried out at 4°C. Buffer B, C, or D was used throughout all purification procedures and UGDA assays unless otherwise stated.

mM

Preparation of the Crude Extract

10%

Whole french bean fruits were washed in tap water followed by deionized water, cut into 2- to 3-cm pieces, weighed, and chilled. The beans were homogenized in a Sunbeam blender for 5 s on low followed by 3 s on high in 50 mM potassium phosphate, pH 7.5, containing 0.4 mM DTT and 20% (v/v) glycerol (double concentration of buffer C) or buffer B or D, at a ratio of 0.6 ml of buffer to 1 g fresh weight of tissue. The homogenate was filtered through one layer of muslin and two layers of Miracloth, and the filtrate centrifuged for 20 min at 15,000g in an 872 head in an IEC B 20A centrifuge. The supernatant was retained as the crude extract.

Assays

UGDA Glyoxylate released from ureidoglycolate was determined essentially as described by Vogels and Van Der Drift (20). The reaction mixture contained the enzyme preparation in buffer A, B, or C; 0.2 ml 0.3% (w/ v) phenylhydrazineeHC1 (prepared within 3 h of use and adjusted to pH 7.5 with NaOH); 10 mM Na ureidoglycolate (prepared within 1 h of use and used to initiate the assay) and made up to a total volume of 0.4 ml with buffer A, B, or C, respectively. The reaction mixtures were inrllhntc=d at for neriods of UD to 30 min. After incubation. 0.8 ml

Ammonium sulfate fractionation Powdered ammonium sulfate was added to the crude saturation and the resulting suspension was cent,rifized

extract to 60% at 15.000~ as

UREIDOGLYCOLATE described above. The precipitate was resuspended to about one-twentieth of the volume of the crude was dialyzed against 40 vol of the resuspending

AMIDOHYDROLASE in buffer C, B, or D, extract. The suspension buffer for 2 h and then

centrifuged for 10 min at 15,OOOg in an 870 head in an IEC B 20A centrifuge. The pellet, containing aggregated chlorophyll-binding proteins but no UGDA, was discarded and the supernatant was reserved as the O-60% ammonium sulfate fraction.

Sepharose

CL-6B

Chromatography

The O-60% ammonium sulfate fraction was applied to a Sepharose CL-6B column (40 X 2.6 or 100 X 2.6 cm) and eluted with the appropriate buffer at a flow rate of 0.1 ml/cm/min. Fractions were assayed for UGDA, allantoinase, urease, and protein. Fractions containing UGDA were pooled and concentrated in an Amicon ultrafiltration cell, Model 202, using a PM-10 membrane to give the Sepharose CL-6B concentrate.

DEAE-Sephacel

Chromatography

The Sepharose CL-6B eluate or concentrate was applied to a DEAES:phacel column (10 X 1 or 40 X 1 cm). The column was washed with buffer B, C, or D and eluted with a linear gradient of O-O.3 M NaCl in buffer B, C, or D (300.ml total volume) at a flow rate of 0.7 ml/cm/min. Fractions were assayed for UGDA, allantoinase, urease, protein, and conductivity (using a Radiometer CDM conductivity meter). Fractions containing UGDA were pooled and concentrated as described above to give the DEAE-Sephacel concentrate.

Sephadex

G-200jSephacryl

S-200

Determination of the M, of UGDA. The !vf, of UGDA prepared in buffer C was determined by gel filtration using a Sephadex G-200 column (100 X 1.6 cm) equilibrated with buffer C and calibrated with cytochrome c, ovalbumin, aldolase, catalase, ferritin, and blue dextran.

of

UGDA. The UGDA of the in buffer C was assayed in 50 to the required pH using NaOH phosphate, 0.2 mM DTT, and release of glyoxylate was deterin this determination.

Determination of the K, of CJGDA. The K, for ureidoglycolate was determined for UGDA prepared in different buffers. Enzyme preparations used were the DEAE-Sephacel concentrate prepared in buffer C which was assayed in buffer A, and the Sepharose CL-6B concentrate prepared in buffer B which was assayed in the presence (buffer D) and the absence (buffer B) of 2 mM Mn”. A sample of The effect of EDTA and diualent metal ions on UGDA. the Sepharose CL-6B concentrate prepared in buffer B was dialyzed against 1.2 mM EDTA and then dialyzed again to remove EDTA. UGDA was determined after each dialysis. Reaction mixtures were set up containing aliquots of the undialysed enzyme with EDTA, Ca” (CaSO,2H,O), Fe’+ (FeSO, * H,O), Mg” (MgCl,. 6H,O), Ni” (NiC1,.7H,O), Zn2+ (ZnCl,), or Mn2+ (MnCl, .4H,O) at various concentrations in a total volume of 0.19 ml. The reaction mixtures were incubated at room temperature for 10 min. Phenylhydrazine hydrochloride (0.2 ml of 0.3% (w/v) adjusted to pH 7.5 with NaOH) was added

Phaseolus

followed by ureidoglycolate colate of 10 mM in a reaction were incubated for 15 min previously termined

described. for each

153

vulgaris

to give a final concentration of ureidoglymixture of 0.4 ml. The reaction mixtures at 25°C and glyoxylate released assayed as

The nonenzymatic release concentration of EDTA and

of glyoxylate was demetal ion tested. The

stability of this UGDA was determined at temperatures of 30, 40, 50, 60, and 70°C in the presence and the absence of 2 mM Mn’+. Aliquots of the enzyme prepared in buffer B, were incubated with either buffer B or buffer D for 0, 2.5, 5, 10, and 15 min at temperatures from 30 to 7O”C, in a total volume of 0.19 ml, and after rapid cooling were assayed for UGDA as described above. Determination of the nitrogen-containingproduct assay the UGDA of the DEAE-Sephacel concentrate B was used. Nitrogen was determined as ammonia,

of UGDA.

For this prepared in buffer either produced di-

rectly

by the UGDA or released by the action of urease added to the reaction mixture, using a microdiffusion assay carried out in Conway vessels (24). Reaction mixtures were set up as shown in Table I. Aliquots of UGDA (100 and 200 al) were incubated at 25°C with 10 mM ureidoglycolate and phenylhydrazine in buffer B for 45 min. Jack bean urease (6.82 pM units in 50% glycerol) was added to the reactions which were incubated for a further 15 min at 25°C. Controls without IJGDA were included in the series of assays. Additional controls were included to confirm the absence of urease activity in the UGDA preparation, to monitor the nonenzymatic breakdown of ureidoglycolate in the assays and to ascertain whether ureidoglycolate or phenylhydrazine

TABLE

Determination of UGDA: and

Chromatography

A 2-ml sample of the DEAE-Sephacel concentrate prepared in buffer C was applied to a Sephadex G-200 column (95 X 1.6 cm) and eluted with phosphate buffer at a flow rate of 0.25 ml/cm/min and 3-ml fractions were collected and assayed for UGDA and protein. DEAE-concentrates prepared in buffer B or D were applied and eluted from Sephacryl S200 in a similar manner to the Sephadex G-200 chromatography. Fractions containing UGDA were pooled and concentrated to give the Sephadex G-ZOO/Sephacryl S-200 concentrate.

Determination of the pH optimum DEAE-Sephacel concentrate prepared mM Taps, Mes, Tes, and Ches adjusted or HCl and containing 25 mM potassium 10% (v/v) glycerol. The nonenzymatic mined for each buffer at each pH used

FROM

I

of the Nitrogen-Containing Content of Reaction Mixtures Amount of Ammonia Detected

Reaction mixture

Contents

+ Urease

Product Used

Ammonia (nmol)

1

100 ~1 UGDA 10 mM ureidoglycolate

-

117

2

100 ~1 UGDA 10 mM ureidoglycolate

+

610

3

200 ~1 UGDA 10 mM ureidoglycolate

4

200 ~1 UGDA 10 mM ureidoglycolate

5

10 mM

ureidoglycolate

6

10 mM

ureidoglycolate

7

100 ~1 UGDA 10 mM urea

8

10 mM

ureidoglycolate

+

483

9

500 nmol urea 10 mM ureidoglycolate

+

1455

10

100 /.d UGDA 500 nmol urea 10 mM ureidoglycolate

+

1461

203

+

727

0 +

491 0

Note.All reaction mixtures contained buffer B and 0.2 ml 0.3% v) phenylhydrazine, pH 7.5. Jack bean urease (6.82 fiM units) in glycerol was added after incubation at 25°C (reactions 2, 4, and 6) immediately after the reactions were set up (reactions 8, 9, and Assays were performed in duplicate and UGDA was the DEAE-Sephacel concentrate prepared in buffer B.

(w/ 50% and 10).

154

WELLS

AND

caused any inhibition of the jack bean urease. The reaction mixtures were then transferred, with rinsing with buffer B to give a total volume of 0.5 ml, to the outer compartments of the Conway vessels. One milliliter of 0.2% (w/v) boric acid containing bromocresol green was placed in the inner well, and 1.0 ml of saturated potassium carbonate was added to the outer compartment separated from the reaction mixture. The vessels were sealed, solutions in the outer compartments mixed, and the units allowed to stand at room temperature for 45 min. Titration of the contents of the inner well with 1.162 mM H2S04 allowed the determination of the amount of ammonia released during the assays. Subcellular Localization of UGDA. Whole french bean pods were washed, cut into 2- to 3-cm pieces and chilled as described for enzyme purification. The beans were then finely chopped using a razor blade in a prechilled petri dish with 50 mM potassium phosphate, pH 7.5, containing 0.2 mM DTT, 0.4 M sucrose, and BSA (50 mg/ml), using 1 ml buffer to 1 g fresh weight until a macerate was formed. The macerate was allowed to stand for 10 min before being filtered through two layers of Miracloth. A lo-ml aliquot of the crude extract was layered onto 24 ml of a linear sucrose density gradient from 25 to 55% (w/w) sucrose containing 20 mM potassium phosphate, pH 7.5, and 0.2 mM DTT. The gradient was then centrifuged for 180 min at 25,000 rpm at 5°C in a Beckman SW 27 rotor in a Beckman Model L-2 preparative ultracentrifuge. The gradients were fractionated into l-ml aliquots by displacing the contents of the tubes with 65% (w/w) sucrose. UGDA was assayed as described in buffer A. Catalase was assayed by incubating aliquots of the fractions with H,OP in 100 mM Tris-HCl, pH 7.5, and observing the decrease in the AZaO of the reactions (25). Fumarase was assayed by incubating aliquots of the fractions with 50 mM I,-malate in 100 mM Tris-HCl, pH 7.5, and observing the increase in absorption at 240 nm

TABLE Purification

of UGDA

from

Developing

UGDA prepared in buffer C and assayed in buffer A

Fraction Crude extract O-60% ammonium sulfate precipitate Sepharose CL-6B concentrate DEAE-Sephacel concentrate Sephadex G-200/ Sephacryl s-200 concentrateb

LEES

(26). The density of the fractions was determined by measuring the refractive index of the samples using a Bellingham Stanley refractometer.

RESULTS UGDA

Assay

Standard curves for glyoxylate prepared in buffers A, B, C, and D were identical and showed that neither EDTA nor Mn2+ affected the standard curve and that phosphate was not required for reaction of glyoxylate with phenylhydrazine (20). Preliminary studies indicated that when a crude extract of french bean fruits was incubated with ureidoglycolate, in the absence of phenylhydrazine, no enzyme-dependent release of glyoxylate could be detected. However, when phenylhydrazine was included in the reaction mixture (19) the low level of activity present in the crude extract could be determined. Subsequently phenylhydrazine was included in all UGDA assays described in this report. Preliminary assays to determine the nitrogenous product of the UGDA reaction were carried out in the absence of phenylhydrazine and unsuccessful attempts were made to determine ammonia released by coupling with glutamate dehydrogenase. Ammonia release was thus determined by the microdif-

11 French

UGDA

Bean

Fruits

in Various

Buffers

prepared and assayed in buffer B

Purification (fold)

Total activity NJ)

Specific activity W/w protein)

2.6

1.0

16,500’

830

3.5

1.3

330

5.8

247d

250’

UGDA

prepared and assayed in buffer D

Purification (fold)

Total activity NJ)

Specific activity W/w protein)

Purification (fold)

4.3

1.0

6975”

3.3

1.0

12,000

7.7

1.7

5075

4.5

1.4

2.2

6,000

10.0

2.2

8550

26.8

8.2

10.3

4.0

4,oood

37.7

8.5

6555d

84.5

25.9

40.0

15.4

2,300’

194.9

43.9

2255’

157.1

48.2

Total activity UJ)

Specific activity W/mg protein)

950”

Note. U, nmoles glyoxylate released per minute. ’ Prepared from 150 g french bean fruits. * Calculated from aliquot of DEAE-Sephacel concentrate ’ Prepared from 800 g french bean fruits. d DEAE-Sephacel concentrate (urease not detectable). ’ Sephadex G-200 concentrate. i Sephacryl S-200 concentrate.

applied

to the Sephadex

G-200

or Sephacryl

S-200

column.

UREIDOGLYCOLATE

I

AMIDOHYDROLASE

w

I

FROM

Phase&s

155

uulgaris

in buffer D throughout the purification procedure the elution profile of the enzyme on Sepharose CL-6B indicated that this preparation also had a molecular weight of about 300.000.

6-

Determination

of the pH Optimum

of UGDA

UGDA prepared in buffer C was found to have a broad pH optimum in the range from 7.0 to 8.5 (Fig. 3). The rate of nonenzymatic breakdown of ureidoglycolate (to urea and glyoxylate), which increases rapidly above pH 9.0 and below pH 7.5, is shown in Fig. 3. The Effect of EDTA and Divalent Metal Ions on UGDA

0

100 Elution

200 volume

300

(ml)

FIG. 1. DEAE-Sephacel elution profile of the Sepharose CL-6B concentrate prepared from french bean fruits in Buffer C. The distribution of b axis) [JGDA (m), pm01 glyoxylate/ml/30 min; urease (0, pmol ammonia/ml/30 min; allantoinase (A), pm01 allantoate/ml/30 min; protein (O), mg/ml; and conductivity (&), mS/cm/lO, are presented. The column was eluted with a linear O-0.3 M NaCl gradient in buffer C, followed by 1 M NaCl in the same buffer.

fusion assay after incubation of UGDA colate in the presence of phenylhydrazine.

with

ureidogly-

Purification of UGDA Glycerol and DTT were found to be necessary for the stability of UGDA in cell-free extracts and were therefore included in all buffers used. When UGDA was purified in buffer C and assayed in buffer A a 15.4-fold increase in specific activity over that found in the crude extract was achieved. This UGDA, prepared using ammonium sulfate fractionation, gel filtration, and ion-exchange chromatography (Table II), had a specific activity of 40 nmol glyoxylate released/min/mg protein. Allantoinase was separated from the higher molecular mass urease and UGDA on Sepharose CL-6B chromatography. UGDA, eluted at 7 mS/cm, and urease, eluted at 12 mS/cm, were clearly separated on DEAE-Sephacel chromatography (Fig. 1). Allantoinase and urease could not be detected in the DEAE-Sephacel, Sephadex G-200, and Sephacryl S200 concentrates. When UGDA was purified and assayed in buffer D a 48.fold increase in specific activity was achieved over that in the crude extract to give a final specific activity of 157 nmol of glyoxylate released/min/ mg protein (Table II). Determination of the M, of UGDA The molecular mass of UGDA prepared in buffer C was found to be 300,000 (Fig. 2). When UGDA was prepared

UGDA was inhibited by EDTA. After dialysis against 1.2 mM EDTA activity was reduced to about 5% of the original level. Further dialysis to remove EDTA resulted in recovery to about 15% of the original level. When this preparation was assayed in the presence of 2 mM Mn2’ activity was about 170% of the original. Assay of aliquots of the undialyzed enzyme after incubation with EDTA showed that 50% inhibition was achieved by preincubation with 0.02 mM EDTA. Of the divalent metal ions (concentration 0.2 mM) tested for possible activation of the enzyme only Mn”+ and Zn2’ resulted in a substantial increase in the UGDA activity of the preparation. Maximal activity was achieved at Mn2+ concentrations above 0.2 mM whereas activity was stimulated by Zn’+ to a concentration of 0.2 mM, above which inhibition was observed (Fig. 4). Activity was increased slightly by Fe”+,

h

0.8

0.6

-

0.4

-

0.2

-

q

Sta

n

UCiDA

n

2 Y

0.0

4

5

6

log Mr FIG. 2. Determination of the M, of UGDA from french bean fruits prepared in buffer C. The calibration curve was prepared using Sephadex G-200, equilibrated, and eluted with buffer C. Standards were cytochrome c (12,X)0), ovalbumin (43,000), aldolase (158,000). catalase (232,000), and ferritin (440,000).

156

WELLS

4oi

“’ 5

I

I

6

7

i

d

1’0

PH FIG. 3. The effect of pH on the activity of UGDA from french beans and nonenzymatic breakdown of ureidoglycolate. The DEAE-Sephacel concentrate prepared in buffer C was used. The buffers used were 50 mM Mes (pH 5.7 to 7.1), 50 mM Tes (pH 7.0 to 8.1), 50 mM Taps (pH 7.9 to 9.2), and 50 mM Ches (pH 9.0 to 9.9) all containing 25 mM potassium phosphate, 0.2 mM DTT, and 10% (v/v) glycerol. UGDA (A) [y axis] nmoles glyoxylate released/min/mg protein (values presented have been corrected for nonenzymatic breakdown of ureidoglycolate for each buffer at each pH value); (Cl) nonenzymatic breakdown of ureidoglycolate, A 520 nm/lO min (X10).

AND LEES

are shown in Table I. Reaction 7 confirms the absence of urease activity in the UGDA preparation, and reaction 5 confirms that ammonia is not released during the nonenzymatic breakdown of ureidoglycolate. Reactions 9 and 10 show that phenylhydrazine (reactions 9 and lo), ureidoglycolate (reaction 9), and UGDA (reaction 10) do not inhibit urease activity in this assay, as the theoretical amount of ammonia (1000 nmol) is released from 500 nmol of urea when the 491 or 483 nmol ammonia produced by the action of high levels of urease on UGDA (reactions 6 or 8) is subtracted. Reactions 1 (117 nmol) and 3 (203 nmol) show that ammonia is released directly from ureidoglycolate in proportion to the amount of UGDA used in the assay (100 yl in reaction 1, and 200 ~1 in reaction 3). Reactions 2 and 4 confirm the results from reactions 1 and 3. Thus the net production of ammonia released by 100 ~1 UGDA from ureidoglycolate is 610 - 491 = 119 nmol as 491 nmol are released from ureidoglycolate by the urease, and the net production of ammonia from 200 ~1 of UGDA is 727 - 491 = 236 nmol. These results show that the products of the enzymatic breakdown of ureidoglycolate by UGDA are glyoxylate, ammonia, and presumably C02. The UGDA activity in french bean fruits may therefore be described as ureidoglycolate amidohydrolase (UGAH). Subcellular Location of UGDA Figure 5 shows the distribution of UGDA, catalase, fumarase, and sucrosedensity throughout the gradient. The

Mg’+,

and Ca2+ and inhibited slightly by Ni’+. In the presence of 2 mM Mn’+, full enzymatic activity of UGDA was retained after incubations for 15 min at 40, 50, and 60°C. At 7O”C, 3% of original activity remained after a 15 min incubation. In the absence of 2 mM Mn2+, enzymatic activity was not maintained with heating. The percentages of original activity remaining after incubation at 40, 50, and 60°C for 15 min were 40, 12, and 2%, respectively. Determination

20 ‘1

of the Km of UGDA

The K, for ureidoglycolate for UGDA prepared in buffer C was determined to be 5.4 mM. The Km for ureidoglycolate for UGDA prepared in buffer B was dependent on the assay buffer. When assayed in buffer B (no Mn2+) the Km was 5.5 mM and when assayed in buffer D (with Mn2’) the K,,, was 2 mM. The V,,, values determined were 105 nmol glyoxylate released/min/ml without added Mn2+ and 182 nmol/min/ml with Mn2+. of the Nitrogen-Containing Product of UGDA

0

1 Divalent

Determination

The results of the assays to determine the ammonia released from ureidoglycolate on incubation with UGDA

FIG.

4.

The

effect

2

cation (mM)

of varying

concentrations of Mn2+ (0) and Zn*+ CL-6B concentrate prepared in buffer B. Results have been corrected for nonenzymatic breakdown of ureidoglycolate for each concentration of cation.

(0) on the UGDA of the Sepharose

UREIDOGLYCOLATE

Fraction

AMIDOHYDROLASE

number

FIG. 5. Sucrose density gradient fractionation of a crude extract of french beans fruits. The crude extract, was prepared in 50 mM potassium phosphate, pH 7.5, containing 0.2 mM DTT, 0.4 M sucrose, and BSA (50 mg/ml). The linear sucrose density gradient, from 25 to 55% (w/w) sucrose contained 20 mM potassium phosphate, pH 7.5, and 0.2 mM DTT. The distribution of (.v axis) UGDA (m) nmol glyoxylate/90 ~1/60 min; catalase (A) A, 240 nm/50pl/min (X100); fumarase (0) A, 240 nm/ 5O~l/min (X1000); and sucrose density (-), g/ml is presented.

mitochondrial marker, fumarase, was located at a density of 1.16-1.18 g/ml and the peroxisome marker, catalase, at a density of 1.19-1.22 g/ml. UGDA was also detected at a density of 1.19-1.22 g/ml indicating that it was associated with the peroxisomes of french beans and not with the mitochondria. DISCUSSION Assay of UGDA

The assay method described here proved to be reliable and reproducible for the determination of UGDA in french beans. For crude extracts containing high levels of protein and low activity, extraction of the formazan into a small volume of an organic solvent enabled the activity to be determined accurately without interference from the precipitated protein. It has been reported that the interaction of phenylhydrazine with glyoxylate is catalyzed by phosphate ions (20) and preliminary experiments to characterize this enzyme in french bean extracts were carried out in phosphate buffers A and C. In later experiments

FROM

Phaseolus

uulgaris

157

to examine the role of manganesein the activity of UGDA, buffers B and D were used to avoid the precipitation of added Mn2+ by phosphate. Standard curves for glyoxylate in buffers A, B, C, and D were identical and showed that under these assay conditions phosphate was not essential for glyoxylate assay. The inclusion of EDTA had no effect on the standard curve for glyoxylate. Careful determination of the nonenzymatic breakdown of ureidoglycolate was necessary for the successful examination of UGDA. Breakdown of the substrate to glyoxylate and urea was greater in buffer C than in TEA (buffer B) and was increased by the presence of metal ions. UGDA was routinely assayed at pH 7.5 which permits determination of activity at an optimal pH with minimum nonenzymatic breakdown of substrate (Fig. 3). UGDA-dependent release of glyoxylate from ureidoglycolate could not be detected unless phenylhydrazine was included in the reaction mixture for the duration of the incubation. In crude extracts this could be attributed to further metabolism of the low levels of glyoxylate released by enzymes such as glycolate oxidase or aminotransferases, but the failure to detect UGDA in purified extracts in the absence of phenylhydrazine cannot be explained in this way. Reports of substantial purification of UGDA from fish liver (19) and Candida tropicalis (17) describe the assay of the enzyme by incubation with phenylhydrazine, and do not comment on the activity of the enzyme if phenylhydrazine is added after completion of the period of incubation. Our results suggest the removal of one or all of the products of the reaction may be necessary for the metabolism of ureidoglycolate in the tissue. This is consistent with our experiments to determine the nitrogenous compound released by UGDA. No enzyme-catalyzed release of ammonia could be detected unless the initial incubation was carried out in the presence of phenylhydrazine. The breakdown of ureidoglycolate to release two molecules of ammonia, one molecule of COZ, and one molecule of glyoxylate is not a simple reaction and further investigations are required to enable us to fully understand the mechanism of this enzyme. Purification of UGDA Developing french bean fruits proved to be a satisfactory source of material for the purification of UGDA. In legumesin which ureides are a major source of transported nitrogen, such as soybean, cowpea, and french bean, the ureides are produced in the nodules, transported throughout the plant, and metabolized when required to provide nitrogen for protein synthesis. Ureide breakdown takes place in the developing fruits to supply nitrogen for the production of storage proteins. Whereas soybean and cowpea are harvested at the mature seed stage, the commercial product of the french bean is the developing fruit, which is available throughout the year in Sydney in retail fruit and vegetable outlets. The level of the enzyme in

158

WELLS

crude preparations varied considerably. This may result from the use of different varieties of beans, grown under a range of environmental conditions with variable postharvest storage. However, irrespective of the initial enzyme activity of the crude preparations, UGDA was consistently purified 15-fold in buffer C, 44-fold in buffer B (-Mn’+), and 48-fold in buffer D (+Mn”). The most important step in the purification procedure for UGDA from french bean fruits was DEAE-Sephacel chromatography, which clearly separated the UGDA from the urease activity of the extract (Fig. 1). The initial ammonium sulfate precipitation served primarily to concentrate the low level of activity in the crude preparation and the Sepharose CL-6B chromatography allowed separation of the membrane-bound broken chloroplast material from the UGDA and other soluble enzymes. It has been suggested that allantoate and ureidoglycolate degrading activity may be catalyzed by urease (27). This purification procedure in either phosphate or TEA buffer shows clearly that UGDA is a separate enzyme activity. The UGDA preparation obtained by DEAE-Sephacel chromatography and further purified on Sephadex G-200/ Sephacryl S-200 had no detectable urease or allantoinase activity. The pH optimum of the UGDA of 7.0 to 8.5 (Fig. 3) is similar to that reported for the enzyme from other sources (12,X, 18,19,28). In contrast, the M, of the french bean UGDA, which was found to be 300,000, differs from that reported for the enzyme from sardine liver (19). The Role of Metal Ions in UGDA

from French Beans

Inhibition by EDTA and reduced specific activity of preparations in phosphate buffers (which could remove metal ions from the enzyme) suggest that UGDA requires metal ions for optimal activity. However, EDTA inhibition of the enzyme could be overcome by removal of the EDTA by dialysis and the addition of 2 mM Mn2’ to the assay buffers. It is possible that the metal ion required by the enzyme is tightly bound to the protein and is not removed during EDTA inhibition. Similarly, the reduced specific activity of the enzyme prepared in phosphate may be caused by interaction of the metal ion with the phosphate which reduces activity without complete denaturation of the enzyme. The results of the determination of the K,,, and V,,, values of the enzyme, prepared in phosphate and TEA buffers and assayed in the presence and the absence of Mn2+, show that Mn*+ could have a major effect on the activity of the enzyme in the tissue. The K,,, for ureidoglycolate of 2 mM in the presence of Mn2+ is within the very wide range of values from 2.5 PM (12) to 92 mM (29) reported for other organisms. Stabilization of the enzyme to temperatures of up to 70°C in the presence of Mn2+ was consistent with the response of the enzymes from frog liver and kidney (18) and Pseudomonas aeruginosa (16) to similar high temperatures.

AND

LEES

The Nitrogen-Containing Product of UGDA The absence of detectable urease activity in this preparation of UGDA has enabled the unequivocal identification of the nitrogenous product of the enzyme. The urease inhibitors used in several in viva (30) and in vitro studies (9), such as acetohydroxamate and phenyl phosphordiamidate, are known to act by forming complexes with metalloenzymes (31). Evidence presented here, and in other reports (17, 19), that UGDA is also a metalloenzyme means that these inhibitors could also inhibit UGDA. Separation of the ammonia produced by microdiffusion allowed determination of ammonia with and without the presence of urease, and demonstrated that ammonia is released directly from the ureidoglycolate by the action of UGDA. This result is consistent with a report (32) that nickel-starved soybean suspension cell cultures, which do not contain active urease, could be grown on media containing allantoin but not urea as the sole nitrogen source. It has been suggested (6, 30) that urea is produced during allantoin degradation from both allantoate and ureidoglycolate, but very low levels of activity were detected. Our results are in agreement with data which showed that a crude cell-free preparation of soybean seed coats released COZ from allantoate and ureidoglycolate in the presence of urease inhibitors (9). The low activity of this preparation may have resulted from inhibition of the allantoate degrading activity by the urease inhibitor used. Intracellular Location of UGDA UGDA from french bean fruits was shown to be located in peroxisomes like that in fish liver (19) and C. tropicalis (17). Peroxisomal metabolism of ammonia and glyoxylate has been described (33,34). The direct releaseof ammonia from ureidoglycolate allows further metabolism of the ammonia to take place within the peroxisome. When [4,514C]allantoin was fed to soybean leaves 14C-iabel was found in allantoate, COz, glyoxylate, and serine (8). The proposal that the two labeled non-ureido carbon atoms of allantoin had entered the photorespiratory pathway is consistent with the location of UGAH in the peroxisomes. REFERENCES 1. Thomas, 371.

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