Purification and properties of inosine monophosphate oxidoreductase from nitrogen-fixing nodules of cowpea (Vigna unguiculata L. Walp)

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 236, No. 2, February 1, pp. 807-814, 1985

Purification and Properties of lnosine Monophosphate Oxidoreductase from Nitrogen-Fixing Nodules of Cowpea (Vigna unguiculata L. Walp)’ C. A. ATKINS,2 Botany

Department, Received

University May

B. J. SHELP,3 of

Western

AND

Australia,

14, 1984, and in revised

form

P. J. STORER Nedhuis

h’(x)9 WA, Awtralia

September

14, 1984

Using ammonium sulfate precipitation, gel filtration, and affinity chromatography, inosine monophosphate (IMP) oxidoreductase (EC 1.2.1.14) was isolated from the soluble proteins of the plant cell fraction of nitrogen-fixing nodules of cowpea (Vigna unguiculata L. Walp). The enzyme, purified more than 140-fold with a yield of ll%, was stabilized with glycerol and required a sulfydryl-reducing agent for maximum activity. Gel filtration indicated a molecular weight of 200,000, and sodium dodecyl sulfate-gel electrophoresis a single subunit of 50,000 Da. The final specific activity ranged from 1.1 to 1.5 pmol min-’ mg protein-‘. The enzyme had an alkaline pH optimum and showed a high affinity for IMP (K, = 9.1 X lop6 M at pH 8.8 and NAD levels above 0.25 mM) and NAD (K, = 18-35 X 10e6 M at pH 8.8). NAD was the preferred coenzyme, with NADP reduction less than 10% of that with NAD, while molecular oxygen did not serve as an electron acceptor. Intermediates of ureide metabolism (allantoin, allantoic acid, uric acid, inosine, xanthosine, and XMP) did not affect the enzyme, while AMP, GMP, and NADH were inhibitors. GMP inhibition was competitive with a Ki = 60 X low6 M. The purified enzyme was activated by K+ (Km = 1.6 X 10e3 M) but not by NH:. The K+ activation was competitively inhibited by MgZ+. The significance of the properties of IMP oxidoreductase for regulation of ureide biosynthesis in legume root nodules is discussed. 0 1985 Academic Press. Inc.

In many tropical legumes, fixed nitrogen is exported from nodules primarily as the ureides (1,2), allantoin and allantoic acid, which are formed by a pathway involving de nova purine synthesis (3) followed by purine oxidation (4). Recently the occurrence of NAD-dependent IMP oxidoreductase (EC 1.2.1.14) in cowpea nodules was demonstrated (5). The enzyme was extremely labile in crude extracts, but a preliminary kinetic study indicated that

IMP oxidation, rather than its dephosphorylation, might be the predominant pathway leading to xanthine and the formation of ureides in v&o (5). In the present study IMP oxidoreductase from cowpea nodules has been purified to homogeneity and some kinetic and regulatory properties of the enzyme investigated. MATERIALS

AND

METHODS

Reagents. In all cases analytical reagent-grade chemicals were used without further purification. IMP, XMP, AMP, GMP, ADP, ATP, xanthosine, inosine, xanthine, hypoxanthine, NAD, NADH, NADP, allopurinol, allantoin. allantoic acid, and uric acid were obtained from Sigma; Sephacryl S-200 and Sephadex G-25 from Pharmacia; Affi-Gel Blue-agarose, protein molecular weight standards, and Coomassie blue R-250 from Bio-Rad Laboratories; cat-

i Supported by funds from the Australian Research Grants Scheme. s To whom correspondence should be addressed, at International Atomic Energy Agency, Wagramerstrasse 5, P.O. Box 200, Vienna A-1400, Austria. ’ Recipient of NSERC (Canada) Postdoctoral Fellowship. Present address: Department of Horticulture, University of Guelph, Guelph. Ontario, Canada. 807

0003-9861/85 Copyright All rights

$3.00

0 1985 by Academic Press. Inc. of reproduction in any form reserved.

808

ATKINS,

SHELP,

alase (bovine

liver), albumin (bovine serum, crystalline), and cytochrome c (horse heart) from Calbiochem; and alcohol oxidoreductase from Boehringer. Plant material Cowpea (Vigna unguiculata (L.) Walp cv. Caloona) plants, effectively nodulated with Rhiwbium strain CB756, were grown in sand culture with nitrogen-free nutrient solution in a naturally lighted glasshouse. Nodules were harvested from 4to B-week-old plants and washed with deionized water before use. Enzyme assays. IMP oxidoreductase activity (EC 121.14) was routinely measured as continuous, IMPdependent NAD (or NADP) reduction at 30°C by the change in absorbance at 340 nm. The l-ml reaction mixture contained 50 mM Tricine*-KOH (pH 8.8), 1 mM dithiothreitol, 50 mM KCI, 1.25 mM NAD (or NADP), 1 mM allopurinol, 1.35 mM IMP, and lo-100 ~1 enzyme solution. Assays of the purified enzyme did not include allopurinol. Xanthine oxidoreductase (EC 1.2.1.37) was measured in the same way using the reaction mixture outlined above for IMP oxidoreductase, but replacing IMP with 1 mM xanthine and omitting allopurinol and KCl. Kinetic studies were carried out by varying conditions as specifically indicated in the text. In all cases linear reciprocal plots were defined by simple regression analyses of the data. Two buffer mixtures were used to determine the relationship between enzyme activity and pH: 50 mM Tris-glycine adjusted to pH 10.2 with KOH and readjusted to the appropriate pH (10.2-7.0) with HCI; and 50 mM MesHepes adjusted to pH 6.5-7.5 with KOH. Where the effect of an added compound on enzyme activity was investigated, the addition was made before the reaction was initiated with substrate. Analytical methoo!.s. Polyacrylamide gel electrophoresis of native protein was carried out in 4.8% resolving gels according to Ornstein (6) and Davis (7), and in the presence of SDS in 10% gels as described by Laemmli (8). Protein was monitored in column eluates following TCA precipitation, and in enzyme preparations at all stages of purification by the method of Lowry et al (9), using bovine serum albumin as standard. RESULTS

Enzyme PurQicatim

Operations at all steps in the purification procedure were carried out at 4°C. 4 Abbreviations used: Tricine, N-[2-hydroxy-l,lbis(hydroxymethy)ethyllglycine; Mes, 4-morpholineethanesulfonic acid; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid.

AND

STORER

About 30-40 g fresh wt of detached nodules was crushed in 1.5 ~0150 mM TricineKOH buffer (pH 8.8) containing 15% (v/ v) glycerol and 10 mM mercaptoethanol (Buffer A). The crude nodule extract was filtered through 100~pm nylon mesh and the soluble fraction collected by centrifugation (30,OOOg for 10 min at 4°C). The pellet, which contained the bacteroids, showed negligible IMP oxidoreductase activity and was discarded. Total activity in the soluble fraction was determined after a portion was passed through a Sephadex G-25 column equilibrated with 50 mM Tricine-KOH (pH 8.8) containing 10 mM mercaptoethanol (Buffer B). The fraction precipitating between 25 and 35% saturation of the soluble extract with ammonium sulfate was redissolved in 2 ml buffer B and applied to an Affi-Gel Blue-agarose column (2 X 20 cm) equilibrated with the same buffer. The column was developed at a flow rate of 13 ml/h and 5-ml fractions were collected (Fig. 1). After washing with 100 ml buffer B to remove most of the protein from the column, a second

FIG. 1. Elution profiles for the affinity chromatography step (Affi-Gel Blue) in the purification of IMP oxidoreductase (IMPOR) and its separation from xanthine oxidoreductase (XOR) in extracts of cowpea nodules. After sample application, the column (2 X 20 cm) was washed with 100 ml Tricine-KOH (pH 8.8) buffer containing 10 mM p-mercaptoethanol, followed by 100 ml buffer + 10 mM NAD, and 100 ml buffer containing 15% (v/v) glycerol + 10 mM NAD, 1 mM IMP. Dashed lines indicate times of buffer change. Fractions (5 ml) were collected at a flow rate of 13 ml/h. Protein values are on a fraction basis.

IMP

OXIDOREDUCTASE

group of proteins, including xanthine oxidoreductase, were eluted by 100 ml buffer B containing 10 mM NAD. IMP oxidoreductase was eluted with buffer A containing 10 mM NAD and 1 mM IMP. Active fractions were combined, and the protein was concentrated by precipitation with saturated ammonium sulfate (in 0.1 mM Tricine-KOH, pH 8.2) added to a final concentration of 60%. Following centrifugation, the pellet was resuspended in a final volume of 1.5 ml with 50 mM TricineKOH (pH 8.8) containing 10 mM mercaptoethanol and 5% glycerol and applied to a Sephacryl S-200 column (2 X 25 cm) equilibrated with the same buffer. The column was developed at a flow rate of 30 ml/h and the eluate was collected in 3-ml fractions (Fig. 2). The major IMP oxidoreductase-containing fractions from the Sephacryl column were combined (those with less than 10% of the total activity being discarded) and, following concentration of the protein by ammonium sulfate precipitation, the preparation (final volume, 1.5 ml) was frozen in liquid Na. Although frozen preparations remained stable for up to 3 months under liquid Na, repeated freezing and thawing resulted in progressive loss of activity. Accordingly, the purified enzyme was frozen in 20-~1 I

FIG. 2. Elution profile for the gel-filtration step (Sephacryl S-200) in the purification of IMP oxidoreductase from cowpea nodules. After application of enzyme, the column (2 X 25 cm), which was equilibrated with 50 mM Tricine-KOH (pH 8.8) containing 10 mM mercaptoethanol and 5% (v/v) glycerol, was developed at a flow rate of 30 ml/h and 3-ml fractions were collected. Protein values are on a fraction basis.

IN

809

COWPEA

droplets, which could readily be sampled whenever enzyme was required for characterization, without repeated thawing and freezing of the rest of the preparation. A typical result for the stepwise purification procedure is shown in Table I. Although generally less than 20% of the initial activity was recovered, the final specific activity indicated a purification of around 150-fold compared to the crude nodule extract. The purified enzyme was completely free of xanthine oxidoreductase activity and showed a single band of protein following polyacrylamide gel electrophoresis (Fig. 3). The final specific activity of preparations made during this study ranged from 1.1 to 1.5 pmol rnin~’ mg protein-‘. Properties

of PuriJied

Enzyme

Comparison of the elution of nodule IMP oxidoreductase with a series of protein standards of known size from a gelfiltration column (Fig. 4) indicated a molecular weight of around 200,000. SDSpolyacrylamide gel electrophoresis yielded a single subunit of molecular weight 50,000 (Fig. 5). The purified enzyme showed a markedly alkaline pH optimum (Fig. 6A) and, in addition to an effect on V,,,,,, pH altered the K, (IMP) of the enzyme (Fig. 6B), indicating uncompetitive inhibition of H+ or a protonated form of the substrate. Full activity at both optimum and suboptimal pH values required the addition of dithiothreitol to the assay mixture. The enzyme displayed a high degree of specificity for both IMP and NAD. Other purine derivatives (inosine, hypoxanthine, xanthine, and GMP) were not effective substrates and NADP, at concentrations up to 1.35 mM, was reduced at rates less than 10% of those with NAD. Molecular oxygen did not serve as an electron acceptor. Although the equilibrium position of the catalyzed reaction with IMP and NAD was not established, there was no oxidation of NADH (up to 0.1 mM) in the presence of XMP (up to 1 mM). With NAD levels above 0.02 mM and as high as 1.25 mM, kinetic analysis provided

810

ATKINS,

SHELP, TABLE

PURIFICATION

OF IMP

OXIDOREDUCTASE

I

FROM THE SOLUBLE

PROTEINS

OF COWPEA

Total activity (nmol mini)

NODULES Specific activity (nmol min-’ mg protein-‘)

Protein

(ml)

(md

58

380

3000

100”

7.9

55

212

3006

100

14.2

2331

‘78

34.0

Crude extract of nodules Soluble fraction collected by centrifugation 25-35% Ammonium sulfate precipitate Pooled fractions from Blue A&Gel column precipitated with 60% ammonium sulfate Pooled fractions from Sephacryl S-200 column precipitated with 60% ammonium sulfate

“Recovery assays.

STORER

Volume Step

Note. Results

AND

5.0

68.5

Yield (%I

1.5

0.95

627

20

660

1.5

0.30

337

11

1123

are for a typical preparation at each step is corrected for

from 37 g fresh portions removed

wt of nodules. and used for

enzyme

activity

and

protein

a relatively constant Km (IMP) value minations of 9.1 X 10m6M at pH 8.8. Howranging from 8.1 X 10m6M to 10.4 X 10e6 ever, as indicated in Fig. ‘7, the Km M, with a mean from 10 separate deter(IMP) increased progressively at NAD levels below 0.02 mM, reaching values of 25 X 10m6M at 0.005 mM NAD, the low-

0.4-

: 8 -2 0.2-

L

40

ELUTION

Y

2

4

DISTANCE

6 km)

8 +

FIG. 3. Polyacrylamide disc gel electrophoresis of purified IMP oxidoreductase from cowpea nodules. Resolving gels (4.8%) were developed for 6 h at 2 mA/tube with 0.05 M Tris, 0.38 M glycine, pH 8.3, buffer at 4°C. After staining with 0.125% (w/v) Coomassie blue R-250 and destaining, the gels were scanned at 595 nm.

120

so

VOLUME

(ml)

FIG. 4. Estimation of molecular weight of purified IMP oxidoreductase (IMPOR) from cowpea nodules by Sephacryl-200 gel filtration. Slope was based on the elution volumes of the standard proteins: catalase (A& 250,000), alcohol oxidoreductase (ikf, 141,000), bovine serum albumin (M, 66,200), and cytochrome c (A4,12,400). Chromatography was carried out on a column (2 X 25 cm) equilibrated with 50 mM TricineKOH (pH 8.8) containing 5% (v/v) glycerol, and developed at a flow rate of 30 ml/h. Protein in the eluate was determined at 280 nm.

IMP

2

4

6

OXIDOREDUCTASE

6

0.6.

IN

COWPEA

811

ductase was unaffected by 1 mM concentrations of XMP, xanthosine, inosine, xanthine, hypoxanthine, uric acid, allantoin, or allantoic acid in assays containing 0.1 mM each of IMP and NAD. The adenylates (AMP, ADP, and ATP) GMP and NADH were all inhibitory, reducing activity by up to 60% at 1 mM. Inhibition of reaction with IMP by GMP was competitive, kinetic analysis indicating a Ki value of 60 X lO-‘j M (Fig. 8). When the purified enzyme was transferred from Tricine-KOH (pH 8.8) to TrisHCl (pH 8.8) buffer by passage through G-25 Sephadex, catalytic activity was markedly reduced. Addition of K+ restored activity and kinetic analysis showed the KD (K+) to be 1.6 X lop3 M (Fig. 9). Addition of NH: did not stimulate the activity of enzyme freed of other inorganic cations, while Mg2+ not only failed to be an activator of the enzyme but it competitively inhibited activation by K+ (Fig. 9). DISCUSSION

0.6-

0.4.

2

4 DISTANCE

6

0 (cm)

FIG. 5. SDS-gel electrophoresis of (A) protein standards of known molecular weight, (B) partially purified IMP oxidoreductase following the affinity chromatography step, and (C) purified IMP oxidoreductase from cowpea nodules after the final gelfiltration step. Proteins were denatured by boiling for 5 min, and were run with bromphenol blue as a marker dye in 10% gels in the presence of SDS and under reducing conditions for 3 h at 1.5 mA/tube. After development the gels were stained with 0.125% (w/v) Coomassie blue R-250, destained, and then scanned at 595 nm.

est level employed. From the data of Fig. ‘7 values for Km (NAD) ranged from 18 X 10e6 M at 0.08 mM IMP to 35 X 10m6 M at IMP levels below 0.02 mM. The activity of purified IMP oxidore-

The purification procedure outlined in Table I relied heavily on selective retention and subsequent elution of IMP-oxidoreductase from an agarose-Affi-gel column. This step alone achieved a 20-fold enrichment of the enzyme and, although a number of proteins were eluted with IMP oxidoreductase (see Fig. 5B), these were readily removed by the final gelfiltration step. In preliminary studies the 25-35% ammonium sulfate-precipitated fraction (see Table I) was applied first to the Sephacryl column, followed by the Affi-gel column. Both IMP- and xanthineoxidoreductases were eluted as a mixture by gel filtration and, although xanthine oxidoreductase could be subsequently collected from the Affi-gel column, there was a very poor yield (less than 10%) of IMP oxidoreductase under these conditions. Binding of IMP-oxidoreductase to the Alligel Blue-agarose only occurred in the absence of glycerol and, unless glycerol was included in the eluting buffer, negligible activity could be recovered from the column. The enzyme from Escherichia coli has also been purified by affinity chro-

812

ATKINS,

7

8

SHELP,

9

AND

10

STORER

-20

PH

FIG. 6. Effect of assay pH on (A) activity from cowpea nodules. In (A) both IMP and NAD was constant at 1.25 mM.

0

20

l!

[IMP]

and (B) K,,, (IMP) NAD were saturating

matography using a column in which GMP was coupled to aminohexyl-Sepharose (10). This procedure afforded a similar relative purification to that described here for Blue Affi-gel, but resulted in a greater yield of enzyme than was the case for cowpea nodules. Highly purified preparations taken from the final gel-filtration step (Table I) and used for characterization of the enzyme were apparently homogenous, showing a single protein band for the native enzyme

40

60

80

(mM-‘I

of IMP oxidoreductase purified at all pH values, while in (B)

on polyacrylamide gel electrophoresis (Fig. 3) and a single subunit band in the presence of SDS (Fig. 5). The cowpea nodule enzyme resembled the IMP oxidoreductases isolated previously from pea seeds (ll), E. coli (10, 12-14), Aerobactw aeroI

20.

16-

120

80

40

0 1 I [IMP]

40

80

(mti’)

FIG. ‘7. Lineweaver-Burk plots for the relationship between IMP concentration and velocity of reaction of purified IMP oxidoreductase from cowpea nodules at five NAD concentrations.

[GMP]

(mM)

FIG. 8. Inhibition by GMP of IMP oxidoreductase purified from cowpea nodules. The data are plotted according to Dixon (25), the dotted line indicating a K, (GMP) = 60 X 1O-6 M.

IMP

OXIDOREDUCTASE

I

I

0.2

0.4

0.6

0.6

I/[K+](IIIM-‘1

FIG. 9. Lineweaver-Burk plots for the activation of purified IMP oxidoreductase from cowpea nodules by K+ (0 mM Me), and competitive inhibition of the K+ activation by 5 mMMe. The plot indicates KD (K+) = 1.6 X 10-a M.

genes (15, 16), and Bacillus subtilis (17, 18)

in having a relatively low K, (IMP), a significantly higher Km (NAD), an alkaline pH optimum, greater activity with NAD compared to NADP, sensitivity to inhibition by GMP, activation by K+, apparent irreversibility, and the requirement of a sulfhydryl reducing agent for maximum activity. There has been considerable uncertainty about the molecular weight of isolated bacterial IMP oxidoreductases. Purified preparations typically yield multiple bands of activity on polyacrylamide gel electrophoresis (10) and at least two active proteins following ultracentrifugation (13). In a number of cases (10, 14, 19-23), functional subunits have been isolated which subsequently form functional oligomerit enzymes, having a range of molecular weights from less than 100,000 to 400,000. The purified cowpea enzyme, on the other hand, showed no evidence of size heterogeneity with a single peak of activity (Fig. 2) corresponding to 200,000 Da (Fig. 4) on gel filtration and a single band on electrophoresis (Fig. 3). The subunit molecular weight of 50,000 (Fig. 5) was similar to that for the enzyme from E. coli [54,000 Da; (lo)], and suggests that the native enzyme in the nodule is probably a tetramer.

IN

813

COWPEA

In cell-free extracts from cowpea nodules two possible routes for IMP metabolism to ureides have been detected (5): One via inosine and hypoxanthine, relying on the functioning of xanthine oxidoreductase; and the second via XMP and xanthine, utilizing both xanthine and IMP oxidoreductases. The latter pathway predominated in cell-free extracts at low IMP levels (5) as well as in viva (2), and it was suggested that this was probably due to the favorable kinetic properties of IMP oxidoreductase (5). The preliminary study of the properties of a partially purified enzyme (5) is supported by the kinetic characteristics of the purified enzyme described in this study. Competitive “feedback” inhibition by GMP, together with the irreversibility of the catalyzed reaction with IMP, are observations consistent with the idea that IMP oxidoreductase may act as a regulatory enzyme in purine nucleotide biosynthesis. While this might be the case in pathways of nucleotide metabolism, in legume nodules IMP oxidation leads to allantoin formation and, although GMP may serve as a substrate for ureide synthesis (4), GMP formation from XMP has not been detected to any significant extent in cell-free extracts (4, 5). IMP synthesis occurs within the plastids of nodule cells (24) so that an irreversible first step of IMP utilization in the cytosol, catalyzed by an enzyme with a high affinity and specificity for substrate, would help ensure continued transfer of IMP across the plastid outer membrane. Recently, one of the soybean nodulespecific host proteins, Nodulin-35 (26), has been identified as the 35-kDa polypeptide of uricase (27), an enzyme of purine oxidation. In view of the relative abundance of the IMP oxidoreductase protein (approx. 0.7%) in cowpea nodules, it is possible that the 50-kDa subunit of this enzyme might also correspond to a specific nodulin. REFERENCES 1. ATKINS, C. A. (1982) in Advances in Agricultural Microbiology (Subba Rao, N. S., ed.), pp. 5388, Oxford and IBH, New Delhi.

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