Formation of pyridine nucleotides under symbiotic and non-symbiotic conditions between soybean nodules and free-living rhizobia

Article abstract of DOI:10.1016/S0031-9422(02)00364-3

Enzymatic regulation of pyricline nucleotide formation, under symbiotic and non-symbiotic conditions, was analyzed using soybeans (Glycine max L, cv. 'Akisengoku') and rhizobia (Bradyrhizobia japonicum strain A1017), respectively. It was found that levels

Full text of DOI:10.1016/S0031-9422(02)00364-3

Phytochemistry 61(2002) 637–644
www.elsevier.com/locate/phytochem
Formation of pyridine nucleotides under symbiotic and
non-symbiotic conditions between soybean nodules and
free-living rhizobia
Takafumi Tezuka *, Yuko Murayamab
a,
aGraduate School of Human Informatics, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
bGraduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
Received in revised form 16 July 2002
Abstract
Enzymatic regulation of pyricline nucleotide formation, under symbiotic and non-symbiotic conditions, was analyzed using
soybeans (Glycine max L. cv. ‘Akisengoku’) and rhizobia (Bradyrhizobia japonicum strain A1017), respectively. It was found that
levels of pyridine nucleotides in bacteroids in root nodules were different from those in free-living cells of rhizobia. This difference
was associated with differences in activities of enzymes involved in the pathway from l-tryptophan to NAD and NADP. That is,
these activities were lower in bacteroids than in free-living bacteria and lower in the nodule cytosol than in root extracts. The
optimum pH for NAD synthetase in bacteroids, was 9.0. Additionally, the optimum pH for ATP-nicotinamide mononucleotide
(
m
NMN) adenyltransferase, final step enzyme in NAD formation, was estimated to be 7.6. In the bacteroid fraction, the K of NAD
synthetase (22 mM) was ꢀ1/22 of that of ATP-NMN adenyltransferase (482 mM). Vmax values were estimated to be almost in the
same order for both NAD synthetase and ATP-NMN adenyltransferase. This is the first report on the formation of pyridine
nucleotides originating from l-tryptophan in bacteroids in soybean nodules and free-living bacteria.
#
2002 Elsevier Science Ltd. All rights reserved.
Keywords: Bradyrhizobium japonicum; Glycine max; Leguminosae; ATP-nicotinamide mononucleotide (NMN) adenyltransferase; Bacteroids; Free-
living rhizobia; NAD synthetase; Nodules; Pyridine nucleotides [NAD)(H)/NADP(H)]
1
. Introduction
nodules in the symbiotic system between soybean roots
and rhizobia.
Leguminous plants have nodules, a symbiotic system
According to Nishizuka and Hayaishi (1971), trypto-
phan serves as a precursor of NAD in animals and in
certain microorganisms, such as Neurospora crassa and
Xanthomonas pruni. Magni et al. (1999) described the
involvement of quinolinate phosphoribosyltransferase
and ATP-NMN adenyltransferase in pyridine nudeotide
metabolism, beginning with a detailed consideration of
the anaerobic and aerobic pathways leading to quino-
linate, a key precursor of NAD in animals. NAD syn-
thetase, as ubiquitous enzyme catalyzing the last step in
the biosynthesis of NAD from deamide-NAD, has been
characterized using Bacillus subtilis (Rizzi et al., 1996).
To our knowledge, the pathways for NAD formation
between nodule bacteroids and free-living rhizobia have
not been compared. In the present work, a comparison
of the NAD synthetic system in free-living cells of
Bradyrhizobia, bacteroids and cytosol from soybean
between rhizobia and root tissues. The nodules are
composed of bacteroid zones and cortex. The bacteroid
zone is mostly occupied by transforming bacteria
derived from rhizobia (Ching and Hedtke, l977; Vasse et
al., 1990) and bacteroids, with nitrogen being fixed by
the latter (Peterson and LaRue, 1981). In the nitrogen
fixation system, both NAD and NADP are essential
receptors of reducing power arising from substrates such
as sugars, organic acids, aldehydes and alcohols (Peter-
son and LaRue, 1982; LaRue et al., 1984). In this study,
the formation of NAD and NADP was investigated to
attempt to understand the involvement of bacteroids in
*
Corresponding author. Tel.: +81-52-789-5022; fax: +81-52-789-
230.
E-mail address: tezuka@info.human.nagoya-u.ac.jp (T. Tezuka).
4
0
031-9422/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(02)00364-3

638
T. Tezuka, Y. Murayama / Phytochemistry 61 (2002) 637–644
nodules, and root extracts of soybeans was undertaken.
The results were discussed with reference to the bio-
chemical difference between symbiotic and non-symbio-
tic conditions.
Table 1
Specific activities of various enzymes involved in the pathway for the
formation of pyridine nucleotides in bacteroids from nodules of Gly-
cine max and free-living cells of Bradyrhizobium japonicum
Bacteroids Free-living
bacteria
2
. Results
À1
[
nkat (mg protein)
]
NADPH-kynurenine hydroxylase
NADH-kynurenine hydroxylase
60.0Æ1.7
41.7Æ3.3
13.3Æ0.0
5.0Æ0.0
1200.0Æ10
2301.7Æ285.0
2900.0Æ8.3
380.0Æ23.3
2
.1. Levels of pyridine nucleotides
Bacteroids isolated from soybean nodules contained a
3-Hydroxyanthranilic acid oxygenase
Quinolinate phosphoribosyltransferase
considerable quantity of the pyridine nucleotides, NAD,
NADH, NADP and NADPH (Fig. 1a), with the levels
of the oxidized forms (NAD and NADP) being higher
than those of the reduced forms (NADH and NADPH).
By contrast, in the free-living cells of B. japonicum strain
A1017 used in this study, the NADP and NADPH
levels were about 10 times higher than NAD and
NADH (Fig. 1b).
À1
[pkat (mg protein)
NAD synthetase
]
0.8Æ0.0
1.9Æ0.16.2
35.9Æ1.7
12.1Æ0.4
Æ0.1
ATP-NMN* adenyltransferase
NAD kinase
82.6Æ1.1
The activities of several key enzymes in the pathway for the formation
of NAD and NADP from l-tryptophan were measured. Bacteroids
and free-living bacteria were prepared as described in the text. Values
are represented as the meansÆS.E. (n=3). *NMN: nicotinamide
mononucleotide.
2.2. Activities of enzymes associated with the formation
of pyridine nucleotides
lower than those in free-living bacteria. Furthermore, in
bacteroids, the activity of NADPH-kynurenine hydroxy-
lases was higher than that of NADH-kynurenine
hydroxylases and in the free-living bacteria, the activity
of both enzymes was just the opposite.
The activity of 3-hydroxyanthranilic acid oxygenase
was relatively lower in the bacteroids than in the free-
living bacteria, like the case of quinolinate phosphor-
ibosyltransferase. NAD synthetase and ATP-NMN
adenyltransferase in bacteroids showed lower activities
than those in the free-living bacteria. Furthermore, in
the bacteroids, ATP-NMN adenyltransferase showed
higher activities than NAD synthetase, whereas in the
free-living bacteria NAD synthetase was about 2 times
more active than ATP-NMN adenyltransferase. The
NAD kinase activity in bacteroids was lower than that
in free-living bacteria.
The activities of some enzymes involved in the for-
mation of NAD from l-tryptophan, and NAD kinase
which converts NAD into NADP were examined. Spe-
cific activities of NADPH- and NADH-kynurenine
hydroxylases, 3-hydroxyanthranilic acid oxygenase,
quinolinate phosphoribosyltransferase, NAD synthe-
tase, ATP-NMN adenyltransferase and NAD kinase in
fractions prepared from the bacteroids and free-living
bacteria are shown in Table 1. The activities of NADPH-
and NADH-kynurenine hydroxylases in bacteroids were
In roots, the NADH-kynurenine hydroxylase showed
higher activity (about 14 times) than NADPH-kynur-
enine hydroxylase (Table 3), as in free-living bacteria
(
Table 1). The other enzymes such as 3-hydroxy-
anthranilic acid oxygenase, quinolinate phosphori-
bosyltransferase, NAD synthetase, ATP-NMN
adenyltransferase and NAD kinase in the pathway of
NAD and NADP formation showed higher activities in
root extract than in nodule cytosol (Table 3).
2.3. pH dependence of NADPH-kynurenine hydroxylase,
NAD synthetase and ATP-NMN adenyltransferase from
bacteroids
The pH dependence of NADPH-kynurenine hydroxy-
lase, NAD synthetase and ATP-NMN adenyl-
transferase in the bacteroids is shown in Fig. 2. The pH
optima for NADPH-kynurenine hydroxylase, NAD
Fig. 1. The levels of pyridine nucleotides in bacteroids of Glycine max
root nodules and in free-living cells of Bradyrhizobium japonicum.
Vertical bars represent the meansÆS.E. (n=3).

T. Tezuka, Y. Murayama / Phytochemistry 61 (2002) 637–644
639
Table 2
Kinetic parameters of some enzymes in bacteroids from nodules of
Glycine max
Bacteroids
NADPH-kynurenine hydroxylase
K
V
m
NADPH (mM)
max (mmol NADP formed mg protein min
11.0Æ0.4
3.6Æ0.3
À1
À1
)
NAD synthetase
K
V
m
Deamide NAD) (mM)
max (nmol NAD formed mg protein
22.0Æ0.7
3.1Æ0.2
À1 À1
h
)
ATP-NMN* adenyltransferase
K
V
m
Nicotinamide mononucleotide (mM)
max (nmol NAD formed mg protein
482.0Æ37.3
7.4Æ0.4
À1 À1
h
)
The activities of some key enzymes in the pathway for the NAD from
l-tryptophan were measured. Bacteroid fraction was prepared as
described in the text. Values are represented as the meansÆS.E.
(
n=3). *NMN: nicotinamide mononucleotide.
Table 3
Specific activities of various enzymes in the pathway for the formation
of NAD in nodule cytosol and root extract of Glycine max plants
Nodule
cytosol
Root
extract
À1
nkat (mg protein) ]
[
NADPH-kynurenine hydroxylase
NADH-kynurenine hydroxylase
56.7Æ1.7
100.0Æ3.3
10.0Æ0.0
21.7Æ0.0
91.7Æ3.3
1266.7Æ31.7
26.7Æ1.7
3-Hydroxyanthranilic acid oxygenase
Qunolinate phosphoribosyltransferase
138.3Æ1.7
À1
[
pkat (mg protein)
NAD synthetase
ATP-NMN* adenyltransferase
]
0.3Æ0.0
1.2Æ0.0
6.5Æ0.1
5.6Æ0.1
The activities of several key enzymes in the pathway for the formation
of NAD from l-tryptophan were measured. Nodule cytosol and root
extract were prepared as described in the text. Values are represented
as the meansÆS.E. (n=3). *NMN: nicotinamide mononucleotide.
synthetase, and ATP-NMN adenyltransferase were
estimated to be 8.0, 9.0 and 7.6, respectively.
2
.4. Kinetic analysis
Lineweaver–Burk plots were made from the isotherms
of NADPH-kynurethne hydroxylase, NAD synthetase
and ATP-NMN adenyltransferase (Table 2). The Km
and Vmax values of NAD synthetase for NAD forma-
tion were smaller than those of ATP-NMN adenyl-
transferase for NAD formation.
Fig. 2. The pH dependence of NADPH-kynurenine hydroxylase,
NAD synthetase and ATP-NMN adenyltransferase in the enzyme
fraction of bacteroids from root nodules of Glycine max. The enzyme
fraction was prepared as described in the text. Vertical bars represent
the meansÆS.E. (n=3). NMN: nicotunamide mononucleotide.
used cv. Akisengoku at 7 weeks when at the flowering
stage.
3
. Discussion
Bacteroids contained NAD and NADP at high levels,
whereas in free-living cells (bacteria) NADP and
NADPH were more predominant (Fig. 1). The levels of
pyridine nucleotides in nodule cytosol were similar to
those in free-living bacteria (data not shown). These
The soybeans (cv. ‘Akisengoku’) used in this experi-
ment were late cultivars. Although soybean cultivars
other than late cultivars are generally used for biochem-
ical analysis at 4–5 weeks (at anthesis) after sowing, we

640
T. Tezuka, Y. Murayama / Phytochemistry 61 (2002) 637–644
phenomena suggest that the pyndine nucleotide level is
regulated differently in free-living bacteria and bacter-
oids. Indeed, the activities of the enzymes in the path-
way from l-tryptophan to NM) and NADP in
bacteroids were different from those in free-living bac-
teria (Table 1).
l-tryptophan, as well as animals and some microorgan-
isms such as N. crassa and X. pruni (Nishizaki and
Hayaishi, 1971).
The activities of NAD synthetase and ATP-NMN
adenyltransferase in the nodule cytosol were similar to
those in bacteroids, whereas those in roots were higher
than those in bacteroids (Tables 1and 3). Therefore, it
appears that these enzymes have lower activities in
symbiotic than non-symbiotic conditions. Similarly, the
activity of NAD kinase which forms NADP from NAD
in bacteroids and free-living bacteria was also lower in
bacteroids than in free-living bacteria (Table 1). Hence,
these phenomena may be common to enzymes in the
pathway for the formation of pyridine nucleotides.
The activities of other enzymes, which are not con-
cerned in the formation of pyridine nucleoticles, also
differ between symbiotic and non-symbiotic conditions.
For example, lipoxygenase and hydroperoxide lyase
from soybeans (cv. ‘Wilkin’), were higher in the root
extract than in the nodule cytosol (data not shown),
similar to what was observed with the activities of
6-phosphogluconate dehydrogenase in soybean (Cope-
land et al., 1989) and aldolase and malate dehy-
drogenase in pea (Smith, 1985). Furthermore, the
activities of isocitrate dehydrogenase in soybean (Suga-
numa and Yamamoto, 1987) and alfalfa plants (Iri-
goyen et al., 1990), as well as glucose-6-phosphate
dehydrogenase, alcohol dehydrogenase, aldolase and
pyruvate kinase in the latter (Irigoyen et al., 1990) were
also higher in free-living bacteria than in bacteroids. By
contrast, according to Copeland et al. (1989), the activ-
ities of sucrose synthetase, glyceraldehyde-3-phosphate
dehydrogenase, phosphoglycerate kinase, enolase pyr-
uvate kinase, phosphoenolpyruvate carboxylase and
a,b-amylase were lower in the root extract than in the
nodule cytosol. Moreover, malate dehydrogenase in
soybean (Suganuma and Yamamoto, 1987) and alfalfa
plants (Irigoyen et al., 1990) were also lower in free-living
bacteria than in bacteroids. All of these phenomena
may be caused by differences in symbiotic and non-
symbiotic metabolism in leguminous plants.
Given that bacteroids are derived from free-living
bacteria (Ching and Hedtke, 1977) and associated with
nodule cells (of plant origin), pyridine nucleotides in
bacteroids may be involved in symbiotic work between
nodule cells and bacteroids, such as nitrogen fixation.
The levels of NADH and NADPH in bacteroids were
lower than those of NAD and NADP, probably because
mtrogenase converts N into NH . The ꢀ2-fold lower
2
3
levels of pyridine nucleotides [NAD(P)+NAD(P)H] per
g fr. wt of bacteroids than that of free-living cells may
also be due to their consumption for nitrogen fixation.
The ratios of NAD/NADH and NADP/NADPH in
the bacteroids were 5.56 and 3.26, which are higher than
the values reported by Tajima and Kouzai (1989), i.e.
2
.17 and 0.37, respectively. The difference between their
NAD(P)/NAD(P)H ratios and ours may be due to dif-
ferences in the procedures used for the extraction and
assay of pyndine nucleotides in bacteroids. In the earlier
work, pyridine nucleotides in bacteroids were extracted
with 40 mM NaOH and the oxidized form [NAD(P)]
ꢁ
was destroyed by heating for 10 min at 60 C to esti-
mate the amount of only the reduced form [NAD(P)H].
To estimate the amount of only NAD(P), pyridine
nucleotides were extracted with the acidic solution of 20
mM H SO /0.1M Na SO and heated for 30 min at
2
4
2
4
ꢁ
6
0 C. The amount of pyridine nudeoticles was esti-
mated by the enzymatic cycling assay (Passonneau and
Lowry, 1974) after incubation for 60 min. In contrast,
our procedure (see Experimental) was based on a
method described elsewhere (Nisselbaum and Green,
1
969; Tezuka et al., 1994; Shiozaki, 2000).
In the pathway for formation of NAD from l-trypto-
phan, in bacteroids the activity of NADPH-kynurenine
hydroxylase was higher than that of NADH-kynurenine
hydroxylase, while that in free-living bacteria was quite
the reverse (Table 1). This phenomenon suggests that
the activities of NADPH- and NADH-kynurenine
hydroxylase under symbiotic conditions are regulated
differently from those under non-symbiotic conditions.
According to Saito et al. (1957), kynurenine hydroxy-
lase in animals is NADPH-dependent. In bacteroids and
free-living bacteria, kynurenine hydroxylase was not
only NADPH-dependent but also NADH-dependent
As shown in Fig. 2, the optimum pH for NADPH-
kynurenine hydroxylase and ATP-NMN adenyl-
transferase was in the region of relatively weak alkali
(pH 8.0 and ꢀ7.6, respectively). However, the optimum
pH of NAD synthetase was different from that of the
above enzymes. That is, the optimum pH was in the
region of relatively strong alkali (pH 9.0). Kinetic ana-
lysis suggests that the formation of NAD in bacteroids
may be dominated by NAD synthetase, as compared
with ATP-NMN adenyltransferase, because the Km of
NAD synthetase, (22 mM) was ꢀ1/22 of that of ATP-
NMN adenyltransferase (482 mM), as shown in Table 2.
In conclusion, the levels of pyridine nucleotides in bac-
teroids and free-living cells of rhizobia depended on the
activities of enzymes in the pathway from l-tryptophan
(
Table 1). Furthermore, kynurenine hydroxylase in the
nodule cytosol and root extract showed activities of
both NADPH- and NADH-enzymes (Table 3). Thus,
organisms other than animals and certain micro-
organisms seem to have both NADPH- and NADH-
kynurenine hydroxylases. This suggests that bacteroids,
free-living rhizobia and soybean roots form NAD from

T. Tezuka, Y. Murayama / Phytochemistry 61 (2002) 637–644
641
to NAD and NADP. A possible pathway for the for-
mation of NM) and NADP in bacteroids and free-living
bacteria, i.e., in symbiotic and non-symbiotic systems, is
shown in Fig. 3. Since the principal function of bacter-
oids in soybean nodules is nitrogen fixation which needs
NADH and NADPH as reducing power, the formation
of NAD and NADP under symbiotic conditions (bac-
teroids) is different from that under non-symbiotic con-
ditions (free-living bacteria). Accordingly, the activities
of enzymes involved in the formation of pyridune
nucleotides differ between symbiotic and non-symbiotic
systems. This is the first report on the formation of
Fig. 3. A possible pathway for the formation of NAD and NADP from l-tryptophan in bacteroids from root nodules of Glycine max and free-living
cells of Bradyrhizobium japonicum. Enzymes in six rectangles were assayed in the present study.

642
T. Tezuka, Y. Murayama / Phytochemistry 61 (2002) 637–644
NAD and NADP originating from l-tryptophan in
symbiotic bacteroids in soybean and non-symbiotic
free-living bacteria.
The supernatant (1.5 ml) was loaded onto a Sephadex
G-25 column (1 Â 14 cm) with and eluted with TES/
KOH (50 mM, pH 7.5). After discarding 20 drops of the
eluate, the protein-rich (enzyme) fraction (3 ml) was
collected to assay enzyme activities.
4
. Experimental
4.3. Free-living bacteria
4
.1. Plant culture
Free-living cells of B. japonicum, strain A10l7 were
cultured in a liquid culture medium (Rowsthorn and
LaRue, 1986) by shaking for 10 days at 30 C. The cul-
ture medium with the free-living bacteria was cen-
Soybean (Glycine max L. cv. ‘Akisengoku’) seeds were
ꢁ
sown in pots containing a mixture of fine vermiculite
and coarse sand (1:1, v/v). Each pot contained 135 mg
N, as KNO , added at planting to promote early growth
trifuged for 10 min at 20,000
g
to remove
3
and good nodulation, and was watered with a nutrient
solution, pH 5.8–6.2, lacking N containing salts (Sum-
merfield et al., 1977). Seeds were inoculated with Bra-
dyrhizobium japonicum strain A1017 that had been
cultured in liquid culture medium (Rowsthorn and
polysaccharides exuded from bacteria during culture.
The pellet (collected free-living bacteria) was washed
with TES/KOH (50 mM, 30 ml, pH 7.5), then sus-
pended in TES/KOH (50 mM, 20 ml, pH 7.5) and cen-
trifuged for 10 min at 10,000 g. The resulting pellet
(washed free-living bacteria) was used for extraction of
pyridine nucleotides and preparation of enzymes. For
preparation of enzymes, the above pellet (washed free-
living bacteria) was suspended in TES/KOH (50 mM, 2
ml, pH 7.5), sonicated in the same way as bacteroid
suspension mentioned above, and centrifuged at 20,000
g for 20 min. The supernatant was treated in the same
way as the preparation of enzymes from bacteroids,
affording the enzyme fraction of free-living bacteria.
The fraction was used to assay enzyme activities.
ꢁ
LaRue, 1986) with shaking for 5 days at 30 C. Plants
were grown in a glasshouse [with a fan (50 Â 50 cm)
ꢁ
operating automatically at a temperature of 35 C or
ꢁ
above] at 14–35 C from April to the end of June and at
ꢁ
7
–35 C from October to December. After emergence,
seedlings were thinned to one per pot and watered with
nutrient solution via a basal saucer as required until 14
days after planting. Pots then received nutrient solution
with free drainage each day. Plants were harvested at 7
weeks after planting. The yield of nodule was about 2–3
À1
g plant
.
4.4. Extraction and quantitation of pyridine nudeotides
4
.2. Isolation of bacteroids and root extracts
ꢁ
All procedures were carried out below 4 C and under
Ar which had been purged of O (Peterson et al., 1982).
ꢁ
All procedures were carried out below 4 C and under
Ar which had been purged of O (Peterson et al., 1982).
2
Nodules (6 g) were homogenized with potassium phos-
phate buffer (5 mM, 18 ml, pH 6.8) in a mortar with a
pestle on ice, squeezed through 4 layers of cheesecloth
and centrifuged at 370 g for 5 min. The supernatant was
recentrifuged at 5000 g for 10 min. The resulting pellet
was suspended in potassium phosphate buffer (5 mM,
18 ml, pH 6.8) and again centrifuged at 5000 g for 10
min. This step was repeated twice. The final pellet was
resuspended in the same buffer (5 ml) as a bacteroid
fraction. The bacteroid fractions were divided into two
rations of equal wet weight to isolate the oxidized
(NAD and NADP) and reduced (NADH and NADPH)
forms of pyridine nucleotides, respectively. The washed
free-living bacteria mentioned above was also weighed
and divided into two rations of equal weight (free-living
cell fraction), one for the extraction of NAD and
NADP, and the other for the extraction of NADH and
NADPH.
2
Nodules (10 g) were ground in a mortar with a pestle
with potassium phosphate buffer (50 mM, 30 ml, pH
7
.4) containing Na-ascorbate (200 mg) and poly-
vinylpolypyrrolidone (2 g) (Polyclar AT) in an ice bath.
The homogenate was squeezed through four layers of
cheesecloth and centrifuged for 5 min at 370 g to
remove starch and cell debris. The supernatant was
again centrifuged for 10 min at 5000 g. The resulting
supernatant was used as nodule cytosol. The pellet con-
taining bacteroids was resuspended three times in
washing buffer [50 mM N-Tris(hydroxymethyl)methyl-
2
-aminoethanesulfonic acid (TES)/KOH, pH 8.2] and
again centrifuged for 10 min at 5000 g to provide the
washed bacteroids. Final pellet (washed bacteroids) was
resuspended in TES/KOH (100 mM, 3.3 ml, pH 8.2)
and stored at À80 ꢁC until use. Root extracts were
obtained from roots (10 g) by the same procedures as
used for the preparation of the nodule cytosol.
Pyridine nucleotides were extracted from bacteroid
and free-living cell fractions by a modified version of the
method of Tezuka et al. (1994). One part of each frac-
After thawing, the bacteroid suspension was sonicated
for (15Â30 s) in an ice bath using a Branson Sonifer
ꢁ
(Model 200, Branson Sonic Power Co., USA) with an
output of 20 W and centrifuged for 20 min at 20,000 g.
tion was homogenized at 90–95 C for 2 min with 0.1N
HC1( 10 ml) to extract oxidized coenzymes (NAD and

T. Tezuka, Y. Murayama / Phytochemistry 61 (2002) 637–644
643
NADP). The other part of each fraction was similarly
treated with 0.1N NaOH to extract reduced coenzymes
a modified version of the method of Tezuka and
Yamamoto (1972). Immediately after the assay, the
mixture was heated at 95 C for 2 mm, cooled in an ice
ꢁ
(
NADH and NADPH). Each of the four homogenates
was then rapidly chilled in an ice bath and the extracts
for oxidized and reduced pyridune nudeotides were
adjusted to pH 6.5 with NaOH and pH 7.5 with HC1,
respectively, and then 0.2 M glycylglycine buffer (0.5
ml) at pH 6.5 and pH 7.5, respectively, were added.
Total volumes were measured, and then each homo-
bath and centrifuged at 10,000 g for 10 min. Finally,
NAD formed by NAD synthetase and ATP-NMN ade-
nyltransferase and NADP formed by NAD kinase were
measured by the method of Tezuka et al. (1994). Protein
was determined according to Lowry et al. (1951) using
bovine serum albumin as the standard. All experiments
were repeated three times with similar results and
representative results are shown.
ꢁ
genate was centrifuged at 10,000 g for 20 min at 4 C.
ꢁ
Supernatants were stored at À80 C prior to the assay
of pyndine nucleotides. To estimate possible losses of
dinucleotides during extraction, authentic standards
(
NADH: 2 nmol; others: 10 nmol) were added to the
Acknowledgements
alkaline and acidic extracts before homogenization.
Recovery of NAD(P) and NAD(P)H was 98–103 and
We thank Messrs. K. Sakakibara and S. Trii for help
in cultivating the soybean plants. Soybeans and rhizobia
were provided by courtesy of Dr. H. Kouchi, Principal
Researcher, National Institute of Agrobiological
Resources, Tsukuba, Ibaraki, Japan. The culture and
cell counting of Rhizobia were carried out by courtesy
of Dr. N. Kido and Miss R. Oshima, Graduate School
of Human Informatics, Nagoya University.
7
5–88%, respectively. Pyridine nucleotides were quanti-
fied by a modified version (Tezuka et al., 1994) of the
method of Nisselbaum and Green (1969).
4
.5. Enzyme assays
ꢁ
Kynuremine hydroxylase was assayed at 25 C in a
reaction mixture (0.6 ml) that contained 0.1M TES/
KOH (pH 8.0), 10 mM KC1, 0.33 mM NADH or
NADPH, 3.33 mM l-kynurenine and an enzyme frac-
tion by a modified version of the method of Okamoto et
al. (1967). 3-Hydroxyanthranilic acid oxygenase was
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¨
after the assay, 0.18 N HCl (0.42 ml) was added to the
ꢁ
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