From the
Root nodules of adzuki bean plant ( Vigna angularis)
contained a novel polyamine. The chemical structure of the new
polyamine was determined to be
NH
The polyamines, putrescine (Put),
Since the occurrence of unusual polyamines in the extreme
thermophile, Thermus thermophilus, and their possible
functional roles in the thermostability of such microorganism has been
reported
(17, 18, 19, 20) , many
researchers have attempted to find novel polyamines in various living
systems. As a result of a broad survey, a number of uncommon polyamines
including long chain and branched polyamines have been found from a
variety of natural sources (see the tables of Refs. 5 or 7). With
regard to triamines, except for commonly found Spd, only three
polyamines have been known to occur naturally, i.e. Norspd,
Homospd, and 3-APcad. The present investigation adds a new triamine,
4-ABcad, to the list of natural polyamines. In contrast to Norspd or
Homospd, which show relatively widespread distributions from
microorganisms to higher plants and animals, 3-APcad has been
recognized only in specific cells, such as polyamine-dependent mutants
of Esherichia coli (21) or Neurospora crassa (22) , and polyamine-depleted animal cells pretreated with
an inhibitor of polyamine biosynthesis
(23, 24) . As is
the case for 3-APcad, the occurrence of 4-ABcad appears to be very
specific, since this triamine was detected exclusively in adzuki bean
root nodules and not in other root or stem nodules tested (22 species
of legumes and five species of non-legumes). The adzuki bean plant was
characterized by its high Cad content within the root nodules differing
from other legumes
(8) . In the wild progenitor species of
adzuki bean (ACC 1238), in which root nodules contained very low Cad,
4-ABcad was not present (). Together with the fact that
B. japonicum A1017 cells produced 4-ABcad only in the growth
medium supplemented with Cad (data not shown), a Cad-rich environment
is found to be necessary for the production of this novel polyamine.
In E. coli, Cad is inducibly formed
(25) when grown
on medium containing lysine, a precursor for Cad synthesis. B.
japonicum A1017, however, showed no ability to form Cad even
though lysine was added at 2 mM into the growth medium. Our
previous study
(11, 26) , and analytical data of other
investigators
(27) also indicated that growing cells from
various Bradyrhizobium and Rhizobium species produced
little Cad as compared with Put and Homospd, implying the lack or very
low activity of lysine decarboxylase in Rhizobiaceae. The data of
systematic analyses of polyamine pattern in proteobacteria
(28) are of special interest because Cad was rarely detected in
the groups containing Homospd as a major polyamine. These facts
probably explain why 4-ABcad has not been detected so far in spite of
the extensive survey of microbial polyamines. In this regard, 4-ABcad
is considered to be an unusual polyamine occurring in rhizobial cells
under a specific environment.
Legume root nodules are specially
differentiated complex tissues, which consist of bacteroids (symbiotic
form of rhizobial cells) and surrounding host legume cells as a result
of plant-microbe symbiotic associations
(29) . After invasion
into the host plant cells, rhizobia differentiate to become bacteroids
which can fix atmospheric nitrogen
(16, 29) . In adzuki
bean root nodules, large quantities of Cad were detected in host plant
cells but not in bacteroids, whereas most of the Homospd and 4-ABcad
were localized in bacteroids (). The results from in
vitro experiment (I) suggested that Homospd synthase,
detected in bacteroids but not in plant cytosol fraction, was a
responsible enzyme for 4-ABcad synthesis since 1) NAD
A
variety of leguminous plants usually contain copper-containing diamine
oxidases which can oxidize both Put and Cad
(34, 35, 36, 37, 38) .
Aminoaldehydes are generally known to occur as an intermediate in the
oxidation process of polyamines
(39) , and the enzymes related
to polyamine catabolism have been detected in various living systems
(40, 41) . These facts offer an another possibility that
aminoaldehydes resulting from the oxidative deamination of Put and Cad
by diamine oxidase might have also been utilized for 4-ABcad synthesis
via the Shiff-base formation between aminoaldehydes and amino-terminal
of diamines. The involvement of diamine oxidase in the synthesis of
4-ABcad, however, seems unlikely because a
Bacteroids
and plant cytosol fraction were separated from the root nodules
harvested from 67 days-old adzuki bean plant as described under
``Materials and Methods.'' An aliquot of each fraction was
extracted with 0.5 M PCA and analyzed for polyamines. Another
aliquot was served for the determination of protein content.
The cell-free extract of bacteroids,
which was thoroughly dialyzed for removal of cellular polyamines, was
employed for this assay. The complete reaction mixture contained 50
mM Tris-HCl buffer, pH 8.7, 50 mM KCl, 0.5 mM NAD
We thank Dr. S. Yamamoto (Okayama University, Japan)
for kindly providing authentic 4-ABcad and 5-APcad, and S. Hiradate
(NIAES, Japan) for his assistance in GC-MS analysis. We are also
grateful to Dr. Mark Taylor (Scottish Crop Research Institute, UK) for
helpful comments and critical reading of the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(CH
)
NH(CH
)
NH
(4-aminobutylcadaverine) based on gas chromatography-mass
spectrometry. The occurrence of 4-aminobutylcadaverine was specific to
the root nodules, since the unusual triamine was not detected in other
organs of the adzuki bean plant. Bacteroids, isolated from root
nodules, contained both sym-homospermidine and
4-aminobutylcadaverine, whereas the plant cytosol fraction contained
large quantities of putrescine and cadaverine. A cell-free extract of
bacteroids showed the ability to form this triamine from putrescine and
cadaverine under the presence of NAD
and
K
. 1,3-Diaminopropane and NADH were inhibitory for the
synthesis of both sym-homospermidine and
4-aminobutylcadaverine. [1,4-
N]Putrescine was
incorporated not only into sym-homospermidine but also into
4-aminobutylcadaverine by the cell-free extract of bacteroids when
incubated with excess cadaverine. Analysis of the fragment ion peaks in
the
N-enriched 4-aminobutylcadaverine indicated the
transfer of a aminobutyl moiety to the amino terminus of cadaverine.
These results suggest that, in adzuki bean, 4-aminobutylcadaverine is
formed through the action of homospermidine synthase in nodule
bacteroids under a cadaverine-rich environment.
(
)
spermidine (Spd), and spermine (Spm) are ubiquitously found
in higher cells. Their physiological and biochemical significance in
the process of cell growth and differentiation has been repeatedly
reviewed
(1, 2, 3) . During the last two
decades, a number of unusual polyamines including long chain and
branched polyamines have been found in a variety of organisms
(4, 5, 6, 7) . Recently, we investigated
the distributions of polyamines in various root or stem nodules
collected from a variety of leguminous and nonleguminous plants
(8) . During the course of the analyses of polyamines by high
performance liquid chromatography (HPLC), we encountered the presence
of several unknown peaks on the elution chromatograms of the extracts
from various Bradyrhizobium-infected legume root nodules.
These compounds, like polyamines, were highly cationic in nature, being
strongly adsorbed on a Dowex 50W-X8 column, and one of these organic
cations was found to be specifically present in root nodules of adzuki
bean ( Vigna angularis). From the structural analysis based on
gas chromatography-mass spectrometry (GC-MS), it was identified as a
new polyamine, 4-aminobutylcadaverine (4-ABcad), which has not yet been
recognized in any of the living systems so far. We describe here the
data showing a natural occurrence of 4-ABcad in adzuki bean and provide
the evidence that bacteroids, isolated from root nodules, or
Bradyrhizobium japonicum, which establishes a symbiotic
relationship with adzuki bean plant, has an ability to synthesize this
unusual polyamine under the cadaverine (Cad)-rich environment. Based on
the results from in vitro experiment using cell-free extract
of isolated bacteroids and from a
N tracer experiment, a
possible mechanism for the biosynthesis of 4-ABcad in adzuki bean root
nodule has also been presented.
Plants and Microbial Growth
Seeds of adzuki bean
plant ( V. angularis cv. Erimoshoudzu, cv. Kamuidainagon, cv.
Tsurugi-3, and var. nipponennsis ACC1238) were obtained from Hokkaido
Prefectural Tokachi Agricultural Experiment Station, Japan. The seeds
were sown and grown in a field of National Agriculture Research Center
at Tsukuba, Japan. Plants were harvested during the vegetative to early
reproductive stage, separated into leaf, stem, root, and nodule, and
stored below 30 °C until use. B. japonicum A1017
(MAFF303005), obtained from the Laboratory of Soil General Microbiology
(National Institute of Agro-Environmental Sciences, Tsukuba, Japan),
was transferred into a liquid medium (pH 6.4) containing 0.06% yeast
extract (Difco), 0.5% K
HPO
, 0.02%
MgSO
7H
O, 0.01% NaCl, and 0.5% sucrose, and
grown aerobically in the presence or absence of 1 mM Cad or 2
mM lysine. The cells were harvested at the early stationary
phase of growth. Nodule formation on the roots of adzuki bean with
B. japonicum A1017 was ascertained by the inoculation test
under sterile growth condition.
Preparation of Bacteroids and Plant Cytosol Fraction from
Root Nodules
Frozen nodules were gently crushed with a pestle
and mortar in a cold grinding medium
(9) . After removal of
nodule debris by passage through four layers of cotton gauze, the
filtrate was centrifuged at 300 g for 4 min. The
resulting supernatant fluid was centrifuged at 5,000
g for 10 min. The pellet was washed twice with the grinding medium
to give the final bacteroid fraction. The supernatant was centrifuged
at 16,000
g for 30 min, and the resulting clear
supernatant was referred to as the plant cytosol fraction. Protein
content in each fraction was determined by the method of Bradford
(10) using bovine serum albumin as a standard.
Analysis of Polyamines by HPLC, Capillary GC, and
GC-MS
Polyamines in plant tissues or bacterial cells were
extracted with 0.5 M perchloric acid and purified as described
previously
(11) . Quantitative analysis of polyamines was
performed with HPLC (Shimadzu LC-9A) equipped with a reversed phase
column (Shim-pack CLC-ODS, 6 150 mm) after derivatization with
benzoyl chloride
(12) . Diaminohexane was used as an internal
standard. Polyamines, after derivatization with pentafluoropropionic
(PFP) anhydride
(13) , were also analyzed with acapillary gas
chromatography (Shimadzu GC-9A) fitted with a split injector and a
flame ionization detector. For the structural analysis of the unknown
compound and for the detection of
N incorporation into the
molecules, GC-MS was performed using a quadrapole GC-mass spectrometer
(Shimadzu QP-5000) equipped with a fused silica capillary column
(CBJ1-M30-025; 30 m
0.25 mm inside diameter, 0.25 µm film
thickness). Electron impact mass spectra were obtained under the
following GC-MS conditions: injection mode and temperature, splitless
(sampling time 0.5 min) at 300 °C; initial column temperature, 50
°C (2 min); temperature program, 5 °C min
;
final column temperature, 270 °C (2 min); carrier gas flow rate,
helium 1.1 ml min
; ion source temperature, 300
°C; ionizing energy, 70 eV.
Assay of Homospd Synthase and 4-ABcad Producing
Activity
Cell-free extract of bacteroids and plant cytosol
fraction were used for the detection of Homospd synthase and 4-ABcad
producing activities. The bacteroids were suspended in 10 mM
sodium phosphate buffer, pH 7.0, containing 1 mM EDTA, 20
mM 2-mercaptoethanol, and 0.05% heparin sodium, sonicated in
an ice bath, and centrifuged at 16,000 g for 30 min.
For complete removal of the cellular polyamines, the supernatant
obtained was dialyzed against 500 volumes of 10 mM sodium
phosphate buffer, pH 7.0, containing 0.5 mM EDTA and 10
mM 2-mercaptoethanol with two changes for 10-16 h. The
plant cytosol fraction was also dialyzed against the same buffer. The
reaction mixture generally contained the following constituents in a
final volume of 1.0 ml: 50 mM Tris-HCl, pH 8.7, 50 mM
KCl, 2 mM dithiothreitol, 0.5 mM
NAD
, and dialyzed cell-free extract of bacteroids or
plant cytosol fraction (0.9-2.3 mg of protein). The reaction was
started by the addition of the substrate (Put or Cad, or both
compounds) to the reaction mixture at 30 °C and terminated by the
addition of 0.1 ml of 20% PCA. After centrifugation, an aliquot of the
acid extract was subjected to the quantitative analysis of Homospd and
4-ABcad by HPLC. The reaction mixture without substrate was also
incubated at 30 °C as a blank.
Cell-free extract of
bacteroids was prepared from 20 g (fresh weight) of frozen nodules,
dialyzed as above, and served for the N Experiment
N experiment.
[1,4-
N]Put (99%
N), in which two
terminal amino groups of Put were labeled with
N, and Cad
were added to the reaction mixture as substrates at final
concentrations of 0.5 mM and 5 mM, respectively. The
reaction mixture (final volume 20 ml) contained 50 mM
Tris-HCl, pH 8.7, 50 mM KCl, 5 mM
NAD
, 2 mM dithiothreitol, and a dialyzed
cell-free extract of bacteroids equivalent to 8.66 mg of protein. The
reaction medium was incubated at 30 °C with gentle shaking for 15
h, and the reaction was terminated by the addition of 2 ml of 20% PCA.
After precipitation of the protein by centrifugation, the supernatant
was neutralized with 10 M KOH and applied to a Dowex 50W-X8
column (H
form, 200-400 mesh, 0.9
4 cm).
After washing the column with 100 ml of distilled water and 50 ml of
0.5 M HCl, the polyamines were eluted with 15 ml of 6
M HCl, evaporated to dryness, converted to their volatile
derivatives by reaction with PFP reagent
(13) , and subjected to
the GC-MS analysis.
Chemicals
Homospd and 3-aminopropylcadaverine
(3-APcad) were chemically synthesized according to the method of Okada
et al. (14) . Authentic 4-ABcad and
5-aminopentylcadaverine (5-APcad) were the generous gift of Dr. S.
Yamamoto (Okayama University, Japan).
[1,4-N]Put,
NH
(CH
)
NH
,
was chemically synthesized from potassium
[
N]phthalimide (99%
N; Shoko,
Tokyo) and 1,4-dibromobutane as starting materials, and its
hydrochloride salt was crystallized from 90% ethanol as described
previously in detail
(15) . Other polyamines and organic and
inorganic chemicals were obtained from commercial sources.
Occurrence of Unknown Compound X in Root Nodules of
Adzuki Bean
Root nodules of adzuki bean ( V. angularis)
contained large quantities of Put, Cad, and Homospd besides small
quantities of Spd and Spm. Fig. 1shows a typical elution
chromatogram of nodule polyamines in HPLC analysis. Generally, two or
more unknown peaks, which could not be assigned to any of the standard
polyamines, appeared between Put and Spm on the elution chromatograms.
These compounds were resistant to acid hydrolysis, since each peak
height was not changed even though the acid extracts of root nodules
were hydrolyzed with 6 M HCl at 100 °C prior to the
derivatization procedure. Among these peaks, the peak posterior to
Homospd (denoted as X in the figure) was detected only in
adzuki bean plant while other peaks were found in the chromatograms of
many other legume root nodules as well. shows distribution
of polyamines in leaf, stem, root, and nodule separated from various
adzuki bean plants (one variety and three cultivars). The peak X in
adzuki bean was found to occur specifically in root nodules and the
unusual peaks were not detected in other organs such as leaf, stem, or
root. A wild type adzuki bean plant (var. nipponennsis ACC1238), which
is a putative wild ancestor of the cultivated adzuki bean, showed
apparently distinct morphology from other adzuki bean cultivars,
e.g. grain size and leaf and stem shape. A characteristic
pattern of polyamine composition was observed in the root nodules of
this wild progenitor species of adzuki bean, showing a very low Cad
content. The peak X was not detected in the root nodules of the wild
type adzuki bean.
Figure 1:
Elution profiles of polyamines in
adzuki bean root nodules ( A) and standard polyamines
( B). Root nodules were collected from a field-grown adzuki
bean plant ( V. angularis cv. Erimoshoudzu) at 46 days after
sowing. Polyamines were extracted from the freeze-dried sample with 0.5
M PCA, purified with Dowex 50W-X8 column, derivatized with
benzoylchloride, and subjected to the HPLC analysis. Diaminohexane was
used as an internal standard. Peak X in Panel A denotes unknown compound. Peaks in Panel B: 1,
Put; 2, Cad; 3, internal standard (diaminohexane);
4, Spd; 5, Homospd; 6,
Spm.
Chemical Structure of the Unknown Compound X
To
determine the chemical structure, the unknown compound X in the nodule
acid extract was separated from other polyamines by ion-exchange
chromatography, and the pooled fraction containing compound X was then
subjected to the HPLC, capillary GC, and GC-MS analysis. Both in HPLC
and capillary GC analysis, the retention time of compound X coincided
with that of authentic 4-ABcad,
NH(CH
)
NH(CH
)
NH
:
the relative retention time against the internal standard
(diaminohexane) was 1.42 in HPLC and 2.01 in capillary GC. The evidence
of identity was further established by GC-MS. Fig. 2shows the
data comparing the mass spectrum of the PFP derivative of compound X
with those of chemically synthesized 4-ABcad and Homospd. Under
electron impact, the mass fragmentation pattern of the compound X
(Fig. 2 C) was identical with that of authentic 4-ABcad
(Fig. 2 B). Very weak molecular ion (M
)
of PFP derivative of the compound X or synthetic 4-ABcad was obtained
at m/e 611 while that of Homospd (Fig. 2 A) was
at m/e 597. The loss of C
F
and
C
F
CO from the parent molecules gave intense
fragment ions both at m/e 492 and m/e 464,
respectively. A characteristic fragment ion peak of 4-ABcad, which was
not obtained in the mass fragment of Homospd, was observed at m/e 407, due to the presence of
NH
(CH
)
NHCH
- moiety in
the molecules.
Figure 2:
Mass
spectra of PFP derivatives of Homospd ( A), 4-ABcad
( B) and unknown compound X ( C). Chemically
synthesized Homospd and 4-ABcad and unknown compound X were converted
to their volatile derivatives with PFP anhydride as described in Flores
and Galston (12). Chemical structures and possible split regions
assigned from the fragment ion peaks are also
shown.
Localization of 4-ABcad within Root Nodule
Tissues
In order to know the localization of this unusual
triamine within the root nodule tissues, root nodules harvested from
the 64-day-old adzuki bean plant were divided into two fractions,
bacteroids (endosymbionts) and a plant cytosol fraction. Bacteroids
contained 4-ABcad besides small quantities of Put and Cad and a large
quantity of Homospd, while plant cytosol fraction contained large
quantities of Put and Cad (). Since both Spd and Spm,
characteristic polyamines of the host plant, were not detected in the
bacteroid fraction, contamination of plant cells to the bacteroid
fraction seems unlikely. By contrast, small quantities of 4-ABcad and
Homospd were found also in plant cytosol fraction, possibly due to the
leakage from bacteroids or rupture of bacteroid cells during the
fractionation procedure, since bacteroids, having a grossly swollen
cell structure within root nodules, are fragile as compared with
cultured rhizobial cells
(16) .
4-ABcad Formation in B. japonicum
To examine
whether the endosymbiont of adzuki bean root nodules participates in
the formation of 4-ABcad, the ability of B. japonicum A1017,
which forms root nodules on the roots of adzuki bean plant, to produce
this unusual polyamine was investigated. It was found that 4-ABcad was
detected only in the cells grown in the culture supplemented Cad (data
not shown). Addition of lysine into the growth medium was not effective
on the production of Cad nor 4-ABcad, suggesting the lack or very low
activity of lysine decarboxylase in B. japonicum A1017.
Biosynthesis of 4-ABcad by Cell-free Extract of
Bacteroids
To confirm the formation of 4-ABcad by bacteroids, an
in vitro experiment was carried out using a cell-free extract
of bacteroid cells, in which cellular polyamines were completely
removed by dialysis (I). Addition of Put to the reaction
mixture including NADand K
resulted
in the production of Homospd. Cad was inhibitive for the synthesis of
Homospd, but 4-ABcad was formed under the presence of both Put and Cad
if Cad was present sufficiently against Put. The peak correspondent to
5-APcad was not found during this experiment even if Cad was used as a
sole substrate. Removal of NAD
or K
from the reaction mixture significantly reduced the formation not
only of Homospd but also of 4-ABcad. Furthermore, addition of NADH or
1,3-diaminopropane, which inhibited Homospd formation, showed strong
repression of 4-ABcad formation as well, implying the involvement of
Homospd synthase on the synthesis of 4-ABcad. The polyamines having
aminopropyl group in the molecules, such as Spd, norspermidine
(Norspd), and 3-APcad, were not produced by the addition of
diaminopropane with Put and Cad. The same experiment was also done with
dialyzed plant cytosol fraction, but neither Homospd nor 4-ABcad was
produced by the plant cytosol fraction. Incorporation of [
N]Put into 4-ABcad-In
order to know the molecular mechanism for biosynthesis of 4-ABcad in
nodule bacteroids, a
N tracer experiment was also carried
out (Fig. 3). [1,4-
N]Put was added into
the dialyzed cell-free extract of bacteroids with excess amount of Cad.
The reaction mixture including NAD
and K
was incubated overnight to obtain sufficient amount of the
reaction products necessary for the analysis of GC-MS.
Fig. 3A shows the total ion monitoring of the sample
after derivatization of polyamines with PFP reagent. Both Homospd and
4-ABcad were detected as reaction products, and no other products
appeared on the elution chromatogram. The positions of
N
incorporated into the reaction products were examined by comparing
their fragment ion peaks under electron impact with those of the
authentic samples without
N shown in Fig. 2. In
Homospd, relatively intense fragment ion peaks observed at m/e 450 and m/e 478 (Fig. 2 A) shifted at
m/e 453 and m/e 481, respectively
(Fig. 3 B), indicating that
N was
incorporated into all of the positions of amino and imino groups in the
Homospd molecules. On the other hand, in 4-ABcad, intense fragment ion
peaks at m/e 464 and m/e 492, due to the loss of
C
F
or C
F
CO from the
parent molecules (Fig. 2, B or C), shifted at
m/e 465 and m/e 493, respectively
(Fig. 3 C). The ion peak at m/e 393, derived
from -CH
NH(CH
)
NH
group,
also showed a shift by 1 mass unit ( m/e 394), whereas the ion
peak at m/e 407, derived from
NH
(CH
)
NHCH
- group,
remained at m/e 407. The data strongly suggest that 4-ABcad is
formed through the transfer of the aminobutyl moiety to the amino
terminus of Cad and not through the transfer of aminopentyl moiety to
the amino terminus of Put.
Figure 3:
Incorporation of
[N]Put into Homospd and 4-ABcad by cell-free
extract of bacteroids. [1,4-
N]Put (99%
N) and Cad were added at final concentrations of 0.5
mM and 5 mM, respectively, to the reaction mixture
including 50 mM Tris-HCl, pH 8.7, 50 mM KCl, 5
mM NAD
, 2 mM dithiothreitol, and
dialyzed cell-free extract of bacteroids (8.66 mg of protein) in a
final volume of 20 ml and incubated at 30 °C with gentle shaking.
After 15 h, polyamines were extracted, purified, derivatized, and
subjected to the GC-MS analysis. Total ion monitoring of the sample
during separation with capillary GC column ( A), mass spectra
of PFP derivatives of the reaction products Homospd ( B), and
4-ABcad ( C) are shown.
and K
, necessary for the full activity of
Homospd synthase
(30, 31, 32, 33) , were
required for the synthesis of 4-ABcad as well, and 2) diaminopropane
and NADH, potent inhibitors of Homospd synthase
(30, 32, 33) , repressed 4-ABcad formation.
N experiment
(Fig. 3) clearly indicated that the transfer of aminopentyl
group, produced by Cad oxidation, to the amino terminus of Put
molecules did not occur. We recently detected the activity of ornithine
decarboxylase in bacteroids (data not shown), implying Put formation
from ornithine as a substrate for Homospd synthase within bacteroid
tissues. In conclusion, as to the occurrence of 4-ABcad within the
adzuki bean root nodules, the present data suggest that this novel
polyamine is formed through a reaction sequence mediated by Homospd
synthase in bacteroid tissues utilizing Cad supplied from the host
legume cells as illustrated in Fig. 4. The recent report which
describes enzymatic formation of 4-ABcad by a purified Homospd synthase
from Acinetobacter tartarogenes (33) might support the
present hypothesis. It is of interest why and how the Cad is produced
abundantly in adzuki bean root nodules differing from other legume
nodules. Together with the physiological traits of adzuki bean, these
are the problems to be elucidated in the future.
Figure 4:
Possible mechanism for the biosynthesis of
4-ABcad in adzuki bean root nodules. 4-ABcad is synthesized from
putrescine and cadaverine supplied from host plant cells under the
presence of NADby the action of Homospd synthase in
bacteroid cells. NADH, produced by the first step reaction (Put
oxidation), is recycled for the subsequent reduction of the putative
intermediate into 4-ABcad.
Table:
Polyamine composition in various organs of
field-grown adzuki bean plants
Table:
Polyamines in bacteroids
and plant cytosol fraction separated from root nodules
Table:
Synthesis of Homospd and 4-ABcad by
cell-free extract of bacteroids
, 2 mM dithiothreitol, and dialyzed
cell-free extract of bacteroids (2.3 mg of protein) in a final volume
of 1.0 ml. The reaction was started by the addition of substrate (Put
or Cad, or both), and incubated at 30 °C for 2 h. Quantitative
analyses of the reaction products were performed with HPLC. The
averages of duplicate determinations are shown.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.