Plant Physiol. (1998) 117: 971-977
Characterization of Spermidine Binding to Solubilized Plasma
Membrane Proteins from Zucchini Hypocotyls1
Annalisa Tassoni,
Fabiana Antognoni,
Maria Luisa Battistini,
Olivier Sanvido, and
Nello Bagni*
Dipartimento di Biologia Evoluzionistica Sperimentale,
Università di Bologna, Via Irnerio 42, 40126 Bologna, Italy
 |
ABSTRACT |
In this work
[14C]spermidine binding to total proteins solubilized
from plasma membrane purified from zucchini (Cucurbita
pepo L.) hypocotyls was investigated. Proteins were solubilized
using octyl glucoside as a detergent. Specific polyamine binding was thermolabile, reversible, pH dependent with an optimum at pH 8.0, and
had a Kd value of 5 µM, as
determined by glass-fiber-filter assays. Sephadex G-25 M gel-filtration
assays confirmed the presence of a spermidine-protein(s) complex with a
specific binding activity. By sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and native polyacrylamide gel electrophoresis of
collected fractions having the highest specific spermidine-binding
activity, several protein bands (113, 75, 66, and 44 kD) were
identified. The specificity of spermidine binding was examined by
gel-filtration competition experiments performed using other polyamines
and compounds structurally related to spermidine. Partial purification
on Sephadex G-200 led to the identification of 66- and 44-kD protein
bands, which may represent the putative spermidine-binding protein(s)
on the plasmalemma.
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INTRODUCTION |
Polyamines are biologically ubiquitous compounds that are
implicated in many aspects of growth and development in a wide range of
organisms (microorganisms, animals, and plants) (Tabor and Tabor, 1985
;
Bagni, 1989; Persson et al., 1996), although their specific mechanism
of action is not well understood. By virtue of their cationic nature at
physiological pH, they can interact with several molecules and thereby
affect their structure and function. Hydrogen binding, ionic and
covalent linkages, and hydrophobic interactions may occur that involve
the charged functional amino and imino groups and the tetra- and
trimethylene chains (Feuerstein and Marton, 1989; Schuber, 1989;
Serafini-Fracassini et al., 1995).
One possible mechanism by which polyamines might act as growth
substances involves their binding to specific regulatory proteins, as
has been observed in many organisms. In both animal and plant systems
it has been postulated that these compounds may play a role in the
posttranslational modifications of proteins (Äschlimann and
Paulsson, 1994; Serafini-Fracassini et al., 1995), as well as in the
modulation of many enzyme activities such as protein kinases,
phosphatases (Datta et al., 1986; Friedman, 1986), and 1,3-
-glucan
synthase (Kauss and Jeblick, 1985). Despite the large amount of
information on the covalent interactions between polyamines and
proteins, little is known about the noncovalent binding of polyamines
to PM proteins, even though this may represent one of the first steps
in their action at the cellular level.
In Escherichia coli two periplasmic polyamine-binding
proteins (PotD and PotF) have been isolated as part of two membrane transport systems, pPT104 and pPT79, respectively (Kashiwagi et al.,
1993; Pistocchi et al., 1993b). In particular, the crystal structure of
PotD, which strongly binds spermidine, was recently determined
(Sugiyama et al., 1996). This crystallographic study led to the
elucidation of the mechanism of spermidine recognition and the
characteristics of the main chain folding at the binding site.
Nevertheless, no information is available on polyamine-binding proteins
of plant membranes. In a previous paper, the main characteristics of
spermidine binding to purified zucchini (Cucurbita pepo L.)
PM vesicles were described (Tassoni et al., 1996). Results indicated
that the specific interaction was saturable, reversible, heat labile,
and detergent- and pronase-sensitive, thus suggesting that the binding
occurred with a protein component of the PM.
In this paper we have defined the features of specific spermidine
binding to solubilized PM proteins. This represents a necessary step
before purification of the specific polyamine-binding protein(s). Moreover, a partial purification of the specific spermidine-binding protein(s) was performed to identify the putative receptor(s) and/or
transporter(s) on the plant plasmalemma.
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MATERIALS AND METHODS |
Seeds of zucchini (Cucurbita pepo L. hybrid Strorr's
Green, Asgrow Co., Lodi, Italy) were planted in moist vermiculite and grown at 22 ± 1°C in total darkness for 7 d. Etiolated
hypocotyls (6-12 cm long) were excised and stored at 
20°C until
use.
Isolation of PM Vesicles, Solubilization of Membrane Proteins, and
Polyamine Analysis
A very pure PM-enriched fraction was obtained after the aqueous
phase-partitioning procedure described by Tassoni et al. (1996). Buffer
A, containing 50 mM Tris-HCl, pH 7.5, 2.0 mM EDTA, 250 mM Suc, and 10 mM
2-mercaptoethanol, was slightly modified by adding a mixture of
protease inhibitors, namely 0.2 mM PMSF, 5 mM
6-amino-n-hexanoic acid (Gray, 1982), 1 mM
benzamidine, 10 µg mL
1
N-
-p-tosyl-L-Arg methyl ester, and
10 µg mL1
N--benzoyl-L-Arg methyl ester (Miller et al.,
1995) (all from Sigma). PM vesicles were resuspended in buffer A at pH
8.0 and stored at 80°C until use. For protein solubilization, 1%
(w/v) OG was added to purified PM vesicles (final protein concentration 2 mg mL1). The solution was sonicated twice for
30 s by means of an ultrasonic disintegrator (20 kHz, amplitude 10 µm peak to peak, model 150-W, Measuring and Scientific Equipment
Ltd., Crawley, UK), left on ice for 30 min, and then centrifuged at
20,000g for 30 min at 4°C. About 95% of the total PM
proteins was recovered in a soluble form. Protein content was
determined by the method of Lowry et al. (1951) with BSA as a standard.
Polyamines noncovalently (PCA-soluble fraction) and covalently bound to
solubilized proteins (about 2 mg mL1) were
determined by HPLC analysis after dansylation, as described by
Scaramagli et al. (1995). In particular, polyamines covalently bound
(PCA-insoluble fraction) were determined after hydrolysis at 110°C
for 20 h with 6 N HCl in flame-sealed vials.
Binding and Dissociation Assay
The standard binding assay (1 mL final volume) contained 0.2 mg of
protein in buffer A, pH 8.0, and 5.55 kBq
[14C]spermidine (specific activity 4.07 GBq
mmol1; Amersham) with or without 1 mM unlabeled spermidine to give total and nonspecific
binding (samples A and B, respectively). Samples were incubated on ice
for 5 min and then directly filtered through glass-fiber filters
(Whatman GF/B) previously soaked for about 2 h in 0.3% (v/v)
polyethylenimine (Sigma) (Bruns et al., 1983), and placed in a
Büchner funnel connected to a vacuum pump. After rapid
filtration, filters were rinsed with 5 mL of buffer A, pH 8.0, to wash
out the unbound spermidine, and then placed overnight in 2 mL of
scintillation cocktail (Ultima Gold, Canberra Packard, Groningen, The
Netherlands) before radioactivity was determined in a scintillation
counter (model LS 1800, Beckman). Specific binding was obtained by
subtracting the radioactivity of sample B (nonspecific binding) from
that of sample A (total binding). All binding experiments were repeated
at least twice with triplicate samples.
To study the dissociation rate, total and nonspecific samples were
incubated until a steady-state level of occupancy was achieved, then 4 mM unlabeled spermidine was added and samples were filtered as described above after different dissociation times.
Gel-Filtration Assays
Solubilized proteins (about 1 mg mL1) were
incubated for 5 min on ice with 7.4 kBq
[14C]spermidine in the absence or presence of
1.7 mM unlabeled spermidine for total and nonspecific
binding assays, respectively (0.6 mL final volume). To verify that a
good separation of bound ligand from free ligand occurred, blank assays
without solubilized proteins were performed. The mixtures were then
applied to Sephadex G-25 medium prepacked columns (9-mL bed volume,
PD-10, Pharmacia) previously equilibrated with about 25 mL of buffer A,
pH 8.0, to which 0.01% (w/v) OG was added to prevent the detergent
concentration from falling below the critical micellar concentration
(Hulme, 1992). Column elution was carried out with buffer A and 20 fractions, each of about 0.4 mL, were recovered. Radioactivity in a
0.2-mL aliquot of each fraction was determined by liquid-scintillation counting, whereas the remainder of the fraction was used for
A280 measurement. Specific binding activity
was calculated as the difference between total and nonspecific values.
Fractions showing the highest specific spermidine-binding activity were
then analyzed by 12% (w/v) SDS-PAGE (Laemmli, 1970) and native PAGE
(Hendrick and Smith, 1968). Gels were stained with silver salts
(Sammons et al., 1981) and then analyzed by densitometry using
Molecular Analyst/PC Image Analysis software (version 1.1.1, 1992-1994, model GS-670, Bio-Rad).
Sephadex G-25 gel-filtration competition experiments were performed
between labeled spermidine and the following analog and polyamines (20 µM final concentration): MGBG, putrescine, spermidine, and spermine. Sephadex G-25 gel-filtration fractions (3.0 mL, approximately 0.4 mg mL1 protein) with the
highest specific spermidine-binding activity were incubated for 5 min
on ice with 18.5 kBq [14C]spermidine. They were
applied to a column (40 cm × 1.6 cm) of Sephadex G-200
equilibrated in buffer A with 0.01% (w/v) OG and maintained at
3.5°C. Elution was carried out with the same buffer. Fractions (0.4 mL) were collected and analyzed for radioactivity and
A280 as described above for Sephadex G-25
fractions. SDS-PAGE (12%, w/v) and silver staining were performed on
fractions that showed the highest spermidine-binding activity.
ATPase, ADC, and ODC Activities
Vanadate-sensitive ATPase activity in PM vesicles, solubilized
proteins, and fractions having maximum spermidine-binding activity was
determined as described by Tassoni et al. (1996). ADC and ODC
activities were assayed as described by Altamura et al. (1993): for a 2 mM final concentration, unlabeled Arg and Orn,
respectively, were added to the assay mixture.
| |
RESULTS AND DISCUSSION |
Solubilization of Membrane Proteins and Polyamine Content
Three detergents (Chaps, Triton X-100, and OG) at 0.3 and 1%
concentration were tested on purified PM vesicles to establish the best
condition for solubilizing zucchini PM proteins. Chaps and OG were more
effective than Triton X-100 at all concentrations tested (data not
shown). Treatment with 0.3% (w/v) Chaps or 1% (w/v) OG led to the
recovery of more than 95% of total PM proteins in the supernatant,
whereas with 0.3% (w/v) OG or 0.3 and 1% (v/v) Triton X-100, the
yield of proteins was less than 90%. OG (1%, w/v) was chosen for
subsequent work because of the greater stability of many membrane
proteins in nonionic detergents (Thomas and McNamee, 1990), and because
this detergent has been successfully used to solubilize other plant PM
proteins in an active form (de Boer et al., 1989).
Polyamines noncovalently (PCA-soluble fraction) and covalently
(PCA-insoluble fraction) bound to solubilized proteins were determined
(Table I). All three polyamines
(putrescine, spermidine, and spermine) were more abundant in the
noncovalently bound form, and the spermidine content was 2- and 16-fold
higher than putrescine and spermine, respectively. This reflects the
pattern of polyamine content previously reported for zucchini PM
vesicles (Tassoni et al., 1996), and suggests that very few polyamines
are bound to acidic PM phospholipids. The content of noncovalently
bound spermidine was about 22-fold higher than that of covalently bound spermidine (Table I).
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Table I.
Noncovalently and covalently bound polyamine content
in proteins solubilized from zucchini PM vesicles
The values are means ± SD of four samples of two
replicate experiments.
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Characteristics of [14C]Spermidine Binding to
Solubilized Proteins
Preliminary results indicated that OG in the range between 0.8%
and 1% (w/v), which coincides with the detergent concentration used
for protein solubilization, gave the maximum specific
spermidine-binding activity (data not shown). Thus, 1% (w/v) OG was
used in subsequent binding assays. Figure
1 shows the association of
[14C]spermidine to total solubilized proteins.
Steady-state binding was reached after 2 min and, upon addition of an
excess of unlabeled spermidine (Fig. 1, arrow), the labeled polyamine
rapidly dissociated and thereafter specific binding remained constant.
This showed that the specific interaction between spermidine and PM
proteins is reversible. In Figure 2 the
effect of pH on spermidine binding, using Tris-Mes buffer in the range
from pH 5.5 to 7.0 and Tris-HCl buffer from pH 7.0 to 9.0, is reported.
Specific spermidine binding to solubilized proteins is pH dependent,
with an optimum at pH 8.0.
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| Figure 1.
Kinetics of specific [14C]spermidine
binding to total proteins solubilized from zucchini PM vesicles. The
assay was performed at pH 8.0. The dissociation of specifically bound
spermidine was started by adding 4 mM unlabeled spermidine
(arrow). The values are means ± SD of triplicate
samples from two replicate experiments. prot, Protein.
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| Figure 2.
Effect of pH on specific
[14C]spermidine binding to total proteins solubilized
from zucchini PM vesicles. The assay was performed with Tris-Mes buffer
from pH 5.5 to 7.0 ( ) and with Tris-HCl buffer from pH 7.0 to 9.0 ( ). The values are means ± SD of triplicate samples from two replicate experiments. prot, Protein.
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Both the reversibility and the pH dependence of specific spermidine
binding reflect properties previously found in PM-enriched fractions
(Tassoni et al., 1996). Heat lability was also similar in solubilized
proteins, specific binding activity being reduced to one-half in assays
performed at 40°C compared with those carried out on ice (data not
shown). Similar pH dependence has been observed with respect to
putrescine and spermidine uptake in several systems, including sugar
beet seedlings (Christ et al., 1989), carrot cell cultures, African
violet petals (Pistocchi et al., 1993a), the lichen Evernia
prunastri (Escribano and Legaz, 1985), and the seaweed Ulva
rigida (Badini et al., 1994). This suggests that binding proteins
in zucchini may be part of a polyamine-transport system in the
plasmalemma that consists of different proteins. It may be analogous to
the system in Escherichia coli, in which PotD protein, a
primary receptor of the spermidine preferential transport system
pPT104, has been characterized and crystallized (Sugiyama et al.,
1996).
The concentration dependence of spermidine binding to solubilized
proteins (Fig. 3) showed that saturation
was reached at 50 µM polyamine concentration with an
apparent Kd of 5 µM (EBDA program, version 3.0, 1983, GA McPherson [Elsevier-Biosoft, Cambridge, UK]). This value is 1 order of magnitude higher in affinity than that
found in PM fractions (Kd approximately 40 µM; Tassoni et al., 1996), but is similar to the
Kd value observed for the PotD protein in
E. coli (Kashiwagi et al., 1993).
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| Figure 3.
Concentration dependence of specific
[14C]spermidine binding to total proteins solubilized
from zucchini PM vesicles. Different amounts of unlabeled spermidine
were added to 5.55 kBq [14C]spermidine to give the final
spermidine concentrations shown. The values are means ± SD of multiple samples from three replicate experiments.
prot, Protein.
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Another approach to the study of spermidine-binding sites on
solubilized proteins involves a gel-filtration technique using prepacked columns (PD-10, Pharmacia). In Figure
4A, the elution pattern of the gel
filtration of the [14C]spermidine-binding assay
is shown. A good separation of bound from free
[14C]spermidine was obtained, indicating that
the gel-filtration technique represents a suitable method for
polyamine-binding studies. In addition, the specific-to-nonspecific
binding ratio was higher than that obtained by filtration with
glass-fiber filters. Fractions 10 to 14 had the highest specific
spermidine-binding activity (Fig. 4B). Total solubilized proteins and
gel-filtration active fractions were analyzed by SDS-PAGE, stained with
silver salts, and compared (Fig. 5, lanes
A and B). Active fractions (Fig. 5, lane B) exhibited bands of 113, 75, 66, and 44 kD, two of which (75 and 44 kD) were enriched compared with
total solubilized proteins (Fig. 5, lane A). Additional bands at 92, 38, and 36 kD were observed on native PAGE (Fig. 5, lane C). The
densitometric analysis of lanes A and B of SDS-PAGE (Fig.
6) revealed that, changing from total
solubilized proteins to gel-filtration fractions, the 44- and 75-kD
protein bands increased in intensity by about 90% and 50%,
respectively, whereas, according to the computer calculation, the
113-kD band did not significantly vary in intensity and the 66-kD band
decreased by about 40%.
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| Figure 4.
Sephadex G-25 M gel-filtration assay of
[14C]spermidine binding to proteins solubilized from
zucchini PM vesicles. A, Blank and standard binding assay elution
patterns. B, Specific binding elution pattern and
A280 protein elution profile. Proteins were eluted with buffer A, pH 8.0, containing 0.01% (w/v) OG and
fractionated in 0.4-mL aliquots. Open symbols, Blank assay; closed
symbols, standard binding assay; circles, total binding; squares,
specific binding; and triangles, nonspecific binding. The values are
means ± SD of triplicate samples of three replicate
experiments. The SD values are contained within the
symbols.
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| Figure 5.
SDS-PAGE of total solubilized proteins (lane A)
and gel-filtration fractions 10 to 14 (lane B), and native PAGE of
gel-filtration fractions (lane C). Protein bands were stained with
silver.
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| Figure 6.
Densitometric profile of SDS-PAGE of total
solubilized proteins (Fig. 5, lane A) and gel-filtration fractions
(Fig. 5, lane B). Continuous line, Total solubilized proteins; broken
line, gel-filtration fractions. OD, Optical density.
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Gel-filtration competition experiments were carried out between
[14C]spermidine and other polyamines and a
spermidine analog. As shown in Figure 7,
MGBG, a competitive inhibitor that resembles spermidine with regard to
the distance between the positively charged amino-terminal groups
(Seiler and Dezeure, 1990), competed for
[14C]spermidine binding as effectively as
unlabeled spermidine. Spermine exerted a lower but still noticeable
inhibitory effect, whereas putrescine did not compete. These results
demonstrate that spermidine-binding protein(s) exhibit considerable
specificity, perhaps attributable to the presence of negatively charged
groups in the binding site, the distance of which strictly resembles
that of positively charged amino-terminal groups of spermidine. In
fact, in PM vesicles, putrescine, in which the two positively charged
amino groups are closer than in spermidine, failed to compete for
specific binding (Tassoni et al., 1996), although spermine competition
was greater then that found with solubilized proteins. Moreover,
transport studies performed on carrot protoplasts confirmed the higher
capacity of spermidine to bind to plasmalemma with respect to
putrescine (Pistocchi et al., 1988).
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| Figure 7.
Effect of different competitors on specific
[14C]spermidine binding to proteins solubilized from
zucchini PM vesicles. The various antagonists were present at 20 µM final concentration. , No antagonist;  ,
putrescine;  , spermine;  , MGBG; and , spermidine. The values are means ± SD of double samples of two replicate
experiments. The SD values are contained within the
symbols.
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Vanadate-sensitive ATPase, a PM marker, and ADC and ODC, two polyamine
biosynthetic enzymes, were assayed in PM vesicles, total solubilized
proteins, and gel-filtration fractions showing the highest
spermidine-binding activity. In E. coli specific transport systems for spermidine and putrescine (pPT104 and pPT79) contain, in
addition to periplasmic substrate-binding proteins (PotD and PotF,
respectively), PotA and PotG proteins that have ATPase activity (Kashiwagi et al., 1993, 1995; Pistocchi et al., 1993b). Moreover, the
latter two proteins have molecular masses of 43 and 45 kD, similar to
our 44-kD protein band found in the SDS-PAGE and native PAGE of active
fractions (Fig. 5, lanes B and C). Because it is known that the loading
through Sephadex gels does not generally interfere with this enzyme
activity (Penefsky, 1977), vanadate-sensitive ATPase activity was also
tested in gel-filtration fractions to determine if this activity is
related to specific spermidine binding. We detected vanadate-sensitive
ATPase activity in PM vesicles (27.11 ± 2.70 nmol
min1 mg1 protein) and
total solubilized proteins (10.73 ± 1.49 nmol
min1 mg1 protein), but
none in gel-filtration fractions. This result shows that ATPase
activity, although present in total solubilized proteins, does not
co-elute during Sephadex G-25 gel filtration with the putative
spermidine-binding proteins. However, the result does not exclude the
possibility that a similar enzyme activity could be correlated with
specific spermidine binding at the plasmalemma level. ADC and ODC
activities were undetectable in PM vesicles, total solubilized
proteins, and gel-filtration fractions, consistent with their reported
subcellular localization in nuclei, mitochondria, and chloroplasts
(Panagiotidis et al., 1982; Torrigiani et al., 1985; Borrell et al.,
1995).
Specific spermidine-binding activity was investigated by gel-filtration
assay of solubilized plasmalemma proteins stored overnight at different
temperatures (4°C, 20°C, and 80°C, respectively) to identify
the optimal storage temperature for subsequent multistep purification
procedures. Storage at 20°C resulted in recovery of the highest
binding activity (80% of fresh control), whereas proteins stored at
4°C and 80°C lost about 50% and 90%, respectively, of their
binding capacity (data not shown).
To achieve partial purification of spermidine-binding proteins,
Sephadex G-25 gel-filtration fractions with the highest specific spermidine-binding activity were applied to a Sephadex G-200 column. The elution profile (Fig. 8) of
[14C]spermidine binding shows the presence of a
radioactivity peak coinciding with the major protein peak
(A280). SDS-PAGE analysis of the most
active spermidine-binding fractions showed two protein bands of 66 and
44 kD (Fig. 8, inset), suggesting that the 113- and 75-kD bands
previously found in Sephadex G-25 gel filtration are not involved in
spermidine binding.
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| Figure 8.
Sephadex G-200 gel-filtration elution profile.
Sephadex G-25 gel-filtration fractions showing the highest specific
spermidine-binding activity were applied to a Sephadex G-200 column
after incubation for 5 min at 5°C with 18.5 kBq
[14C]spermidine. Proteins were eluted by using buffer A,
pH 8.0, with 0.01% (w/v) OG and 0.4-mL aliquots were collected. Closed symbols, radioactivity (dpm fraction1) elution profile;
open symbols, A280 protein elution profile. Inset, Silver-stained 12% (w/v) SDS-PAGE of the most-active
spermidine-binding fractions (fractions 171, 172, and 173).
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In conclusion, the characteristics of spermidine binding to total
solubilized proteins from plant plasmalemma turned out to be similar to
those previously found in PM vesicles. This confirms the hypothesis
that the specific polyamine interaction between spermidine and PM
occurs with the protein component of the membrane. This interaction
could represent one of the first steps by which polyamines act as
growth regulators. If the spermidine-binding protein(s) is a polyamine
receptor, this specific binding could activate the reaction chain
responsible for the range of polyamine effects at the cellular level.
However, if the binding protein is part of a PM carrier(s), polyamine
entry would stimulate the physiological response once inside the cell.
One or both of the two proteins (66 and 44 kD) found after partial
purification with Sephadex G-200 gel filtration are probably involved
in specific spermidine binding to the PM. Work is in progress with the
aim of identifying and characterizing the polyamine-binding protein(s).
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FOOTNOTES |
1
This work was supported in part by funds from
the National Research Council of Italy, special project Ricerche
Avanzate per Innovazioni nel Sistema Agricolo, subproject no. 2, and in
part by funds from the Ministero dell'Università, della Ricerca
Scientifica e Tecnologica. O.S. was a recipient of a grant from the
Roche Research Foundation (Basel, Switzerland).
*
Corresponding author; e-mail bagninel{at}kaiser.alma.unibo.it; fax
39-51-242576.
Received January 2, 1998;
accepted April 9, 1998.
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ABBREVIATIONS |
Abbreviations:
ADC, Arg decarboxylase.
Chaps, 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propane-sulfonate.
MGBG, methylglyoxal-bis(guanylhydrazone).
ODC, Orn decarboxylase.
OG, octyl glucoside.
PCA, perchloric acid.
PM, plasma membrane.
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ACKNOWLEDGMENTS |
We are very grateful to Prof. M.A. Venis (Horticulture Research
International, Wellesbourne, UK) for useful scientific discussion and
help in preparing the manuscript, and to Prof. Patrizia Aducci (University of Rome "Tor Vergata," Italy) for her most valuable suggestions.
| |
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Abstract
Introduction
