One source of NO is nitrite, which can be converted to NO Keywords Arabidopsis; argininosuccinate lyase; citrulline; nitric oxide Correspondence N.. In this article, we report that when
Trang 1assay by argininosuccinate lyase activity in Arabidopsis extracts
Rudolf Tischner1,*, Mary Galli2,*, Yair M Heimer3,*, Sarah Bielefeld1, Mamoru Okamoto2,
Alyson Mack2and Nigel M Crawford2
1 Albrecht von Haller Institut fur Pflanzenwissenschaften, University of Gottingen, Germany
2 Section of Cell and Developmental Biology, Division of Biological Sciences, University of California at San Diego, La Jolla, CA, USA
3 Department of Dryland Biotechnologies, J Blaustein Institute for Desert Research, Ben-Gurion University, Sede-Boker, Israel
Nitric oxide (NO) serves as a central signal in a wide
variety of processes, including vasodilation, neural
communication and immune function in animals [1],
and defense responses, hormonal signaling and
flower-ing in plants [2–6] The primary mechanism for NO
synthesis in animals involves oxidation of l-arginine to
l-citrulline and NO, and requires NADPH and oxygen
[7–9] This reaction is catalyzed by nitric oxide
syn-thase (NOS) enzymes, which require
tetrahydrobiopter-in (BH4), FMN, FAD, calmodulin (CaM), and Ca2+
Three isoforms of highly conserved NOS enzymes have
been identified in mammals: neuronal NOS (nNOS
or NOS-I), inducible NOS (iNOS or NOS-II), and
endothelial NOS (eNOS or NOS-III) NOS enzymes
contain an N-terminal oxygenase domain and a
C-terminal reductase domain connected by a CaM-binding hinge region NOS enzymes are also found in specific species of fish, invertebrates, protozoa and fungi [10–12] Even bacteria contain genes coding for truncated NOS proteins with homology to the oxygen-ase domain of mammalian NOS, and these enzymes have nitration or NO synthesis activity [13–16]
Despite the high degree of conservation found among NOS enzymes, no protein with significant sequence similarity has been identified in plants, including Arabidopsis [17] and rice [18], the genomes of which have been sequenced Plants can produce and release significant amounts of NO, especially under hypoxic conditions or during infection [2,3,19–27] One source of NO is nitrite, which can be converted to NO
Keywords
Arabidopsis; argininosuccinate lyase;
citrulline; nitric oxide
Correspondence
N M Crawford, Section of Cell and
Developmental Biology, Division of
Biological Sciences, University of California
at San Diego, La Jolla, CA 92093-0116, USA
Fax ⁄ Tel: +1 858 534 1637
E-mail: ncrawford@ucsd.edu
*These authors contributed equally to this
work
(Received 23 February 2007, revised
24 May 2007, accepted 20 June 2007)
doi:10.1111/j.1742-4658.2007.05950.x
There are many reports of an arginine-dependent nitric oxide synthase activity in plants; however, the gene(s) or protein(s) responsible for this activity have yet to be convincingly identified To measure nitric oxide syn-thase activity, many studies have relied on a citrulline-based assay that measures the formation of l-citrulline from l-arginine using ion exchange chromatography In this article, we report that when such assays are used with protein extracts from Arabidopsis, an arginine-dependent activity was observed, but it produced a product other than citrulline TLC analysis identified the product as argininosuccinate The reaction was stimulated by fumarate (> 500 lm), implicating the urea cycle enzyme argininosuccinate lyase (EC 4.3.2.1), which reversibly converts arginine and fumarate to argi-ninosuccinate These results indicate that caution is needed when using standard citrulline-based assays to measure nitric oxide synthase activity in plant extracts, and highlight the importance of verifying the identity of the product as citrulline
Abbreviations
ADF, Arabidopsis-derived factor; ASL, argininosuccinate lyase; BH 4 , tetrahydrobiopterin; CaM, calmodulin; NO, nitric oxide; NOS, nitric oxide synthase.
Trang 2by: (a) plant nitrate reductase [22,28–30]; (b)
mito-chondria [31–33]; and (c) nonenzymatic processes
[34,35] There is also ample evidence from biochemical
and pharmacological data that an arginine-dependent
mechanism analogous to animal NOS reactions exists
in plants [26,36–43]; however, the identity of the
argi-nine-dependent activity in plants has yet to be
conclu-sively determined
Some of the evidence supporting an
arginine-depen-dent mechanism in plants comes from commercially
available ‘NOS assay kits’ (citrulline-based assays) that
measure the conversion of arginine to citrulline using
ion exchange chromatography [44] Radiolabeled
argi-nine is provided as a substrate, and is then separated
from reaction products by cation exchange
chromato-graphy Positively charged arginine binds the ion
exchange resin but citrulline does not The unbound
fraction, which is generally assumed to be citrulline, is
measured in a scintillation counter Examples of the
use of this assay include the analysis of NOS activity
in aluminum-treated Hibiscus [45], in pea peroxisomes
[38], and in elicitor-treated Hypericum cells [46]
Although the assay is quick and sensitive, it does not
identify the product as citrulline; any arginine
deriva-tive that does not bind to the cation exchange resin
will give a signal The discovery of a product from a
typical NOS reaction that is not citrulline was reported
in a mammalian system [47]
There have been several attempts to identify the
source responsible for arginine-dependent NOS activity
in plants The most recent attempt, which identified
the gene AtNOS1 [42], has subsequently been
chal-lenged [48–50], leading to the proposal that the gene
be renamed AtNOA1 for nitric oxide-associated [48]
Thus, a renewed effort was made to determine the
source of arginine-dependent NOS activity in plants,
using crude protein extracts from Arabidopsis leaves
By employing the citrulline-based NOS assay, an
argi-nine-dependent activity was discovered that was
strongly stimulated by an extract of low molecular
weight compounds from Arabidopsis leaves and
pro-duced argininosuccinate rather than citrulline These
results identify a reaction that is catalyzed by an
activ-ity unrelated to NOS and that can interfere with or
mask authentic NOS activity
Results and Discussion
As a first approach to search for NOS activity in
Arabid-opsis, the citrulline-based NOS assay was used to test
extracts from Arabidopsis leaves Crude protein extracts
(supernatant from a 2· 104gcentrifugation) were
incu-bated with [14C]arginine, NADPH and mammalian
NOS cofactors (BH4, FMN, FAD, Ca2+and CaM) At the end of the reaction, unreacted arginine was removed from the assay mixture with a cation exchange resin Radioactive material that did not bind the resin, pre-sumably citrulline, was measured by scintillation count-ing The signal obtained from a complete reaction (Fig 1, lane 1) was up to 20 times higher than that from the control, which was a complete reaction terminated immediately after the addition of radiolabeled arginine
To determine potential cofactor requirements for the observed activity, leaf extracts were desalted using G-25 Sephadex to remove low molecular weight compounds The low molecular weight compounds retained by the G-25 column were also collected by further elution of the column as described in Experimental procedures Desalted protein extracts alone had greatly reduced levels of activity (Fig 1, lane 2), indicating that a low molecular weight compound(s) from the extract was necessary for activity Adding back the low molecular weight fraction from the G-25 column to the desalted protein extract restored activity (Fig 1, lane 3) We named the low molecular weight fraction ADF, for
0 2000 4000 6000 8000 10000 12000 14000 16000 18000
-1 h
Fig 1 Detection of arginine-dependent activity in Arabidopsis extracts Reactions measured the conversion of [14C]arginine to a product that did not bind a cation exchange resin The data are pre-sented as delta c.p.m.Æmg)1proteinÆh)1, which refers to the c.p.m value of the test reaction minus the c.p.m value from the control reaction (reaction terminated immediately after the addition of [ 14 C]arginine) The average c.p.m for the control reaction was approximately 1800 Reactions were performed using the com-plete, initial buffer containing NOS cofactors as described in Experi-mental procedures Reactions also contained the following components: lane 1, crude protein extract from Arabidopsis leaves; lane 2, desalted protein extract; lane 3, desalted protein extract plus low molecular weight fraction (ADF); lane 4, same as lane 3 except that the desalted protein extract was boiled before the assay; lane 5, same as lane 3 except that the ADF was boiled before the assay Data are averages from 10 reactions; error bars indicate SDs.
Trang 3Arabidopsis-derived factor The stimulation of activity
by ADF was positively correlated with the amount of
ADF added (Fig 2) Boiled protein extract showed very
little activity in the presence of ADF (Fig 1, lane 4),
whereas boiled ADF (Fig 1, lane 5) stimulated
activ-ity as much as untreated ADF (Fig 1, lane 3) when
added to the desalted extract, indicating that ADF is
heat stable
These results suggested that an arginine-dependent
activity was present in protein extracts of Arabidopsis
leaves, and that a low molecular weight molecule(s)
was required for this activity To determine whether
this activity was similar to that of mammalian NOS
enzymes, two experiments were performed First,
cofactors essential for NOS activity (BH4, FMN,
FAD, Ca2+ and CaM) were omitted from the
reac-tion Robust activity was still observed for crude
pro-tein extract and desalted propro-tein extract to which ADF
was added (Fig 3A, lanes 1–4) For these reactions,
partially purified ADF preparations were used
(purifi-cation involved boiling leaf extracts and then passing
them through two gel filtration columns and an anion
exchange column, as described in Experimental
proce-dures) We performed an additional experiment to test
for flavin-dependent activity, using diphenylene
iodoni-um (an inhibitor of flavoproteins including animal
NOS), and found no inhibition of the activity at
con-centrations of diphenylene iodonium up to 10 lm (data
not shown) Second, the products of the reaction were
analyzed by one-dimensional TLC followed by
radiography No citrulline was detected on the
auto-radiograms; instead, an unidentified compound was
observed as the major reaction product (Fig 3B) Together, these results showed that the reaction had
no requirement for known NOS cofactors and did not produce the NOS coproduct citrulline, indicating that
it was not a typical NOS reaction
To identify the unknown compound, the reaction products were analyzed by two-dimensional TLC on silica gel plates 14C-Labeled argininosuccinate was the only radiolabeled product identified (Fig 4) No radio-labeled products comigrating with citrulline, ornithine, urea, valine, hydroxyarginine, agmatine, spermine, spermidine, putrescine or proline were detected (Fig 4 and data not shown)
0
2000
4000
6000
8000
10000
12000
14000
16000
-1 h
µL ADF
Fig 2 Dependence of arginine-dependent reaction on ADF
Desalt-ed protein extracts were incubatDesalt-ed with [ 14 C]arginine and
increas-ing amounts of partially purified ADF, and then assayed for activity
as described in Fig 1 Data points are averages from five
repli-cates; error bars indicate SDs.
A
B
Fig 3 The arginine-derived reaction product is not citrulline Crude protein extracts (lanes 1 and 2), desalted protein extracts (lanes 3 and 4), boiled extracts (lanes 5 and 6) and no extract (lane 7) were assayed with [14C]arginine and 50 m M NaPO 4 (i.e with no NOS co-factors) in 50 lL as described in Experimental procedures Partially purified ADF (37 lg) was included in lanes 2, 4 and 6 Reaction products (radioactive material that did not bind the cation exchange column) were analyzed by scintillation counting (A) and TLC (B) The TLC plate was developed with acetonitrile ⁄ ammonium hydrox-ide ⁄ water (4 : 1 : 1) and then autoradiographed.
Trang 4Argininosuccinate is the immediate precursor to arginine in the urea cycle, and is converted to arginine and fumarate by argininosuccinate lyase (ASL;
EC 4.3.2.1; Fig 5) Argininosuccinate is normally made from citrulline and aspartate by argininosuccin-ate synthetase, but it can also be produced by ASL in
a reverse reaction ASL is found in plants, animals and bacteria, and requires no external cofactors or metal ions for catalytic activity [51] The forward reaction (argininosuccinate to arginine and fumarate) is favored; reported Km values for argininosuccinate range from 0.13 mm in jack bean [52] to 0.2 mm in human liver [53], whereas the reported Km values for the reverse reaction are 5.3 mm for fumarate and 3.0 mm for arginine [53]
If argininosuccinate synthesis is being catalyzed by ASL in the Arabidopsis protein extracts, then fumarate would be needed as a cosubstrate, and fumarate would
be the active component in the ADF preparation Therefore, partially purified ADF was treated with fumarase, which converts fumarate to malate After fumarase treatment, ADF no longer enhanced the pro-duction of argininosuccinate (Table 1) Next, fumarate was tested as a replacement for ADF in the reactions Desalted protein extracts from Arabidopsis were incu-bated with either ADF or fumarate; both reactions produced the same product, which comigrated with argininosuccinate by TLC analysis (Fig 6) When maleic acid (the cis-isomer of fumarate) was used
Origin 1-D
2-D
Ornithine
Argininosuccinate
Hydroxyarginine Citrulline
Fig 4 Two-dimensional TLC analysis of reaction product A
reaction with [ 14 C]arginine, desalted protein extract and ADF was
performed as described in Fig 1, and then treated with cation
exchange resin A portion of the unbound material (5 lL out of a
total of 100 lL) was spotted together with unlabeled markers onto
a silica TLC plate The TLC plate was developed with two solvent
systems as follows: first dimension, n-butanol ⁄ methanol ⁄
ammo-nium hydroxide ⁄ water (33 : 33 : 24 : 10); second dimension,
chloroform ⁄ methanol ⁄ acetic acid (2 : 4 : 4) The plate was then
autoradiographed The markers were visualized by ninhydrin
stain-ing, and then marked as dashed lines on the autoradiogram The
origin was marked with an ink spot.
Fig 5 Urea cycle (A) Schematic diagram of
the urea cycle (B) Structures of the
sub-strates and products for ASL.
Trang 5instead of fumarate, no activity was detected (data not
shown) When the amount of product produced was
measured as a function of fumarate concentration
using desalted Arabidopsis extracts, the data showed a
saturation curve (Fig 7), which yielded a Km
(fuma-rate) of 4.5 mm, similar to what is reported for human
liver [53] The reaction could be strongly inhibited (by
97%) by 0.3 mm argininosuccinate (data not shown),
the substrate for the favored forward reaction Desalted
protein extracts from Escherichia coli were also tested,
and the same argininosuccinate product was produced
with ADF or fumarate (Fig 6)
Our results show that when the citrulline-based
assay is employed, protein extracts from Arabidopsis
catalyze a reaction with arginine that mimics an NOS
reaction This reaction, however, produces
argininosuc-cinate, not citrulline, and requires fumarate, indicating
that ASL is catalyzing the reaction Because
arginino-succinate does not bind the cation exchange column,
the signal from the reaction could be mistaken for
NOS activity Initially, it was puzzling why activity
was obtained in crude Arabidopsis extracts without
added fumarate (ADF); however, several articles have
reported that fumarate levels can be quite high in plants, especially in Arabidopsis, where it is reported to
be one of the most abundant organic acids [54,55] The same activity can also be observed in protein extracts of E coli, but only if fumarate or low mole-cular weight compounds from Arabidopsis leaves are added to the E coli extracts
These results demonstrate the importance of verify-ing the identity of the products in standard citrulline-based NOS assays of plant and, especially, Arabidopsis extracts Until such tests are performed, the results from such assays cannot be used to support the exis-tence of arginine-dependent NOS activity in plants
Experimental procedures
Plant material and protein extractions Leaves from 3-week old Arabidopsis plants (ecotype Columbia) grown under 16 h light conditions were har-vested and ground in liquid N2 with a mortar and pestle Extraction buffer (2.5 mL of 50 mm Hepes, pH 7.4, 1 mm EDTA, 10 mm MgCl2, 1 mm b-mercaptoethanol, 1 mm
Table 1 Fumarase destroys ADF activity [ 14 C]Arginine was incubated with desalted protein extracts from Arabidopsis leaves and partially purified ADF that was untreated or treated with fumarase as indicated Treated ADF (500 lL) was incubated with 50 U of fumarase, and an aliquot was used in the assay after fumarase was inactivated by heat Activity is presented as delta c.p.m.Æmg)1proteinÆh)1with SDs.
Fig 6 Fumarate can replace ADF as a cosubstrate for the reaction.
Desalted protein extracts from Arabidopsis leaves or from E coli
pellets were incubated with [14C]arginine in 50 m M NaPO 4 with or
without partially purified ADF (37 lg) or fumarate (final
concentra-tion of 12.5 m M ) as indicated The reaction products were treated
with cation exchange resin, and unbound material was spotted
onto a silica TLC plate as described The one-dimensional TLC was
developed with acetonitrile ⁄ ammonium hydroxide ⁄ water (4 : 1 : 1)
and then autoradiographed.
Fig 7 The fumarate-dependent reaction follows Michaelis–Menten kinetics Reactions were performed with [14C]arginine (20 l M ), de-salted Arabidopsis protein extract and fumarate as described above The amount of product (shown as delta c.p.m.) was determined as
a function of fumarate concentration The inset shows the double reciprocal plot used to calculate Km.
Trang 64-(2-aminoethyl)-benzolsulfonylfluorid, 1· Roche Protease
Inhibitor cocktail per gram fresh weight) was mixed with
the ground plant material, and samples were centrifuged
(2· 104g) for 10 min at 4C (Beckman J2-HS, rotor
JA-20, Palo Alto, CA, USA) The supernatant (crude
pro-tein extract) was either used directly or further desalted on
a G-25 Sephadex gel filtration column (PD-10 column from
GE Healthcare, Piscataway, NJ, USA), according to the
manufacturer’s instructions Briefly, 2.5 mL protein extract
was applied to a PD10 column of 10 mL bed volume and
then washed with extraction buffer The first 2.5 mL of
elu-ant was discarded, the next 3.5 mL (excluded volume) was
collected (called desalted protein extract), and the next
3.5 mL (included volume) was collected and contained
small molecules Extracts were concentrated in a
Centricon-30 filter device (Millipore, Bedford, MA, USA) at 4C
For E coli protein extracts, cell pellets were resuspended in
lysis buffer (25 mm Hepes, 0.7 mm Na2HPO4, 137 mm
NaCl, 5 mm KCl, pH 7.4), incubated on ice for 20 min
with 1 mgÆmL)1 lysozyme, and sonicated Lysate was
cen-trifuged at 100 000 g for 1 h (Beckman ultracentrifuge L7,
rotor SW51), desalted on a PD-10 column, and
concen-trated with a Centricon-30 filter device Protein
concentra-tions were determined using the Bradford Assay (Biorad,
Hercules, CA, USA)
ADF preparation
Leaf tissue (50 g) from 3-week-old Arabidopsis plants was
boiled for 15 min in 100 mL of water containing 1 mm
b-mercaptoethanol The boiled extract was centrifuged at
2· 104
g at room temperature (Beckman J2-HS, rotor
JA-20), and the supernatant was lyophilized Resuspended
material was used directly or partially purified on a
72 cm· 1.5 cm column containing G-15 Sephadex (Sigma,
St Louis, MO, USA) in water Fractions were assayed for
activation of desalted protein extracts Active fractions were
subsequently pooled and applied to a Q-Separose FF
column (Amersham) equilibrated with 50 mm NaPO4
(pH 7.4) The column was eluted with increasing
concentra-tions of NaCl Active fracconcentra-tions eluted between 0.4 m and
0.5 m NaCl These fractions were pooled, lyophilized, and
separated on the same G-15 Sephadex column as described
previously Fractions were assayed for activation potential,
combined, lyophilized, and resuspended into 100 lL of
water
Enzyme assays and cation exchange
chromatography
Thirty to 150 lg of protein extract (either desalted or
crude) was used for each assay The initial assay buffer
with NOS cofactors contained 1 mm NADPH, 130 lm
BH4, 520 lm FMN, 200 lm FAD, 1 lm CaM, 1 mm
CaCl2, 50 mm Hepes (pH 7.4), and 10 lm [14C]arginine
(Amersham) Assays with desalted extracts were supplied with ADF (1–5 lL) unless indicated otherwise Subse-quently, the initial assay buffer was replaced with 50 mm NaPO4 buffer (pH 7.4) (i.e with no NOS cofactors) and
10 lm [14C]arginine Reactions were incubated at 30C for
1 h, and terminated by boiling or immediately applying the reaction to spin columns (Corning, NY, USA) containing DOWEX 50WX8-400 (Sigma) cation exchange resin DOWEX columns were prepared as previously described [56], and the flow-through was counted in a scintillation counter
TLC Following treatment with the cation exchange resin, 10% of the unbound material was counted in a scintillation counter and the remaining 90% was used for TLC analysis as fol-lows The unbound material was washed with four volumes
of cold acetonitrile and centrifuged for 10 min at 15 000 g (Eppendorf 5415C centrifuge; Brinkmann, Westbury, NY, USA) to precipitate large molecular weight compounds The supernatant was evaporated to dryness in a speedvac, and resuspended in 10% of the original volume with 10% aceto-nitrile in water For one-dimensional TLC, 1 lL was spot-ted on silica gel TLC plates (Whatman #4420221, Clifton,
NJ, USA) and developed with acetonitrile⁄ water ⁄ ammo-nium hydroxide 4 : 1 : 1 For two-dimensional TLC, 4 lL
of this mixture was spotted on silica gel TLC plates (Whatman #4420221) and developed with n-butanol⁄ methanol⁄ ammonium hydroxide ⁄ water (33 : 33 : 24 : 10) in the first dimension After drying, the plates were developed
in the second dimension with chloroform⁄ methanol ⁄ acetic acid (2 : 4 : 4) Standards of known amines and amino acids were run in parallel; they were spotted with the radioactive material and detected by spraying with ninhydrin Radioac-tive arginine derivaRadioac-tives were detected directly on the TLC plates by autoradiography (Hyblot CL, Denville Scientific, Metuchen, NJ, USA)
Acknowledgements
We thank Dr Fujinori Hanawa for his excellent techni-cal advice This work was funded by grant from the National Institutes of Health (GM40672)
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