Báo cáo khoa học: Interference with the citrulline-based nitric oxide synthase assay by argininosuccinate lyase activity in Arabidopsis extracts docx

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Interference with the citrulline-based nitric oxide synthaseassay by argininosuccinate lyase activity in ArabidopsisextractsRudolf Tischner1,*, Mary Galli2,*, Yair M. Heimer3,*, Sarah Bielefeld1, Mamoru Okamoto2,Alyson Mack2and Nigel M. Crawford21 Albrecht von Haller Institut fur Pflanzenwissenschaften, University of Gottingen, Germany2 Section of Cell and Developmental Biology, Division of Biological Sciences, University of California at San Diego, La Jolla, CA, USA3 Department of Dryland Biotechnologies, J. Blaustein Institute for Desert Research, Ben-Gurion University, Sede-Boker, IsraelNitric oxide (NO) serves as a central signal in a widevariety of processes, including vasodilation, neuralcommunication and immune function in animals [1],and defense responses, hormonal signaling and flower-ing in plants [2–6]. The primary mechanism for NOsynthesis in animals involves oxidation of l-arginine tol-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 havebeen identified in mammals: neuronal NOS (nNOSor NOS-I), inducible NOS (iNOS or NOS-II), andendothelial NOS (eNOS or NOS-III). NOS enzymescontain an N-terminal oxygenase domain and aC-terminal reductase domain connected by a CaM-binding hinge region. NOS enzymes are also found inspecific species of fish, invertebrates, protozoa andfungi [10–12]. Even bacteria contain genes coding fortruncated NOS proteins with homology to the oxygen-ase domain of mammalian NOS, and these enzymeshave nitration or NO synthesis activity [13–16].Despite the high degree of conservation foundamong NOS enzymes, no protein with significantsequence similarity has been identified in plants,including Arabidopsis [17] and rice [18], the genomes ofwhich have been sequenced. Plants can produce andrelease significant amounts of NO, especially underhypoxic conditions or during infection [2,3,19–27]. Onesource of NO is nitrite, which can be converted to NOKeywordsArabidopsis; argininosuccinate lyase;citrulline; nitric oxideCorrespondenceN. M. Crawford, Section of Cell andDevelopmental Biology, Division ofBiological Sciences, University of Californiaat San Diego, La Jolla, CA 92093-0116, USAFax ⁄ Tel: +1 858 534 1637E-mail: ncrawford@ucsd.edu*These authors contributed equally to thiswork(Received 23 February 2007, revised24 May 2007, accepted 20 June 2007)doi:10.1111/j.1742-4658.2007.05950.xThere are many reports of an arginine-dependent nitric oxide synthaseactivity in plants; however, the gene(s) or protein(s) responsible for thisactivity have yet to be convincingly identified. To measure nitric oxide syn-thase activity, many studies have relied on a citrulline-based assay thatmeasures the formation of l-citrulline from l-arginine using ion exchangechromatography. In this article, we report that when such assays are usedwith protein extracts from Arabidopsis, an arginine-dependent activity wasobserved, but it produced a product other than citrulline. TLC analysisidentified the product as argininosuccinate. The reaction was stimulated byfumarate (> 500 lm), implicating the urea cycle enzyme argininosuccinatelyase (EC 4.3.2.1), which reversibly converts arginine and fumarate to argi-ninosuccinate. These results indicate that caution is needed when usingstandard citrulline-based assays to measure nitric oxide synthase activity inplant extracts, and highlight the importance of verifying the identity of theproduct as citrulline.AbbreviationsADF, Arabidopsis-derived factor; ASL, argininosuccinate lyase; BH4, tetrahydrobiopterin; CaM, calmodulin; NO, nitric oxide; NOS, nitric oxidesynthase.4238 FEBS Journal 274 (2007) 4238–4245 ª 2007 The Authors Journal compilation ª 2007 FEBSby: (a) plant nitrate reductase [22,28–30]; (b) mito-chondria [31–33]; and (c) nonenzymatic processes[34,35]. There is also ample evidence from biochemicaland pharmacological data that an arginine-dependentmechanism analogous to animal NOS reactions existsin 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 commerciallyavailable ‘NOS assay kits’ (citrulline-based assays) thatmeasure the conversion of arginine to citrulline usingion exchange chromatography [44]. Radiolabeled argi-nine is provided as a substrate, and is then separatedfrom reaction products by cation exchange chromato-graphy. Positively charged arginine binds the ionexchange resin but citrulline does not. The unboundfraction, which is generally assumed to be citrulline, ismeasured in a scintillation counter. Examples of theuse of this assay include the analysis of NOS activityin aluminum-treated Hibiscus [45], in pea peroxisomes[38], and in elicitor-treated Hypericum cells [46].Although the assay is quick and sensitive, it does notidentify the product as citrulline; any arginine deriva-tive that does not bind to the cation exchange resinwill give a signal. The discovery of a product from atypical NOS reaction that is not citrulline was reportedin a mammalian system [47].There have been several attempts to identify thesource responsible for arginine-dependent NOS activityin plants. The most recent attempt, which identifiedthe gene AtNOS1 [42], has subsequently been chal-lenged [48–50], leading to the proposal that the genebe renamed AtNOA1 for nitric oxide-associated [48].Thus, a renewed effort was made to determine thesource 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 wasstrongly stimulated by an extract of low molecularweight compounds from Arabidopsis leaves and pro-duced argininosuccinate rather than citrulline. Theseresults identify a reaction that is catalyzed by an activ-ity unrelated to NOS and that can interfere with ormask authentic NOS activity.Results and DiscussionAs a first approach to search for NOS activity in Arabid-opsis, the citrulline-based NOS assay was used to testextracts from Arabidopsis leaves. Crude protein extracts(supernatant from a 2 · 104g centrifugation) were incu-bated with [14C]arginine, NADPH and mammalianNOS cofactors (BH4, FMN, FAD, Ca2+and CaM). Atthe end of the reaction, unreacted arginine was removedfrom 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 fromthe control, which was a complete reaction terminatedimmediately after the addition of radiolabeled arginine.To determine potential cofactor requirements for theobserved activity, leaf extracts were desalted using G-25Sephadex to remove low molecular weight compounds.The low molecular weight compounds retained by theG-25 column were also collected by further elution ofthe column as described in Experimental procedures.Desalted protein extracts alone had greatly reducedlevels of activity (Fig. 1, lane 2), indicating that a lowmolecular weight compound(s) from the extract wasnecessary for activity. Adding back the low molecularweight fraction from the G-25 column to the desaltedprotein extract restored activity (Fig. 1, lane 3). Wenamed the low molecular weight fraction ADF, for020004000600080001000012000140001600018000delta cpm mg protein-1 h-112345Fig. 1. Detection of arginine-dependent activity in Arabidopsisextracts. Reactions measured the conversion of [14C]arginine to aproduct 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 controlreaction (reaction terminated immediately after the addition of[14C]arginine). The average c.p.m. for the control reaction wasapproximately 1800. Reactions were performed using the com-plete, initial buffer containing NOS cofactors as described in Experi-mental procedures. Reactions also contained the followingcomponents: lane 1, crude protein extract from Arabidopsis leaves;lane 2, desalted protein extract; lane 3, desalted protein extractplus low molecular weight fraction (ADF); lane 4, same as lane 3except that the desalted protein extract was boiled before theassay; lane 5, same as lane 3 except that the ADF was boiledbefore the assay. Data are averages from 10 reactions; error barsindicate SDs.R. Tischner et al. Argininosuccinate lyase and NOS assayFEBS Journal 274 (2007) 4238–4245 ª 2007 The Authors Journal compilation ª 2007 FEBS 4239Arabidopsis-derived factor. The stimulation of activityby ADF was positively correlated with the amount ofADF added (Fig. 2). Boiled protein extract showed verylittle 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) whenadded to the desalted extract, indicating that ADF isheat stable.These results suggested that an arginine-dependentactivity was present in protein extracts of Arabidopsisleaves, and that a low molecular weight molecule(s)was required for this activity. To determine whetherthis activity was similar to that of mammalian NOSenzymes, 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 protein extract to which ADFwas added (Fig. 3A, lanes 1–4). For these reactions,partially purified ADF preparations were used (purifi-cation involved boiling leaf extracts and then passingthem through two gel filtration columns and an anionexchange column, as described in Experimental proce-dures). We performed an additional experiment to testfor flavin-dependent activity, using diphenylene iodoni-um (an inhibitor of flavoproteins including animalNOS), and found no inhibition of the activity at con-centrations of diphenylene iodonium up to 10 lm (datanot shown). Second, the products of the reaction wereanalyzed by one-dimensional TLC followed by auto-radiography. No citrulline was detected on the auto-radiograms; instead, an unidentified compound wasobserved as the major reaction product (Fig. 3B).Together, these results showed that the reaction hadno requirement for known NOS cofactors and did notproduce the NOS coproduct citrulline, indicating thatit was not a typical NOS reaction.To identify the unknown compound, the reactionproducts were analyzed by two-dimensional TLC onsilica gel plates.14C-Labeled argininosuccinate was theonly 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. 4and data not shown).0 5 10 15 200200040006000800010000120001400016000delta cpm mg protein-1 h-1µL ADFFig. 2. Dependence of arginine-dependent reaction on ADF. Desalt-ed protein extracts were incubated with [14C]arginine and increas-ing amounts of partially purified ADF, and then assayed for activityas described in Fig. 1. Data points are averages from five repli-cates; error bars indicate SDs.ABFig. 3. The arginine-derived reaction product is not citrulline. Crudeprotein extracts (lanes 1 and 2), desalted protein extracts (lanes 3and 4), boiled extracts (lanes 5 and 6) and no extract (lane 7) wereassayed with [14C]arginine and 50 mM NaPO4(i.e. with no NOS co-factors) in 50 lL as described in Experimental procedures. Partiallypurified ADF (37 lg) was included in lanes 2, 4 and 6. Reactionproducts (radioactive material that did not bind the cation exchangecolumn) 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.Argininosuccinate lyase and NOS assay R. Tischner et al.4240 FEBS Journal 274 (2007) 4238–4245 ª 2007 The Authors Journal compilation ª 2007 FEBSArgininosuccinate is the immediate precursor toarginine in the urea cycle, and is converted to arginineand fumarate by argininosuccinate lyase (ASL;EC 4.3.2.1; Fig. 5). Argininosuccinate is normallymade from citrulline and aspartate by argininosuccin-ate synthetase, but it can also be produced by ASL ina reverse reaction. ASL is found in plants, animals andbacteria, and requires no external cofactors or metalions for catalytic activity [51]. The forward reaction(argininosuccinate to arginine and fumarate) isfavored; reported Kmvalues for argininosuccinaterange from 0.13 mm in jack bean [52] to 0.2 mm inhuman liver [53], whereas the reported Kmvalues forthe reverse reaction are 5.3 mm for fumarate and3.0 mm for arginine [53].If argininosuccinate synthesis is being catalyzed byASL in the Arabidopsis protein extracts, then fumaratewould be needed as a cosubstrate, and fumarate wouldbe the active component in the ADF preparation.Therefore, partially purified ADF was treated withfumarase, which converts fumarate to malate. Afterfumarase treatment, ADF no longer enhanced the pro-duction of argininosuccinate (Table 1). Next, fumaratewas tested as a replacement for ADF in the reactions.Desalted protein extracts from Arabidopsis were incu-bated with either ADF or fumarate; both reactionsproduced the same product, which comigrated withargininosuccinate by TLC analysis (Fig. 6). Whenmaleic acid (the cis-isomer of fumarate) was usedOrigin1-D2-DOrnithineArgininosuccinateHydroxyarginineCitrullineFig. 4. Two-dimensional TLC analysis of reaction product. Areaction with [14C]arginine, desalted protein extract and ADF wasperformed as described in Fig. 1, and then treated with cationexchange resin. A portion of the unbound material (5 lL out of atotal of 100 lL) was spotted together with unlabeled markers ontoa silica TLC plate. The TLC plate was developed with two solventsystems 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 thenautoradiographed. The markers were visualized by ninhydrin stain-ing, and then marked as dashed lines on the autoradiogram. Theorigin was marked with an ink spot.Fig. 5. Urea cycle. (A) Schematic diagram ofthe urea cycle. (B) Structures of the sub-strates and products for ASL.R. Tischner et al. Argininosuccinate lyase and NOS assayFEBS Journal 274 (2007) 4238–4245 ª 2007 The Authors Journal compilation ª 2007 FEBS 4241instead of fumarate, no activity was detected (data notshown). When the amount of product produced wasmeasured as a function of fumarate concentrationusing desalted Arabidopsis extracts, the data showed asaturation curve (Fig. 7), which yielded a Km(fuma-rate) of 4.5 mm, similar to what is reported for humanliver [53]. The reaction could be strongly inhibited (by97%) by 0.3 mm argininosuccinate (data not shown),the substrate for the favored forward reaction. Desaltedprotein extracts from Escherichia coli were also tested,and the same argininosuccinate product was producedwith ADF or fumarate (Fig. 6).Our results show that when the citrulline-basedassay is employed, protein extracts from Arabidopsiscatalyze a reaction with arginine that mimics an NOSreaction. This reaction, however, produces argininosuc-cinate, not citrulline, and requires fumarate, indicatingthat ASL is catalyzing the reaction. Because arginino-succinate does not bind the cation exchange column,the signal from the reaction could be mistaken forNOS activity. Initially, it was puzzling why activitywas obtained in crude Arabidopsis extracts withoutadded fumarate (ADF); however, several articles havereported that fumarate levels can be quite high inplants, especially in Arabidopsis, where it is reported tobe one of the most abundant organic acids [54,55].The same activity can also be observed in proteinextracts of E. coli, but only if fumarate or low mole-cular weight compounds from Arabidopsis leaves areadded 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, Arabidopsisextracts. Until such tests are performed, the resultsfrom such assays cannot be used to support the exis-tence of arginine-dependent NOS activity in plants.Experimental proceduresPlant material and protein extractionsLeaves from 3-week old Arabidopsis plants (ecotypeColumbia) grown under 16 h light conditions were har-vested and ground in liquid N2with a mortar and pestle.Extraction buffer (2.5 mL of 50 mm Hepes, pH 7.4, 1 mmEDTA, 10 mm MgCl2,1mm b-mercaptoethanol, 1 mmTable 1. Fumarase destroys ADF activity. [14C]Arginine was incubated with desalted protein extracts from Arabidopsis leaves and partiallypurified ADF that was untreated or treated with fumarase as indicated. Treated ADF (500 lL) was incubated with 50 U of fumarase, and analiquot was used in the assay after fumarase was inactivated by heat. Activity is presented as delta c.p.m.Æmg)1proteinÆh)1with SDs.Treatment Crude extract Desalted extract Desalted extract + ADF Desalted extract + treated ADFActivity 16 354 ± 1267 1762 ± 119 16 085 ± 1440 2583 ± 183Fig. 6. Fumarate can replace ADF as a cosubstrate for the reaction.Desalted protein extracts from Arabidopsis leaves or from E. colipellets were incubated with [14C]arginine in 50 m M NaPO4with orwithout partially purified ADF (37 lg) or fumarate (final concentra-tion of 12.5 mM) as indicated. The reaction products were treatedwith cation exchange resin, and unbound material was spottedonto a silica TLC plate as described. The one-dimensional TLC wasdeveloped with acetonitrile ⁄ ammonium hydroxide ⁄ water (4 : 1 : 1)and then autoradiographed.Fig. 7. The fumarate-dependent reaction follows Michaelis–Mentenkinetics. Reactions were performed with [14C]arginine (20 lM), de-salted Arabidopsis protein extract and fumarate as described above.The amount of product (shown as delta c.p.m.) was determined asa function of fumarate concentration. The inset shows the doublereciprocal plot used to calculate Km.Argininosuccinate lyase and NOS assay R. Tischner et al.4242 FEBS Journal 274 (2007) 4238–4245 ª 2007 The Authors Journal compilation ª 2007 FEBS4-(2-aminoethyl)-benzolsulfonylfluorid, 1 · Roche ProteaseInhibitor cocktail per gram fresh weight) was mixed withthe ground plant material, and samples were centrifuged(2 · 104g) for 10 min at 4 °C (Beckman J2-HS, rotorJA-20, Palo Alto, CA, USA). The supernatant (crude pro-tein extract) was either used directly or further desalted ona G-25 Sephadex gel filtration column (PD-10 column fromGE Healthcare, Piscataway, NJ, USA), according to themanufacturer’s instructions. Briefly, 2.5 mL protein extractwas applied to a PD10 column of 10 mL bed volume andthen washed with extraction buffer. The first 2.5 mL of elu-ant was discarded, the next 3.5 mL (excluded volume) wascollected (called desalted protein extract), and the next3.5 mL (included volume) was collected and containedsmall molecules. Extracts were concentrated in a Centricon-30 filter device (Millipore, Bedford, MA, USA) at 4 °C.For E. coli protein extracts, cell pellets were resuspended inlysis buffer (25 mm Hepes, 0.7 mm Na2HPO4, 137 m mNaCl, 5 mm KCl, pH 7.4), incubated on ice for 20 minwith 1 mgÆmL)1lysozyme, 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 preparationLeaf tissue (50 g) from 3-week-old Arabidopsis plants wasboiled for 15 min in 100 mL of water containing 1 mmb-mercaptoethanol. The boiled extract was centrifuged at2 · 104g at room temperature (Beckman J2-HS, rotorJA-20), and the supernatant was lyophilized. Resuspendedmaterial was used directly or partially purified on a72 cm · 1.5 cm column containing G-15 Sephadex (Sigma,St Louis, MO, USA) in water. Fractions were assayed foractivation of desalted protein extracts. Active fractions weresubsequently pooled and applied to a Q-Separose FFcolumn (Amersham) equilibrated with 50 mm NaPO4(pH 7.4). The column was eluted with increasing concentra-tions of NaCl. Active fractions eluted between 0.4 m and0.5 m NaCl. These fractions were pooled, lyophilized, andseparated on the same G-15 Sephadex column as describedpreviously. Fractions were assayed for activation potential,combined, lyophilized, and resuspended into 100 lLofwater.Enzyme assays and cation exchangechromatographyThirty to 150 lg of protein extract (either desalted orcrude) was used for each assay. The initial assay bufferwith NOS cofactors contained 1 mm NADPH, 130 lmBH4, 520 lm FMN, 200 lm FAD, 1 lm CaM, 1 mmCaCl2,50mm Hepes (pH 7.4), and 10 lm [14C]arginine(Amersham). Assays with desalted extracts were suppliedwith ADF (1–5 lL) unless indicated otherwise. Subse-quently, the initial assay buffer was replaced with 50 mmNaPO4buffer (pH 7.4) (i.e. with no NOS cofactors) and10 lm [14C]arginine. Reactions were incubated at 30 °C for1 h, and terminated by boiling or immediately applying thereaction to spin columns (Corning, NY, USA) containingDOWEX 50WX8-400 (Sigma) cation exchange resin.DOWEX columns were prepared as previously described[56], and the flow-through was counted in a scintillationcounter.TLCFollowing treatment with the cation exchange resin, 10% ofthe unbound material was counted in a scintillation counterand the remaining 90% was used for TLC analysis as fol-lows. The unbound material was washed with four volumesof cold acetonitrile and centrifuged for 10 min at 15 000 g(Eppendorf 5415C centrifuge; Brinkmann, Westbury, NY,USA) to precipitate large molecular weight compounds. Thesupernatant was evaporated to dryness in a speedvac, andresuspended 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 lLof this mixture was spotted on silica gel TLC plates(Whatman #4420221) and developed with n-butanol ⁄methanol ⁄ ammonium hydroxide ⁄ water (33 : 33 : 24 : 10) inthe first dimension. After drying, the plates were developedin the second dimension with chloroform ⁄ methanol ⁄ aceticacid (2 : 4 : 4). Standards of known amines and amino acidswere run in parallel; they were spotted with the radioactivematerial and detected by spraying with ninhydrin. Radioac-tive arginine derivatives were detected directly on the TLCplates by autoradiography (Hyblot CL, Denville Scientific,Metuchen, NJ, USA).AcknowledgementsWe thank Dr Fujinori Hanawa for his excellent techni-cal advice. 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