Właściwości GPOX z korzeni, Publikacje naukowe
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//-->Properties of Guaiacol Peroxidase Activities Isolated fromCorn Root Plasma Membranes1Angela Mika and Sabine Luthje*¨Universitat Hamburg, Institut fur Allgemeine Botanik, Ohnhorststrasse 18, D–22609 Hamburg, Germany¨¨Although several investigations have demonstrated a plasma membrane (PM)-bound peroxidase activity in plants, thisstudy is the first, to our knowledge, to purify and characterize the enzymes responsible. Proteins were extracted from highlyenriched and thoroughly washed PMs. Washing and solubilization procedures indicated that the enzymes were tightlybound to the membrane. At least two distinct peroxidase activities could be separated by cation exchange chromatography(pmPOX1 and pmPOX2). Prosthetic groups were identified in fractions with peroxidase activity by absorption spectra, andthe corresponding protein bands were identified byhemestaining. The activities of the peroxidase enzymes respondeddifferent to various substrates and effectors and had different thermal stabilities and pH and temperature optima. Becausethe enzymes were localized at the PM and were not effected byp-chloromercuribenzoate,they were probably class IIIperoxidases. Additional size exclusion chromatography of pmPOX1 revealed a single activity peak with a molecular massof 70 kD for the native enzyme, whereas pmPOX2 had two activity peaks (155 and 40 kD). Further analysis of these fractionsby a modified sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination withhemestaining confirmed theestimated molecular masses of the size exclusion chromatography.Peroxidases (EC 1.11.1.7, etc.) belong to a largefamily of enzymes that are ubiquitous in fungi,plants, and vertebrates. These proteins usually con-tain a ferriprotoporphyrin IX prosthetic group andoxidize several substrates in the presence of hydro-gen peroxide (H2O2; Penel et al., 1992; Vianello et al.,1997). In higher plants, the number of isoenzymesmay be extremely high, up to 40 genes correspondingto isoperoxidases for each plant, and several otherisoforms can be generated by posttranscriptional andposttranslational modifications (Welinder et al., 1996;De Marco et al., 1999).Although many soluble intracellular and extracel-lular peroxidases have been characterized in detail(for refs., see Gaspar et al., 1982; Hiraga et al., 2001;Shigeoka et al., 2002), less is known about membrane-bound enzymes, in particular the peroxidases ofplant plasma membranes (PMs). Evidence for aPM-bound peroxidase activity in higher plants hasbeen demonstrated frequently. Lin (1982) reported anincreased oxygen consumption by intact corn (Zeamays)root protoplasts in the presence of extracellularNADH. Pantoja and Willmer (1988) obtained similarresults using guard cell protoplasts fromCommelinacommunisin the presence of NAD(P) H. PMs iso-lated from several species and plant parts showedNAD(P) H oxidase activities, which were compara-ble with a peroxidase (Møller and Berczi, 1986;´This work was supported by the Deutsche Forschungsgemein-schaft (grant no. DFG Lu 668/1–2) and by the University of Ham-burg (PhD student’s grant no. HmbNFG to A.M.).* Corresponding author; e-mail s.luthje@botanik.uni-hamburg.de; fax 49 – 40 – 82282–254.Article, publication date, and citation information can be foundat www.plantphysiol.org/cgi/doi/10.1104/pp.103.020396.1Askerlund et al., 1987; Vianello et al., 1990, 1997; DeMarco et al., 1995; Zancani et al., 1995; Sagi andFluhr, 2001). Because the application of detergents didnot significantly affect the activity observed and be-cause activity could be detected with intact proto-plasts, peroxidase activity has been suggested to belocated at the apoplastic surface of the PM.The NADH oxidation by PM from cauliflower(Brassicaoleracea)could be stimulated by phenolicsubstances or inhibited by typical effectors of peroxi-dases like catalase, superoxide dismutase, cyanide, orazide (Askerlund et al., 1987). In PM-enriched frac-tions of Arabidopsis and Chinese cabbage (BrassicacampestrisL. subsp.pekinensis)seedlings, oxidation ofTrp was reported in the presence of H2O2(Ludwig-Muller et al., 1990; Ludwig-Muller and Hilgenberg,¨¨1992). PM isolated from soybean (Glycinemax)rootsshowed a peroxidase activity in the presence of sub-strates likeo-dianisidine,guaiacol, and ascorbate (Vi-anello et al., 1997). The oxidation of ascorbate couldbe strongly stimulated by phenolic acids, like caffeicand ferulic acid. Guaiacol oro-dianisidineoxidationrates were increased by CaCl2and inhibited by po-tassium cyanide and azide. When proteins solubi-lized by SDS from non-washed PM were separatedby SDS-PAGE, two bands (38 and 45 kD) could bedetected byhemestaining. Peroxidase activity ofthese bands was not demonstrated, and only one, lessintensive band remained after partial washing of themembrane vesicles (Vianello et al., 1997).In addition to these experiments, antibodies spe-cific for apoplastic peroxidases were used to detectPM-bound peroxidases by immunogold labeling andelectron microscopy in situ (Hu et al., 1989; Penel andCastillo, 1991; Crevecoeur et al., 1997). However,1489Downloaded from www.plantphysiol.org on March©2015 - Published by www.plant.orgPlant Physiology,July 2003, Vol. 132, pp. 1489–1498, www.plantphysiol.org14,2003 American Society of Plant BiologistsCopyright © 2003 American Society of Plant Biologists. All rights reserved.Mika and Luthje¨Askerlund et al. (1987) demonstrated that the pres-ence of peroxidases in PM preparations dependslargely on the final PM washing procedure, whichdecreases the level of peroxidases significantly. APM-bound peroxidase has not yet been isolated andcharacterized from highly purified and properlywashed PM (Berczi and Møller, 2000).´In the present work, we demonstrate the occur-rence of at least two distinct peroxidase activities(pmPOX1 and 2) in corn root PM. A purificationprotocol for the isolation of these enzymes was de-veloped, and the properties of the partially purifiedproteins were investigated by comparing them withsoluble peroxidase activities.In Arabidopsis, three genes encoding membrane-bound ascorbate peroxidases were found (Jespersenet al., 1997). One of the corresponding proteins isprobably bound to microbodies by a C-terminaltransmembrane domain like membrane-bound intra-cellular peroxidases of other plant species (Bunkel-mann and Trelease, 1996; Nito et al., 2001). However,sequence analysis of these peroxidases revealed thatthey are class I peroxidases, which implies they can-not occur in the PM.PurificationRESULTS AND DISCUSSIONBinding to the PMTo check if peroxidase activities were looselybound to the PM or entrapped inside the vesicles,different washing procedures were carried out. Inde-pendent of the salt concentrations used a maximumof 40% of the activity could be washed off in thepresence of 1 mm EDTA and 0.01% (w/v) TritonX-100, i.e. 79%7.2% (n2) of the activity re-mained in the PM at 150 mm KCl and 60%1.9%(n4) at 500 mm KCl, respectively. Using 1 mmEGTA instead of EDTA did not change this result. Acombination of 150 mm KCl, 1 mm EDTA, 0.01%(w/v) Triton X-100, and 0.1% (w/v) CHAPS (i.e. adetergent:protein ratio of 6:1 [w:w]) removed 62%0.4% (n 2) of the peroxidase activity from the PM.Due to the fact that neither physiological or highsalt concentrations in the presence of detergent andEDTA or EGTA nor high detergent concentrationswere able to remove the activity completely from thePM, we conclude that these enzymes are probablytightly bound to the PM. Salts should have minimaleffects on the micellar size of Triton X-100, whereaseffects on the zwitterionic detergent CHAPS cannotbe excluded. Thus, the presence of higher salt con-centrations could change the critical micellar concen-tration of CHAPS, thereby increasing the proportionof washed off peroxidase activity as a result of partialsolubilization.However, because peroxidase activity remains inthe low detergent phase after Triton X-114 solubili-zation and temperature-induced phase separation(data not shown), the peroxidases were probably notstrongly hydrophobic. Independently of the detergentto protein ratio used, none of the detergents tested(Triton X-114, Triton X-100, CHAPS, or octylglucopy-ranoside) could solubilize the activity completely fromthe PM. The mechanism of the binding to the PM isunknown, but sequence analysis of intracellular per-oxidases indicated that transmembrane domains mayexist in plant peroxidases (Bunkelmann and Trelease,1996; Jespersen et al., 1997; Nito et al., 2001).1490Solubilization by CHAPS yields about 30%1%(n 2) of the activity and increased up to 73% 4%(n5) in the presence of the dipole aminocaproicacid. Two activity peaks (pmPOX1 and pmPOX2)could be separated by cation exchange chromatogra-phy (Fig. 1). Peroxidase activities were eluted at 115and 395 mm KCl. The total activity was divided into59%3% and 41%2% (n4) for pmPOX1 andpmPOX2, respectively. Starting from washed PM(specific activity 401 52 nmol min1mg protein1;n5), a 24.0- and 8.8-fold purification for peakfractions of pmPOX1 and pmPOX2 with an overallyield of 31.4% was achieved. To compare the prop-erties of pmPOX with soluble peroxidases, activitiesof the washing fluid of the PM (wPOX) were concen-trated and separated by the same protocol (Fig. 1). Theelution profile obtained was similar to that from thePM-bound POX. The total activity was divided into30% and 70% for wPOX1 and wPOX2, respectively.Relative Molecular MassAs shown in Figure 2, pmPOX1 displayed a singlepeak after size exclusion chromatography. By modi-Figure 1.Elution profiles of POX after cation exchange chromatog-raphy. Enzyme activities isolated from corn root PM were separatedon a Uno S1 column. Bound proteins were eluted by a KCl gradientfrom 0 to 1M. The flow rate was 1 mL min1, and fractions of 1.0 and0.5 mL were collected. A, Separation of POX activities from washingfluid of PM (wPOX;E).B, Elution profile of PM-bound peroxidaseactivities (pmPOX;F).Enzyme activities were measured in the pres-ence of 8.26 mMguaiacol and 8.8 mMH2O2.Downloaded from www.plantphysiol.org on March 14, 2015 - Published by www.plant.orgPhysiol. Vol. 132, 2003PlantCopyright © 2003 American Society of Plant Biologists. All rights reserved.Plasma Membrane-Bound Peroxidasesthe PM vesicles with NaCl, and several apoplasticperoxidases with these molecular masses were iden-tified in different plant species (Hendriks et al., 1991;Melo et al., 1996; De Marco et al., 1999; Blee et al.,2001). The molecular masses of pmPOX1 andpmPOX2a were different compared with the proteinbands identified in soybean PM. However, this couldbe due to the different material.Prosthetic GroupsFigure 2.Elution profiles of PM-bound POX after size exclusionchromatography. Peak fractions collected from several Uno S runswere combined, concentrated, and applied onto a Superdex 200column. Proteins were separated by a flow rate of 0.5 mL min1. Thefraction size was automatically adjusted between 0.75 and 0.5 mLdepending on increase or decrease inA280(dotted line) Enzymeactivities were measured as described in Figure 1. PM-bound perox-idase activities could be separated into three peaks.fied SDS-PAGE andhemestaining, a protein bandwith an apparent molecular mass of 70 kD could beidentified (Fig. 3). However, pmPOX2 was clearlyseparated into two peaks after size exclusion chro-matography (pmPOX2a and pmPOX2b; Fig. 2). Incomparison with peak fractions eluted during thecation exchange chromatography, analysis ofpmPOX2b showed a significant increase in intensityof a 40-kD band afterhemestaining (Fig. 3). pmPOX2aexhibited a protein band between 100 and 170 kD.Due to the modifications of the SDS-PAGE, these aremolecular masses of whole enzymes, i.e. oligomerswere not separated into subunits.Molecular masses were also calculated by elutionvolumes of the size exclusion purification step incomparison with marker proteins. The native en-zymes revealed apparent molecular masses of 70,155, and 38 kD for pmPOX1, pmPOX2a, andpmPOX2b, respectively, confirming results obtainedby gel electrophoresis and suggesting the presence ofthree distinct peroxidases at the plant PM. On theother hand, the separation of pmPOX2 into two per-oxidase peaks by size exclusion chromatographycould be due to proteins that were not fully solubi-lized and remained as aggregates (i.e. protein deter-gent or protein aggregates). However, the data ob-tained by SDS-PAGE excluded this hypothesis.Known class III peroxidases revealed molecularmasses in a range of 28 to 60 kD (Hiraga et al., 2001)and a 70- or a 155-kD protein have not been describedfor soluble peroxidases from higher plants.In PM isolated from soybean roots, 38- and 45-kDbands were identified by SDS-PAGE andhemestain-ing (Vianello et al., 1997), masses comparable withthat found for pmPOX2b (Fig. 3). However, bothbands decreased in intensity after partial washing ofUV/visible absorption spectra of pmPOX1 andpmPOX2 were almost identical and typical forheme-containing proteins (e.g. Converso and Fernandez,1995; Kvaratskhelia et al., 1997). Both pmPOXs exhib-ited a Soret peak at 416 nm (Fig. 4). In addition tothese, the oxidized enzymes showed - and -absorption bands at 607 and 528 nm, respectively.The Soret peak and the -band shifted to 425 and559 nm when the proteins were reduced by sodiumdithionite. TheA416/A280values, which are a crite-rion of purity andhemecontent, were 0.5 and 0.2 forpmPOX1 and pmPOX2, suggesting that the enzymeswere not purified to homogeneity, which was alsoshown by SDS-PAGE. Thus, the -absorption band at607 nm cannot be definitely ascribed to thehemegroup of the peroxidase. The spectra of both pmPOXsmore closely resemble those of guaiacol rather thanascorbate peroxidases (Chen and Asada, 1989; Con-verso and Fernandez, 1995; Kvaratskhelia et al.,1997). Peroxidase staining of the isolated proteinssuggests a relatively strong binding of thehemeFigure 3.Hemestaining of pmPOX fractions after modified SDS-PAGE. Electrophoresis was performed using a low concentrated SDS-PAGE, i.e. 0.1% (w/v) SDS in all solutions and gels without dithio-threitol or mercaptoethanol. Thus, the oligomers were not separatedinto their subunits.Heme-containingprotein bands were visualizedby their reaction with the peroxidase substrates tetramethylbenzidine(TMB) and H2O2as described in “Materials and Methods.” Left,pmPOX1 (a) and pmPOX2 (b) are shown after cation exchangechromatography. Further purification of these fractions by size ex-clusion is presented on the right: pmPOX1 (c), pmPOX2a (d), andpmPOX2b (e). In addition, f shows pmPOX2b treated with 25 mMdithiothreitol. Bars indicate the corresponding molecular masses.After size exclusion chromatography, the PM-bound enzymes hadapparent molecular masses of 70 and 40 kD for pmPOX1 andpmPOX2b, whereas pmPOX2a exhibited a broad protein band be-tween 100 and 170 kD.1491Downloaded from www.plantphysiol.org on March 14, 2015 - Published by www.plant.orgPlant Physiol. Vol. 132, 2003Copyright © 2003 American Society of Plant Biologists. All rights reserved.Mika and Luthje¨Figure 4.Absorption spectra of partially purified pmPOX1. Samples(1.1 mg protein mL1) containing the native enzyme (dashed line)were measured in 50 mMsodium phosphate buffer (pH 7.0) withbuffer as reference. Ferric enzymes were reduced by the addition ofapproximately 1.5 mMdithionite (straight line). The spectra weremeasured at 50 nm min1.n2 independent preparations showingidentical results. The spectra indicate the presence ofhemegroups asthe prosthetic group.Figure 6.Dependence of the guaiacol peroxidase activity of purifiedpmPOX on temperature (Arrhenius plot). The rates of guaiacol oxi-dation were determined under the standard assay conditions exceptfor temperature. Data presented are average valuesSDofn3experiments.f,pmPOX1;E,pmPOX2.pH Optimum and Kinetic Studiesgroups to the enzymes. Only pmPOX2b could bedetected byhemestaining after treatment with dithio-threitol and revealed the same molecular mass aswithout reducing compounds (Fig. 3). Thus,pmPOX2b was identified as a monomer. pmPOX1and pmPOX2a did not reveal any visible band afterthe same treatment (data not shown). Conforma-tional changes due to the cleavage of disulfidebridges within the molecules possibly resulted in arelease of thehemegroups.The properties of POX, which were separated bycation exchange chromatography, were further char-acterized. As shown in Figure 5, the highest activitywith guaiacol as a substrate was observed betweenpH 4.5 and 5.5 for pmPOX1, whereas pmPOX2 ex-hibited a pH optimum in the range of 5.0 to 6.0. Withguaiacol as substrate, acidic pH optima have oftenbeen reported for the apoplastic peroxidases of sev-eral plant species (Hendriks et al., 1991; Melo et al.,1996; Nair and Showalter, 1996). Variations in pHoptima could represent efficient regulatory means invivo to shift optimal conditions from one isoenzymeto another and thereby favor different processes (DeMarco et al., 1999).Figure 5.Dependence of the guaiacol peroxidase activity of partiallypurified pmPOX on pH. The rates of guaiacol oxidation were deter-mined under the standard assay conditions except that 25 mMso-dium acetate (pH 4.0–5.0), MES (pH 5.5–6.5), or HEPES (pH 7.0–8.0)buffers were used. Data presented are average valuesSDofn3experiments.f,pmPOX1;E,pmPOX2.1492Figure 7.Thermal stability of guaiacol peroxidase activities. Solubleand PM-bound POX were incubated at 50°C at different time slices.Data presented are average valuesSDofn2 experiments.f,pmPOX1;F,pmPOX2; , wPOX1;E,wPOX2.Downloaded from www.plantphysiol.org on March 14, 2015 - Published by www.plant.orgPhysiol. Vol. 132, 2003PlantCopyright © 2003 American Society of Plant Biologists. All rights reserved.Plasma Membrane-Bound PeroxidasesTable I.Guaiacol-dependent activity in the absence or presence of typical peroxidase effectorsPeroxidase activity was measured with the partially purified enzymes after cation exchange chromatography in the presence of 8.26 mMguaiacol and 8.8 mMH2O2at pH 7.0 as described in “Materials and Methods.” Data are given as meanSD(n).SubstanceConcentrationPeroxidase ActivitypmPOX1pmPOX2mol min1wPOX1mg protein1wPOX2ControlControlKCNAzidep-Chloromercuribenzoate(pCMB)5.20.1 (3)1.60.1 (3)12.0100.01.20.299.7101.454.50.3 (3)2.3 (3)1.6 (3)0.3 (2)b9.6 (3)2.0 (3)10.9 (3)16.8100.00.67.496.4102.177.20.1 (3)0.3 (3)0.8 (3)0.9 (2)b4.6 (3)8.3 (3)9.1 (3)(% of control)1 mM1 mM50M200M1 mM100.0 1.5 (3)n.d.a(3)10.6 0.4 (3)b112.5 7.1 (3)105.7 2.3 (3)110.2 4.3 (3)100.0 4.9 (3)n.d. (3)2.9 0.6 (3)b111.1 5.7 (3)102.2 5.0 (3)106.9 0.6 (3)an.d., Not detectable.bpH 5.0.TheKms of both PM-bound peroxidase activitiesfor guaiacol were comparable (12.2 mm for pmPOX1and 14.3 mm for pmPOX2, calculated by Eadie-Hofstee plots).Kmvalues in a millimolar range aretypical for peroxidases with artificial substrates likeguaiacol. For instance, soluble peroxidases from kiwi-fruit (Actinidiadeliciosa)and tomato (Lycopersiconesculentum)fruits hadKmvalues of 7.4 and 10 mm,respectively (Soda et al., 1991).Temperature Optima and Thermal StabilityEffector StudiesAt low temperatures the enzyme activity ofpmPOX2 was about 2-fold lower compared withpmPOX1 (Fig. 6). The activity of both protein frac-tions increased with higher temperatures. Althoughthe activity of pmPOX2 more or less continuouslyincreased in the range of 2°C to 51°C, pmPOX1showed a maximum of activity at 43°C and decreaseddramatically afterward.In a second set of experiments, the thermal stabilityof soluble and PM-bound peroxidases was investi-gated (Fig. 7). All enzymes lost between 40% and 50%of their activities within 5 min at 50°C. During anincubation time of 3 h, the guaiacol peroxidase activ-ities decreased exponentially to values between 5.7%and 34.3%. After 3 h, pmPOX1 showed twice theactivity of pmPOX2. Most peroxidases from plantsand animals seemed to have high temperature op-tima and show high thermal stabilities (Bakardjievaet al., 1996; Madhavan and Naidu, 2000). Apoplastic,cytosolic, and soluble peroxidases of several planttissues showed temperature optima between 30°Cand 60°C, the most between 50°C and 60°C (Soda etal., 1991; Bakardjieva et al., 1996; Nair and Showalter,1996; Bernards et al., 1999; Loukili et al., 1999). Due tothe fact that pmPOX1 had a lower temperature opti-mum than pmPOX2, the latter enzyme seemed to bemore stable (Fig. 6). However, for longer treatmentsof higher temperatures, pmPOX1 revealed a higherthermal stability (Fig. 7).As shown in Table I, classical peroxidase inhibitorslike potassium cyanide or sodium azide caused acomplete loss of the peroxidase activities or de-creased the rates more than 90%. These results wereconsistent with the presence ofhemegroups as pros-thetic groups.The localization of the enzymes at the plant PMsuggests that they may be part of the secretory path-way. According to Welinder et al. (1996), pCMB, asulfhydryl inhibitor, is often used to distinguish be-tween class I and class III peroxidases. As shown inTable I, this inhibitor did not effect the PM-boundactivities of pmPOX1 or pmPOX2, indicating that SHgroups did not participate in the active center ormaintenance of the conformation of the isoenzymes.Thus, the PM-bound peroxidases were probablyclass III peroxidases. In contrast to the pmPOX,wPOX1 and wPOX2 were slightly inhibited in thepresence of 1 mm pCMB.Both PM-bound peroxidase activities were de-creased by distinct concentrations of the lectins con-canavalin A (Con A) and wheat germ agglutinin(WGA; Table II), whereas the Ulex europaeus agglu-tinin (UEA1) was without significant effect (data notshown). Inhibition of wPOX1 and wPOX2 was weakand occurred only at higher concentrations of Con Aand WGA (Table II). The effects of lectins indicateglycosylation of the enzymes. These results are con-sistent with the finding of Vianello et al. (1997) thattreatment of soybean roots with tunicamycin, an in-hibitor of glycoprotein synthesis, reduced the guaia-col peroxidase activity of unwashed PM vesicles by40%. Due to the possible glycosylation, which wasalso indicated by diffuse protein bands in SDS gels(Fig. 3), the real molecular masses of all identifiedproteins may be different from the calculated values.However, the structures of the proteins have to befurther elucidated.1493Downloaded from www.plantphysiol.org on March 14, 2015 - Published by www.plant.orgPlant Physiol. Vol. 132, 2003Copyright © 2003 American Society of Plant Biologists. All rights reserved. [ Pobierz całość w formacie PDF ] |