Purification, Characterization, and Immunological

Plant Physiol. (1990) 94, 1402-14090032-0889/90/94/1 402/08/$01 .00/0

Received for publication April 18, 1990Accepted July 13, 1990

Purification, Characterization, and ImmunologicalProperties for Two Isoforms of Glutathione Reductase from

Eastern White Pine Needles1

James V. Anderson, John L. Hess*, and Boris 1. ChevoneDepartment of Plant Pathology, Physiology and Weed Science (J.V.A., B.l.C.) and Department of Biochemistry and

Nutrition (J.L.H.), Virginia Polytechnic Institute and State University Blacksburg, Virginia 24061

ABSTRACT

Glutathione reductase (EC 1.6.4.2) was purified from Easternwhite pine (Pinus strobus L.) needles. The purification stepsincluded affinity chromatography using 2', 5'-ADP-Sepharose,FPLC-anion-exchange, FPLC-hydrophobic interaction, and FPLC-gel filtration. Separation of proteins by FPLC-anion-exchangeresulted in the recovery of two distinct isoforms of glutathionereductase (GRA and GRB). Purified GRA had a specific activity of1.81 microkatals per milligram of protein and GRB had a specificactivity of 6.08 microkatals per milligram of protein. GRA ac-counted for 17% of the total units of glutathione reductase re-covered after anion-exchange separation and GRB accounted for83%. The native molecular mass for GRA was 103 to 104 kilodal-tons and for GRB was 88 to 95 kilodaltons. Both isoforms ofglutathione reductase were dimers composed of identical subunitmolecular masses which were 53 to 54 kilodaltons for GRA and57 kilodaltons for GR.. The pH optimum for GRA was 7.25 to 7.75and for GR. was 7.25. At 250C the Km for GSSG was 15.3 and39.8 micromolar for GRA and GRB, respectively. For NADPH, theKm was 3.7 and 8.8 micromolar for GRA and GRB, respectively.Antibody produced from purified GR. was reactive with bothnative and denatured GR., but was cross-reactive with only nativeGRA.

Plants have demonstrated altered antioxidant levels whenexposed to air pollutants, low temperature, drought, or xe-nobiotics such as herbicides (1, 28). These plant-stress inter-actions, at one time or another, have been associated withincreased cellular concentrations of superoxide anion radicals(02-s-) and hydrogen peroxide (H202). Superoxide radicals andH202 can directly attack lipid membranes and certain sulfhy-dryl-containing enzymes of the reductive pentose cycle (24),or they can further interact to generate hydroxyl radicals (OH)which also can react with membrane lipids and proteins (2,28). Such alterations to cell metabolism and membrane bio-chemistry would limit normal growth and vigor. Mechanismsfor the scavenging of toxic free radicals have previously beenreviewed in the plant literature (1-3, 17, 28).

' This research was supported by Environmental Protection Agencygrant No. R-814224-01-0 to B. I. C. and J. L. H. This article has notbeen subjected to EPA peer and administrative review and may notnecessarily reflect the views of EPA and no official endorsementshould be inferred.

The univalent reduction of oxygen to 02-'- via the pseudo-cyclic flow of electrons (Mehler reaction) naturally occurs inphotosynthetically active, illuminated chloroplasts. Conse-quently, when the ratio ofNADPH/NADP+ is high, or whencarbon fixation is not optimal, 02, in the presence of thyla-koids, can compete with NADP+ as a Hill reductant (3, 17).C02 fixation can be limited by factors such as short-termexposure to 03 (32) which can result in the inactivation anddecrease of ribulose bisphosphate carboxylase/oxygenase inpotato foliage (8). Also, during water deficits, when stomataclose to limit water loss, the reduction in internal C02 leadsto C02-limited carbon fixation (29). Such conditions decreasethe availability ofNADP+ as an electron acceptor for PSI andthus enhance 02 uptake. Therefore, endogenous antioxidants,and their metabolism, could both reduce the toxic effects ofactivated oxygen species, and maintain a turnover ofNADPH, thereby providing an inherent tolerance mechanismagainst oxidative stress.

Glutathione, a ubiquitous free thiol-containing tripeptidein plants, possesses antioxidant properties which can protectplant membranes and protein thiols against oxidation by 02-'and H202 (1, 28). Glutathione reductase (EC 1.6.4.2) catalyzesthe reduction of oxidized (GSSG) to reduced (2 GSH) gluta-thione in an NADPH-dependent reaction which can maintainGSH/GSSG ratios of 10:1 in the chloroplast (18). Glutathionereductase is also involved in the ascorbate-glutathione cyclefor the scavenging of H202 (18, 24). This pathway functionsin the chloroplast (17) and may also occur in the cytosol (5,6). Increased cellular glutathione and glutathione reductasehave been reported in plants which show tolerance to airpollutants (6, 23, 30), cold stress (9, 11, 15, 16), drought (13),and 02-enriched air (12).

Glutathione reductase has been isolated from plant chlo-roplasts (5, 7, 20, 21, 26), roots (5), whole leaf (18, 31), andseeds (19, 22). Purified or partially purified glutathione reduc-tase generally shows a Km for NADPH between 1 to 13 AMand a Km for GSSG between 10 to 200 AM at 25°C. Thesereports have also indicated large variations among glutathionereductase native molecular masses (106-190 kD), subunitmolecular masses (41-72 kD), and pH optimum (7.6-9.0).

In only a few reports are different isozymes of glutathionereductase described. Guy and Carter (15) reported that cold-hardened spinach showed increased glutathione reductaseactivity and had different kinetic and molecular forms ofglutathione reductase than did nonhardened tissue. Also,

1402

GLUTATHIONE REDUCTASE FROM EASTERN WHITE PINE

plastidic and cytoplasmic isoforms of glutathione reductasehave been separated from mustard cotyledons. These isoformsshow different characteristics when exposed to light and pho-tooxidative conditions (10). These data suggest that plantscontain different isozymes of glutathione reductase whichmay be stimulated by different environmental signals.

In our progress to understand better the role that glutathi-one reductase plays in antioxidative protection of Easternwhite pine (Pinus strobus L.) against 03, we have isolated twodistinct isoforms of this enzyme from whole needles. Wereport the purification and characterization of the two formsofglutathione reductase. The specificity ofantibody producedfrom the major form of the enzyme also has been partiallycharacterized.

METHODS AND MATERIALS

Plant Material

Current-year needles from a 30-year-old Eastern white pine(Pinus strobus L.) growing on the campus of Virginia Poly-technic Institute and State University were collected in Oc-tober.

Enzyme Purification

All steps in the purification were carried out at 4°C exceptfor chromatography by fast protein liquid chromatography(FPLC) and high performance liquid chromatography(HPLC) which were performed at 20 to 22°C.

Needles (300 g fresh weight) were crushed in liquid N2 usinga chilled mortar and pestle. The needles were homogenizedwith a Polytron homogenizer (Brinkmann Instrument Co.) attop speed for 3 min in 1 L of 50 mm Pipes buffer (pH 6.8), 6mM L-cysteine hydrochloride, 10 mM D-isoascorbate, 1 mMEDTA, 0.3% Triton X-100, 1% w/v polyvinylpyrrolidonemol wt 10,000 (PVP-10), and 1% insoluble polyclar-AT. Thehomogenate was filtered through 8 layers of cheesecloth. Theremaining insoluble material was reextracted with an addi-tional 1 L of extraction buffer. The filtrates were pooled andcentrifuged for 30 min at 22,000g in a fixed angle rotor(Beckman JA- 14).The supernatant was brought to 35% saturation with solid

ammonium sulfate and stirred for 30 min. After centrifuga-tion at 22,000g for 30 min the resulting supernatant wasbrought to 75% saturation with additional solid ammoniumsulfate and stirred for 1 h. Following centrifugation at 22,000gfor 35 min the residue was resuspended in 150 mL of bufferA: 50 mM Tris-HCL (pH 7.5), 1 mM EDTA, and 0.1 mMDTT. This suspension was made 1 M with solid ammoniumsulfate and stirred for 15 min. Centrifugation at 22,000g for20 min removed the dark green pigmentation. The resultingsupernatant was brought to 3 M saturation with additionalsolid ammonium sulfate. The pale yellow precipitate wasremoved by centrifugation at 22,000g for 20 min and resus-pended in 30 mL of buffer A.

After desalting the solution on Sephadex G-25 (PharmaciaFine Chemicals), fractions containing glutathione reductaseactivity were applied to a 1.5 x 8 cm column of 2',5'-ADP-Sepharose 4B (Pharmacia) equilibrated with buffer A. Afterwashing the column with 30 mL of buffer A, followed by an

additional 30 mL wash with 25 mm NaCl in buffer A, theNADP+-dependent enzymes were eluted with a 5 mL pulseof 10 mM NADP+. The column was washed with an additional20 mL of buffer A followed by 20 mL of 500 mm NaCl inbuffer A.

Fractions containing glutathione reductase activity werepooled, concentrated (Amicon model 52, having a cut-offfilter of 10 kD), and dialyzed overnight against 4 L of bufferB: 20 mm Tris-HCl (pH 7.5), and 1 mM EDTA. The samplewas filtered through a 22 micron Acrodisk (Gelman SciencesInc., Ann Arbor, MI) and chromatographed on an anion-exchange column, Mono Q (Pharmacia).

Fractions containing individual peaks ofglutathione reduc-tase activity (two peaks, GRA and GRB), eluted from theanion-exchange column, were made 1 M by adding solidammonium sulfate. The samples were individually applied toa hydrophobic interaction column (Phenyl-Superose HR 5/5, Pharmacia) equilibrated with 1 M ammonium sulfate inbuffer B (buffer C).GRA and GRB were individually concentrated to 500 ,uL

and separately applied to a gel filtration column (Superose-12, Pharmacia) equilibrated with buffer D: 50 mM Tris-HCL(pH 7.5) and 10 mm NaCl. After gel filtration, both GRA andGRB were reapplied individually to an anion-exchange col-umn (Mono Q HR 5/5) equilibrated with 20 mm Tris-HCL(pH 9.0), containing 1 mM EDTA (buffer E). Factions con-taining glutathione reductase activity were pooled and storedat -4°C in buffer E.

Protein Determination

Protein was determined by the method of Bradford (4)using lyophilized BSA (Bio-Rad) as a standard.

Glutathione Reductase (EC 1.6.4.2) Assay

Glutathione reductase was assayed spectrophotometricallyby monitoring the GSSG-dependent NADPH oxidation at340 nm. Assays were performed at 25°C in a 1 mL reactionmixture of 50 mm Tris-HCl (pH 7.5), 3.0 mM MgCl2, 150 nMNADPH, and 500 nM GSSG unless otherwise specified.

Preparation of Antisera

Antibody to purified GRB (215 ,ug protein) was preparedby Cocalico Biologicals, Reamstown, PA. The rabbit wasgiven booster injections at 14 and 21 d and serum collected,at 6 and 10 weeks after the initial injection of antigen,contained sufficient levels of antibody.

Electrophoresis

SDS-PAGE (10% gels) and native-PAGE (7.5% gels) weredone as described by Smith (27). Visualization of proteins ongels was accomplished by silver staining as described by Sasse(25).

Glutathione reductase activity was detected on native-PAGE gels by incubating them in 100 mL solution containing10 mg MTT, 10 mg 2,6-dichlorophenolindophenol, 3.4 mMGSSG, 0.4 mm NADPH, and 50 mm Tris (pH 7.5). Duplicategels were assayed for glutathione reductase activity: one withand one without GSSG.

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Plant Physiol. Vol. 94, 1990

Table 1. Purification of Glutathione Reductase from Eastern White Pine

Purification Step Specific Activity Total Purification RecoveryActivity Factor

pKat (mg protein)-' units %

G-25 sephadex 0.01 246 12',5'-ADP sepharose 0.41 253 38.1 103Mono Q (GRA), pH 7.5 0.12 9.2 10.7Mono Q (GRB), pH 7.5 1.05 116 98.7 A + B = 51Phenyl superose (GRA) 0.31 4.6 29.1Phenyl superose (GRB) 1.76 82.2 165 A + B = 35Superose-1 2 (GRA) 0.45 3.2 41.2Superose-1 2 (GRB) 4.8 76 443 A + B = 32Mono 0 (GRA), pH 9.0 1.81 2 170Mono Q (GRB), pH 9.0 6.08 82 570 A + B = 15a

* A total of 12 units of GRA were applied; a total of 167 units of GRB were applied.

Western Blotting

Proteins were transferred from polyacrylamide gels to nitro-cellulose using a Trans-Blot Semi-Dry Electrophoretic Trans-fer Cell (Bio-Rad) at 20 V for 15 to 30 min. Transfers were

done using 48 mM Tris, 39 mm glycine, 0.13 mm SDS, and20% methanol (pH 9.2) as a transfer buffer. Transblots were

probed with antisera produced from purified GRB (anti-GRB)and glutathione reductase was visualized on the membranesusing a 1:1000 dilution of horseradish peroxidase conjugatedgoat anti-rabbit IgG (BioRad). Color development was per-

formed using 2.8 mM 4-chloro-l-naphthol (BioRad) and0.03% H202.

Immunological Detection of Antigen

Antibody sensitivity and cross-reaction of native glutathi-one reductase (antigen) with anti-GRB (antibody) were ex-

amined using both ELISA and slot-blot analysis. Serial dilu-

tions ofGRB were visualized on ELISA plates using anti-GRBand a 1:1000 dilution of horseradish peroxidase conjugatedgoat anti-rabbit IgG. Color development was accomplishedusing 0.18 mM 2,2'-azino-di(3-ethylbenzothiazoline sulfo-nate) and 0.09% H202. Serial dilutions of GRA and GRBbound to nitrocellulose by slot blot application were visualizedas previously described for Western transblot analysis andscanned at 580 nm with a Shimadzu Dual-wavelength, modelCS9000U, densitometer.

Molecular Mass Determination

The subunit molecular mass of glutathione reductase wasdetermined by SDS-PAGE using prestained electrophoresismol wt standards (Diversified Biotech, Newton Centre, MA).The native mol mass of the enzyme was determined by gel

filtration chromatography using a 1.5 x 30 cm Superose 6

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FRACTION NUMBER

Figure 1. Chromatograpic profile of glutathione reductase from East-

ern white pine on 2'5' ADP-Sepharose 4B. Glutathione reductase

activity was first eluted with a 5 mL pulse of 10 mm NADP+ in 50 mMTris-HCI (pH 7.5), 1 mM EDTA, 0.1 mm DTT, and 25 mm NaCI. The

remaining glutathione reductase activity was eluted with 20 mL of

the same buffer containing 500 mM NaCI. Each fraction represents 2

mL of eluant. The flow rate was 1 mL min-'. Fractions 49 through 58

contain 253 total units of activity and fractions 74 through 81 contain

15 total units of activity.

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Figure 2. Chromatograpic profile of glutathione reductase from East-ern white pine on Mono 0 HR 5/5 (FPLC) anion-exchange. Thecolumn was equilibrated with 20 mm Tris-HCI (pH 7.5) containing 1mM EDTA (buffer B). A 4 mL sample of glutathione reductase elutedfrom the 2'5'-ADP-Sepharose 4 B column was applied to the column.The column was washed with 5 mL of buffer B and eluted with 15mL of a linear gradient of 0 to 500 mM NaCI. The solvents weredelivered at a flow rate of 0.5 mL min-1. Each fraction represents500 AL of eluant. Fractions 9 through 15 contain 14.3 total units ofactivity, fractions 27 through 30 contain 9.1 total units and fractions38 through 43 contain 115 total units of activity.

- Am- .0'm

1 404 ANDERSON ET AL.


A B our results and previous reports ( 15, 18, 20). The inability ofscots pine glutathione reductase to elute from the affinity4 3 2 1 1 2 3 4 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnncolumnwith10mmnNADPemay have been a result of using

PVP-360 in the extraction medium (31). When we replacede i t'.-Si'.X;F.PVP-10 with PVP-360 in our extraction buffer we also were

unable to elute glutathione reductase from the affinity columnwith 10 mm NADP+ and found that the enzyme only elutedafter addition of 500 mm NaCl. We also observed a 4-folddecrease in the recovery of glutathione reductase from crudeextracts when we replaced PVP-10 with PVP-360.

A1 2 3 4 5 6 7

kD

*,, 95

Figure 3. Electrophoretic separation of proteins from Eastern whitepine containing glutathione reductase activity on 7.5% native-PAGEgels. (A) Glutathione reductase activity stain; (B) silver stain of gel.Lane 1, 1 jig protein of unbound fraction from anion exchangechromatography; lane 2, 1 jg of protein recovered from 2',5'-ADP-Sepharose 4B; lane 3, 250 ng of protein (GRB) recovered from anion-exchange chromatography; lane 4, 250 ng of protein (GRA) recoveredfrom anion-exchange chromatography.

FPLC column (Pharmacia) and a 7.5 x 300 mm Bio-Sil TSK-250 (Bio-Rad) HPLC gel filtration column equilibrated with0.1Im Na2SO4 and 0.02 m NaH2PO4 (pH 6.8). The columnswere precalibrated with gel filtration mol wt standards (Bio-Rad).

RESULTS AND DISCUSSION

Protein Purification

The protein purification procedure resulted in the isolationof two distinct formns of glutathione reductase from Easternwhite pine needles. These two forms were labeled GRA andGRB. Purified GRA had a specific activity of 1.81 MKat (mgprotein)-' and represented a 170-fold purification (Table I).Purified GRB had a specific activity of 6.08 gKat (mg pro-tein)-' which represented a 570-fold purification (Table I).The specific activity for GRA was more representative ofreported values for glutathione reductase isolated from plants,which range from 0.43 to 4.1 uKat (7, 18, 20-22); however,GRB had a greater specific activity than the maximum thathas been reported in the plant literature.

Affinity chromatography with 2',5'-ADP-Sepharose 4B re-sulted in a 38.1-fold purification step for the isolation ofglutathione reductase (Table I). Approximately 80 to 90% ofthe protein containing glutathione reductase activity waseluted with a 5 mL pulse of 10 mmv NADP' (Fig. 1). Theremaining enzyme activity was eluted with 500 mm NaCl.Wingsle (31) reported that the majority of glutathione reduc-tase from scots pine eluted from 2',5'-ADP-Sepharose 4Bonly after 500 mm NaCl was applied, a result in contrast to

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Figure 4. Electrophorectic separation of purified unbound, GRA andGRB glutathione reductase from Eastern white pine on SDS-PAGEgels and Western transblot. (A) Silver stain of gel; lanes 1 and 6, 150ng GRA; lane 2, 100 ng each of GRA and GRB; lane 3, 200 ng GRB;lanes 4 and 7, 2 ML of standards; lane 5, 200 ng purified unboundfraction from anion-exchange chromatography. (B) Western transblotprobed with antibody produced against purified GRB glutathionereductase from Eastern white pine; lanes 1 and 2, 150 ng purifiedunbound and GRA glutathione reductase respectively; lanes 3 to 7,150, 100, 50, 10, and 5 ng each of GRA and GRB, respectively.

1 405

A. I uj:' ..tfn 'i

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Plant Physiol. Vol. 94,1990

Anion-exchange with a Mono Q HR 5/5 column (FPLC)provided a simple, rapid method for the separation ofdifferentisoforms of glutathione reductase. Approximately 8% of thetotal glutathione reductase activity recovered from anion-exchange was GRA and 83% was GRB (Fig. 2). The remaining9 to 10% of enzyme activity was recovered in the unboundfraction (Fig. 2). This unbound fraction was determined to bethe same GRA isoform which eluted from the anion-exchangecolumn with 150 mM NaCl. This conclusion was obtained bycomparing the migration patterns of the unbound fraction,GRA and GRB on native and SDS-PAGE gels. GRA and theunbound fraction always showed the same migration patternson native gels which were stained for either activity (Fig. 3A)or protein (Fig. 3B). Similar migration patterns also wereobserved for GRA and the unbound fraction on SDS-PAGEgels (Fig. 4A). The specific activities ofGRA and the unboundfraction were also similar throughout all steps in the purifi-cation procedure.Drumm-Herrel et al. (10) also used FPLC anion-exchange

chromatography to isolate two peaks of glutathione reductaseactivity from mustard cotyledons. In contrast to our results,they reported the first eluting peak, 'GR,,' to be the majorpeak of activity and the second eluting peak, 'GR2,' to be theminor peak of activity. They also termed GR2 as a plastidicisoform based on its sensitivity to photooxidation, whereasGR, showed no sensitivity to photooxidation.

After further purification using Phenyl-Superose HR 5/5and Superose-12 (FPLC), GRA and GRB were reapplied in-dividually to a Mono Q HR 5/5 (FPLC) chromatographycolumn equilibrated at pH 9.0. At this point both GRA andGRB were characterized by a single band of glutathione re-ductase activity (Fig. 5A) and a single band of protein onnative polyacrylamide gels (Fig. 5B). SDS-PAGE gels alsoshowed only a single band of protein for both GRA and GRB(Fig. 4A). It was concluded that both GRA and GRB werepurified to homogeneity.

Molecular Mass Determination

The native molecular mass determined by Superose-6(FPLC) chromatography was 103 ± 1.5 kD for GRA and 95± 2 kD for GRB. Native molecular masses were also deter-mined using a TSK-250 gel filtration column (HPLC). Bythis method, the native molecular mass ofGRA was 104 ± 2kD and 88 ± 2 kD for GRB. The subunit molecular mass asdetermined by SDS-PAGE was 53 to 54 kD for GRA and 57kD for GRB (Fig. 4A). The subunit molecular mass forglutathione reductase from Eastern white pine is similar tothat reported from pea chloroplast (7), scots pine (31), andrice kernel (19) (Table II). However, these subunit values arehigher than the 41 and 42 kD subunits reported by Kalt-Torres et al. (20) for pea chloroplast and lower than the 72kD subunits reported by Halliwell and Foyer (18) for spinachleaves. Although Mahan and Burke (21) reported subunitweights of 65, 63, 34, and 32 kD for glutathione reductaseisolated from corn mesophyll chloroplast, most literature onglutathione reductase suggests that this protein is a dimercomposed oftwo identical subunits (7, 18, 19, 3 1). Our resultsfor both GRA and GRB are also consistent with dimericstructures each consisting of identical subunits.

The native molecular masses of glutathione reductase fromEastern white pine (GRA and GRB) are somewhat lower thanreported for most plant glutathione reductases which rangebetween 106 and 190 kD (Table II). That the native molecularmass of GRA is greater than the molecular mass of GRB, yetshows smaller subunit molecular mass than GRB, is somewhatinteresting. These data may suggest that GRA includes a shortsignal sequence which dissociates from the polypeptide duringdenaturation.

Kinetic Parameters

For the substrate NADPH, GRA had a Km of 3.7 zlM andGRB had a Km of 8.8 gM (Table II). For GSSG, GRA had aKm of 15.3 gM, whereas GRB had a Km of 39.8 gM. Althoughthese two isoforms of glutathione reductase show differentkinetic constants, the Km values do agree well with thosepreviously reported from plant tissues (Table II).

pH Optima

The pH optimum ofGRA was 7.25 to 7.75 with 50% of theactivity ranging between 7.0 to 8.0 (Fig. 6). The pH optimumfor GRB was 7.25 with 50% of the activity ranging between6.5 and 8.0. These values are lower than those previouslyreported for plant glutathione reductase which range between7.7 and 9.0 for whole leaf (I 5, 18, 31), root (5), and seeds (19)(Table II).

Immunological Characterization

Antiserum (anti-GRB) from rabbit was tested for cross-reactivity with both purified GRA and GRB. Anti-GRB binding

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Figure 5. Electrophoretic separation of purified GRA and GRB gluta-thione reductase from Eastem white pine on 10% native-PAGE gelsand Western transblot of native-PAGE gel. (A) Glutathione reductaseactivity stain; (B) silver stain of gel; (C) Western transblot probed withantibody produced from GRB. Lane 1, 210 ng GRA; lane 2, 280 ngGRB-

ANDERSON ET AL.1 406


was quantified with ELISA and slot blot analysis. With ELISAthe sensitivity of the reaction between GRB and anti-GRBcould be detected at 150 pg total protein (Fig. 7A). Slot blotanalysis of GRB on nitrocellulose showed a reactivity withanti-GRB at a sensitivity of approximately 80 pg (Fig. 7B).Slot blot analysis of GRA on nitrocellulose showed a cross-

reactivity with anti-GRB at a sensitivity of approximately 10to 20 ng (Fig. 7B).Western transblots of SDS-PAGE gels probed with anti-

GRB resulted in no cross-reactivity with GRA but did showprimary reactivity with GRB (Fig. 4B). The data indicatedthat 5 to 10 ng of protein was the limit for reactivity betweenanti-GRB and denatured GRB. Western transblots of nativegels were also probed with anti-GRB. The native form ofGRAdid show cross-reactivity with anti-GRB (Fig. 5C). Thus, itappears that a determinant or conserved site for cross-reactiv-ity is lost or altered when GRA is denatured.

Conclusions

From these data we conclude that GRB may be a putativeplastidic form of glutathione reductase. GRB represented ap-proximately 80% of the total glutathione reductase recoveredfrom needles, which is in close agreement with Gillham andDodge (14) who reported that 76.8% of the total glutathionereductase recovered from pea leaves was contained in thechloroplast. Further evidence (10), in which the second elutingpeak ofglutathione reductase from FPLC anion-exchange wasreported to be a plastidic form based on its sensitivity tophotooxidation, also suggests that our GRB (the second peakof activity to elute from FPLC anion-exchange, Fig. 2) maybe a plastidic form. In addition, the pH range of 6.5 to 8.0for GRB (Fig. 6) would be optimal for a chloroplastic form ofglutathione reductase to operate efficiently over stromal pH

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Figure 6. pH profile of purified glutathione reductase GRA and GRBfrom Eastern white pine. Reactions at pH 5.5 to 7.0 were done using50 mM Mes buffer and reactions at pH 7.0 to 9.5 were done using50 mM Tris buffer. Each reaction was conducted using 175 ng ofpurified protein for GRA and 25 ng for GRB.

changes that occur during active photosynthesis and in thedark.

Identifying GRB as a putative chloroplastic form is consist-ent with its' greater Km for GSSG and NADPH and specificactivity compared to GRA. The concentration ofNADPH inthe chloroplasts of spinach is approximately 700 ,uM in thedark (17). Although only approximately 10% of the totalglutathione of a leaf is chloroplastic (14), the stromal GSHconcentration generally ranges between 1 to 3.5 mm andGSSG between 100 and 200 ,M (17). Such substrate concen-trations would be more than sufficient to saturate Easternwhite pine glutathione reductase (GRB). Since it has been

Table II. Comparisons of Physical and Kinetic Properties of Purified and Partially Purified Glutathione Reductase from Different Sourcesof Plant Tissues

Source Native Subunit GSSG NADPH pH Ref.Optimummol mass, kD Km, pM

ChloroplastPea 60 62 3.0 7Pea 156 41/42 1 1 1.7 7.6 20Spinach 70 6.5-8.0 26Corn mesophyll 190 32/34/63/65 14 1.7 8.0 21

Whole leafSpinach 145 72 196 2.8 8.5-9.0 18hardened #a 52 6.0 8.0 15nonhardened 38 8.0 8.0 15

RootsPea 10 2.3 7.7 5

SeedsPea 120 17 4.7 22Rice 106 52 34 13.0 7.9 19

Pine needlesScots 59 28 1.0 7.7 31Eastern whiteGRA 103-104 53-54 15.3 ± 1.6b 3.7 ± 0.2 7.2-7.8 **c

GRB 88-95 57 39.8 ± 1.7 8.8 ± 0.8 7.25a #, Multiple isoforms. b Means are results of four replicates (GRA) and five replicates (GRB) ± SE. c This study.

1 407

Plant Physiol. Vol. 94, 1990

Further studies on glutathione reductase in Eastern whitepine will be directed toward the use of anti-GRB to determinethe response of this enzyme to ozone exposure. Tanaka et al.(30) observed an induction of glutathione reductase in spin-ach, which had been exposed to 700 nL L' ozone, usingantibodies to the purified enzyme.

ACKNOWLEDGMENTS

o l oo - Z/ The authors express appreciation to Miles and Ruth Horton forproviding the Miles C. Horton Education Foundation Fellowship to

00001ooo-_____/_ _l_l J. V. A. The authors also gratefully thank Bernadette Mondy andO.O10 O O00 ooo0.010 0.100 1.000 1 0.000 1 00 000 Alice Tira for their technical assistance during this study.

3E4

1 E40 10 0.100 1.000 10.000 100.000

ANTIGEN (ng)

Figure 7. Sensitivity of antibody detection of glutathione reductasefrom Eastern white pine. Antibody to GRB was used in either anELISA assay against GRB with a detection limit of >100 pg (A), orslot blot analysis with detection limits for GRB >80 pg and for GRA>10 ng (B). The sensitivity for GRB was greater than for GRA. Datapresented with a second order regression line.

established that glutathione and glutathione reductase are

components ofthe ascorbate-glutathione cycle for the removalof H202 in the chloroplast ( 18, 24), it should be expected thatthe chloroplastic form of glutathione reductase would have a

greater Km for GSSG and NADPH and a greater turnoverrate to keep the GSH/GSSG at 10:1. Under illumination andlimiting carbon reduction conditions, or when NADPH con-centrations are high, the 3.3-fold greater specific activity (Vmax)of a chloroplastic form of glutathione reductase, compared toa cytosolic form, would be consistent with the function of thisenzyme in maintaining NADP+ availability.

SUMMARY

Based on the data presented in this study, we have isolatedtwo distinct isoforms of glutathione reductase from Easternwhite pine needles. Considering the percentage of recovery,greater Km for GSSG and NADPH, and the greater Vmax forGRB, compared to GRA, we suggest GRB to be a putativeplastidic isoform of glutathione reductase. We believe GRA tobe either a cytosolic isoform or a precursor of the plastidicenzyme.

LITERATURE CITED

1. Alscher RG (1989) Biosynthesis and antioxidant function ofglutathione in plants. Physiol Plant 77: 457-464

2. Alscher RG, Amthor JS (1988) The physiology of free-radicalscavenging: maintenance and repair processes. In S Schulte-Hostede, NM Darrall, LW Blank, eds, Air Pollution and PlantMetabolism. Elsevier, New York, pp 95-110

3. Badger MR (1985) Photosynthetic oxygen exchange. Annu RevPlant Physiol 33: 73-96

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Purification, Characterization, and Immunological