Plant Physiol. (1982) 69, 72-760032-0889/82/69/0072/05/$00.50/0

Oxidation of Reduced Pyridine Nucleotide by a System UsingAscorbate and Hydrogen Peroxide from Plants and Algae'

Received for publication February 16, 1981 and in revised form June 20, 1981

YoKE WAH KoW2, DOUGLAS A. SMYTH3, AND MARTIN GIBBSInstitutefor Photobiology of Cells and Organelles, Brandeis University, Waltham, Massachusetts 02254

ABSTRACT

A NAD(P)H oxiding system (NAAP) was detected and partiallypurife from leaves of spinh and Sedwn praeabu, seeds and leaves ofpea and cells of green and red algae which oxiized NAD(P)H in thepresence of ascorbate and H202.The partially-purified spinach system had substrate K. values of 5

micromolar for NADH, 50 micromolar for H202, and 300 micromolar fori-ascorbic acid at the pH opthnum of 6.8 NADH was a better electrondonor than NADPH. Among other electron donors, isoascorbic acid hadconsiderable activty but hydroquinone and resorcinol had only weakactivities. The enzyme was inhbited by cyanide, a,a'-dipyridyl, an mono-and di-thol reagents. Inhibition by thiol-reagents was partially restored byFe'+ as was enzyic actvity lost following dialysis against buffer.

Subcelular locization studies with spinach and S. praeaiuu leavesindicated that a portion of the cei's NAAP was in the chloroplast fraction.Photosynthetic conditions resulted in a decrease in this actvity solubilizedfrom spinach and S. praealuw chrwoplasts. The presence of 3-(3,4-di-chlorophenyl)-1,1-dimethylurea or Fe2' in the incubation medium elimi-nated the light-mediated inhibition of NAAP.NAAP may function in the recycling ofNAD(P)H generated in the dark

within the chloroplast. Inasmuch as all preparations of NAAP containedascorbate peroxidase activity, the data do not rule out the possibility thatNAAP is the same protein as ascorbate peroxidase or, alternatively, acombination of ascorbate peroxidase and some other enzyme.

Recently, ascorbate peroxidases have been solubilized fromseveral higher plants (15) and algae (15, 22) and an insoluble formhas been localized on the thylakoids of the spinach chloroplast(11). The stoichiometry for the spinach chloroplast enzyme is Imol each of H202 and ascorbate consumed for I mol of dehy-droascorbate formed (11) and AAP4 has been envisioned as a

mechanism of detoxifying H202 through the use of ascorbate, an

abundant constituent in Chl-containing cells. In the chloroplast,at least, the presence of reduced glutathione and glutathionereductase (8) provides a potential means of recycling dehydroas-corbate.

1 Supported by National Science Foundation Grant PCM 79-22612 andDepartment of Energy Grant EY-76-S-3231-14.

2 Prewnt address: Department of Biochemistry, University of Wiscon-

sin, Madison, WI 53706.3 Present address: Department of Agronomy and Soils, Washington

State University, Pullman, WA 99164.4 Abbreviations: AAP, ascorbate peroxidase which catalyzes the reaction

involving ascorbate and H202; NAAP, reoxidation of reduced pyridinenucleotide with a preparation involving ascorbate, H202 and NAD(P)H.Most experiments were carried out with NADH-NAAP.

Ofthe soluble peroxidases, the most characterized is the Euglenagracilis enzyme which is a hemoprotein (21) like many other plantperoxidases (18). Euglena AAP is an unusual hemoprotein in thatit is stabilized by sucrose and by ferrous sulfate. In addition, theenzyme has high affinities for ascorbate and H202, with Km valuesof410 and 56 ILK respectively (21). The insoluble spinach enzymehas similar substrate affinities and also appears to be a hemopro-tein.We report here on a peroxidative activity similar to AAP in

peas, spinach, Sedum praealtum and algae. This enzyme systemdesignated as NAAP uses NAD(P)H, ascorbate, and H202 assubstrates. Furthermore, this system can be solubilized from thechloroplast and is subject to photoregulation.

MATERIALS AND METHODS

Plants and Algae. S. praealtum and peas (Pisum sativum var.Progress No. 9) were grown in the greenhouse. Sedum and peaswere grown in a vermiculite-soil mixture or in vermiculite, respec-tively. Spinach (Spinacia oleracea) was cultivated under controlledconditions (12 h light at 22°C and 12 h dark at 18°C) in avermiculite-soil mixture. The algae with the exception of Chla-mydomonas reinhardii F-60 (9) and Euglena gracilis (10) weregrown photoautotrophically with CO2 as the carbon source.

Cell-Free Preparations. About 2 kg of fully expanded spinachleaves or pea leaves taken from 2-week-old plants were washed,cut into segments after the midribs were removed, and homoge-nized with a Waring Blendor in 50 mM Hepes-NaOH (pH 7.0) at4°C. Pea seed which were imbibed in water overnight was alsodisintegrated in the blender. After the homogenate was filteredthrough cheesecloth, the homogenate was centrifuged at 10,000gfor 10 min to remove debris. The homogenate was then fraction-ated by solid (NH4)2SO4 precipitation. The portion of the homog-enate insoluble in 50 to 80%1o (NH4)2SO4 was resuspended in 50mM Hepes-NaOH (pH 7.0) and absorbed onto calcium phosphategel. After elution from the gel with 0.1 M K-phosphate, thissolution free of catalase was used as the source of higher plantascorbate peroxidases. The result of treatment with (NH4)2SO4and gel was an approximate 5-fold purification. Cell-free algalpreparations were made by grinding the organisms with an equalweight of aluminum oxide for 5 min in a chilled mortar and pestle.About 5 to 10 ml of 50 mm Hepes-NaOH buffer (pH 7.0) wasadded and the slurry was centrifuged at 25,000g for 10 min toremove the cellular debris. The clear supernatant fluid was usedfor enzyme assays.Enzyme Activity. NAAP was assayed at 250C as the oxidation

ofNAD(P)H at 340 um. The 1-ml assay volume contained 50 mMHepes-NaOH (pH 7.0), 0.2 mM NAD(P)H, and 5 mm ascorbate.After a baseline of absorbance change was established, a readingwas taken 1 min after the addition of 0.18 mM H202. AAP wasassayed at 25°C as the oxidation of ascorbate at 265 nm (15). The1-ml assay volume contained 50 mM Hepes-NaOH (pH 7.0) and0.1 mm ascorbate. The reaction was started by adding 0.18 mM

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REDUCED PYRIDINE NUCLEOTIDE, ASCORBATE, H202

H202. Malate dehydrogenase and triose-P dehydrogenase weremeasured as the oxidation of NADPH at 340 rm. Malate dehy-drogenase assays contained, in a final 1.1 ml volume: 50 mmHepes-NaOH (pH 7.0), 0.11 mM NADPH, and 0.22 mm oxalace-tate to initiate the reaction. Triose-P dehydrogenase assays con-tained in a final 1.0 ml volume: 50 mm Hepes-NaOH (pH 7.6), 0.2mm NADPH, 1 mm glycerate-3-P, 1 mm MgCl2, 1 mm DTT, and30 units glycerate-3-P kinase. Glucose-6-P dehydrogenase wasmeasured as the reduction ofNADP at 340 nm. The l.0-ml assaymedium contained 50 mm Hepes-NaOH (pH 7.6), 1 mm MgCl2,2 mm glucose-6-P, and 0.5 mM NADP. Cytochrome oxidase wasmeasured as the oxidation of reduced Cyt c at 550 nm (24).Catalase was monitored as the breakdown of H202 at 240am (16).

Chloroplast Isolation. Spinach leaves were chopped into smallpieces and ground (1.5 s) with a VirTis model 45 in a gindingmedium of 50 mm Hepes-NaOH (pH 6.8), 330 mm sorbitol, 2 mMdisodium EDTA, 1 mm MgCl2, and 1 mm MnCl2. The homogenatewas filtered through two layers ofMiracloth (Chicopee Mills, Inc.,Miltown, NJ) with the filtrate being centrifuged at 750g for 1 min.The chloroplast pellet was resuspended in fresh grinding mediumand recentrifuged. The final washed chloroplast pellet was resus-pended in a minimal volume of grinding medium and used forchloroplast incubations. Chloroplasts were at least 50%7v intact asmeasured by ferricyanide-stimulated 02 evolution.

In some experiments, spinach chloroplasts were prepared fromprotoplasts according to the procedure ofNishimura et aL (17). Inthis procedure, the protoplasts were disrupted by passage througha syringe into the grinding medium used for the spinach leaves.

S. praealtum leaves were sliced and digested enzymically toyield protoplasts as described by Spalding and Edwards (23). Theprotoplasts were gently broken by passage through a syringe andthe homogenate obtained was centrifuged in a flotation gradientto yield a chloroplast fraction. These chloroplasts were at least90% intact as measured by ferricyanide-stimulated 02 evolution.

Chemical Assays. Protein content was determined according toBradford (5), using standards of BSA. Chl was determined ac-cording to Arnon (4).

RESULTS

Comparison of NAAP and AAP Activties. NAAP and AAP(Table I) coexisted in roughly equal levels in the leaf and algalpreparations with the possible exception of pea seed, spinachthylakoids, and C. vulgaris (light and dark grown). Only a trace ofAAP was detected in pea seed while the particulate matter fromthe spinach chloroplast was free of NAAP and with respect toChlorella, the ratio of NAAP:AAP averaged about 2:1. NADH at0.2 mm was twice as effective as NADPH at an equal concentra-tion. In general, NADH was twice as effective as NADPH.Solubilized activities of AAP and NAAP from spinach and pealeaves could be concentrated but not separated by (NH4)2SO4fractionation and treatment with calcium phosphate gel, indicatingthe two enzyme activities may be associated with the same protein.Further attempts to resolve the higher plant enzymes by alcoholprecipitation or by DEAE chromatography resulted in a total lossof peroxidase activity. Attempts to determined stoichiometry forNAAP were unsuccessful due to the presence of AAP activity.

Effect of Substrate Concentration on the Rate of SpinachNAAP. The apparent Km values were 5 ,UM for NADH, 50 um forH202, and 300 um for ascorbate (Fig. 1). The V,,. for ascorbatewas 1.07 psmol/mg protein *min, a value which contrasts with 0.47,0.17, and 0.06 when ascorbate was replaced by 5 mM isoascorbate,hydroquinone, or resorcinol.pH Profile of Spinach NAAP. The pH optimum for enzyme

activity was around 6.8, both for NADH (Fig. 2) and NADPH(data not shown)-linked activity. At pH 8.0 enzyme activity wasless that 25% of the rate at pH 6.8.

Effect of Temperature on the Stability of NAAP and AAP.

Table I. Activity ofNAAP andAAP From Dif7erent SourcesPea, spinach, S. praealtum leaf, and pea seed preparations were partially

purified from crude homogenates by 50 to 80% (NH4)2SO4 precipitation,followed by elution from calcium phosphate gel. Spinach chloroplastparticles were prepared by rupturing intact chloroplasts in 50 mM Hepes-NaOH (pH 7.0). The particulate matter was spun down and washed oncewith the same buffer. To make cell-free algal preparations, the organismswere ground with an equal weight of A1203 for 5 min. Then 5 to 10 mlHepes-NaOH (pH 7.0) buffer was added and slurry was centrifuged at25,000g for 10 min to remove A1203 and cellular debris. The clearsupematant fluid was used for enzyme essays.

NAAPPlant Sources AAP

NADH NADPH

Mmol NAD(P)H/mg umol ascor-bate/mgprotein*mpnproteinmin

Pea leaf 2.95 2.01 4.32Spinach leaf 1.59 0.64 2.51S. praealtum leaf 2.13 0.90 5.32Pea seed 0.25 0.14 0.03Spinach chloroplast particles NDa ND 1.16E. gracilis Z 0.15 0.04 0.32Scenedesmus obliquus 0.23 0.14 0.16Chlamydomonas reinhardii F-60 0.16 0.20 0.68Chlamydomonas reinhardii 1.00 0.16 1.77Chlorella vulgaris (dark) 0.19 0.15 0.09Chlorella vulgaris (light) 0.12 0.10 0.04Porphyridium cnrentum 1.01 0.71 2.17

a Not detected.

z

z

I-

Q.

z

-J0

[mM NADH]rFIG. 1. Lineweaver-Burk plot of spinach NADH-NAAP as a function

of ascorbate, H202, and NADH concentrations. Reaction mixtures were asdescribed in Figure 2 using Hepes-NaOH as a buffer. The concentrationof ascorbate (upper), H202 (middle), and NADH (lower) were varied asindicated.

NAAP isolated from the spinach leaf or from the spinach chlo-roplast was unaffected by heating for 5 min at 40°C. Heating at60°C for 5mincompletely inactivated both preparations. On theother hand, only 50% of the chloroplasts membrane-bound AAPwas destroyed after 5 minat 60°C.

Inhibition Studies with Spinach and Pea NADH-NAAP. Several

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Plant Physiol. Vol. 69, 1982

2.0

Z

1.5w

0~

0

6 7 8 9

FIG. 2. pH profile of spinach NADH-NAAP. Reaction was monitoredby following the oxidation of 0.2 mM NADH in the presence of 5 mmascorbate and 0.18 mm H202 in the appropriate buffer (100 mM) asindicated: (0), phosphate; (M), Hepes buffer, (A), Tricine buffer.

Table II. Inhibitors of Spinach NADH-NAAPSpinach preparations were incubated in the presence of the indicated

compounds for a period (usually 2-5 min) at 25°C prior to enzyme assay.The control rate was 1.3 pmol/mg protein.min.

Inhibitor Activity

Control 100KCN, 0.1 mM 302,3-Dimercaptopropanol, 25 ,UM 30L-Cysteine, 0.2 mm 50Mercaptoethanol, 0.3 mM 30DTT, 0.3 mM 30a,a'-Dipyridyl, 0.2 mM 30Tiron, 0.2 mM 105Diethyldithiocarbamate, 0.2 mM 99Pyrophosphate, 10 mM 18

classes of compounds were tested as inhibitors with the 5-foldpurified spinach NADH-NAAP (Table II). Cyanide at 0.1 mminhibited enzyme activity by 70%o. Incubation with sulfhydrylreagents such as 2,3-dimercaptoethanol, DTT, cysteine, and mer-captoethanol reduced enzyme activity but 2 mm reduced glutathi-one had no effect. The degree of inhibition by these -SH con-taining compounds was dependent upon the order of substrateaddition. For example, 25 ,pm 2,3-dimercaptopropanol and 300p,m 2-mercaptoethanol inhibited the peroxidase by 70%o if the thiolcompound was incubated for 5 min with the enzyme preparationprior to the addition of 5 mM ascorbate. If the ascorbate wasadded first, the inhibitory effect of the thiol reagents were reducedto about 10o.

Several oxidized compounds were tested to determine if theycould reverse the dithiol inhibition of NADH-linked ascorbateperoxidase. Dehydroascorbate, oxidized glutathione, or oxidizedDTT at 1 mm had no effect on the peroxidase. When NAAP wasincubated for 5 min with 0.1 mM DTT it was inhibited 60%o andthe inhibition value was reduced to 30%o on addition of 1 mmdehydroascorbate but 1 mm oxidized DTT or 1 mm oxidizedglutathione was without effect.The pea leaf NADH-linked peroxidase showed similar re-

sponses to the reagents listed in Table II. Furthermore, the pealeaf peroxidase was unaffected by inhibitors like arsenite (1.0 mM),iodoacetamide (5 mM), and N-ethylmaleimide (5 mM) indicatingthat a sul&hydryl group is not required for enzyme activity. Theenzyme was inhibited 50Yo by a 5 mm NaN3 but little affected by

1 mm NaN3.The Fe2+ chelator, a,a'-dipyridyl at 0.2 mm inhibited by 70%o

spinach NAAP while the chelators of Fe3+ (Tiron) and of Cu2+(diethyldithiocarbamate) were completely ineffective at 0.2 mmlevels. The addition of 10 mm pyrophosphate inhibited the enzymeby 80%o.

Effect of Fe2 on Spinach NADH-NAAP. Since inhibition bypyrophosphate and cyanide indicated participation of a metal ionand the finding with a,a-dipyridyl specified Fe2 , this property ofNAAP was studied in some detail.

Half of the NADH-linked peroxidase activity was lost after 1 hdialysis of 1 ml enzyme against 2 liters of 50 mm Hepes-NaOH(pH 7.0). This loss was increased to 90%o if 0.1 mm DTT wasincluded in the dialyzing fluid. The activity ofthe dialyzed enzymewas completely restored on addition of 10 to 25 ,UM Fe2+. Higherconcentrations (50-100 ,iM) of Fe2+ severely inhibited. Bound ironin the form of ferredoxin or ferrocyanide was not active inrestoring activity lost during dialysis. Other ions such as Zn2+Mg2+, Co2+, Ni +, MoO42+ at 25 ,UM had either no effect or wereinhibitory.

Cellular Localization of NAAP and AAP. The data recorded inTable I indicated the presence of AAP but not to NAAP boundto the spinach chloroplastic thylakoids. On lysis of spinach pro-toplasts, about 20 to 30%o of the total NADH-linked ascorbateperoxidase and AAP was found to be associated with a 650g pelletwhich contained the bulk of the NADPH-glyceraldehyde-3-Pdehydrogenase, a chloroplast marker (Table III). Another inter-esting feature of Table III is the finding that repeated washing ofthis fraction in an isotonic medium did not change the specificactivity on a Chl basis of either NADPH-glyceraldehyde-3-Pdehydrogenase or the ascorbate peroxidases while the specificactivity of catalase, the microbody marker and Cyt c oxidase, themitochondrial marker decreased several fold.

Photoregulation of Ascorbate Peroxidases. The amount ofNAAP and AAP solubilized from a chloroplast preparation was

Table III. Enzyme Activities Associated with Particles Sedimenting at650 g

Spinach protoplasts were prepared and then ruptured with a syringe toyield a cell homogenate in a volume of 1.5 to 2 ml. The homogenate wascentrifuged at 650g for 1 min and separated into a supernatant fractionand pellet. In experiment 2, a portion of the pellet was washed by tworesuspensions in fresh medium and recentrifuged at 650g prior to finalresuspension. Samples were then frozen and thawed. In experiment 1, thefrozen and thawed materials were sonicated in a Branson cell disrupted(model 200) for 20 s. The sonicated sample was centrifuged at 2,000g for3 min to remove debris. The samples from the other experiment wereassayed directly without sonication and centrifugation. The data areenzyme activities associated with the 650g pellet, with the value in paren-thesis representing percent of total protoplast enzyme activity associatedwith the pellet.

Activity in 650g Pellet

Enzyme Experiment 2Experiment 1

Initial Washed

munol/mg Chl.hNADH-NAAP 8.8(20) 11.2(17) 22.1AAP 20.8(30) 18.7(32) 28.1Triose-P dehydrogenase(NADPH) 155.3(94) 123.2(65) 123.7

Catalase 6911(17) 8092(8) 2038Cytochrome oxidase 0.07(1) 4.7(17) 0.5Glucose-6-P dehydro-

genase 0 0 0

74 KOW ET AL.

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REDUCED PYRIDINE NUCLEOTIDE, ASCORBATE, H202

found to be dependent upon whether the chloroplasts were pho-tosynthesizing or kept in the dark prior to organelle rupture. Forexample, NADH-NAAP (Fig. 3A) and AAP (Fig. 3B) declined to20 to 30% in photosynthesizing spinach chloroplasts when com-pared to the enzymic activity in dark-kept chloroplasts. Theinhibitory effect of light was reversible inasmuch as NADH-NAAP and AAP increased once the light was turned off. Thelight-dark response was the same regardless ofwhether chloroplastincubation and lysis was carried out at pH 7.1 or 8.1.

Light treatment also reduced the level of NADH-NAAP (Fig.4A) and AAP (not shown) solubilized from S. praealtum chloro-plasts. NADPH-dependent malate dehydrogenase (Fig. 4B) wasassayed as a control in order to demonstrate parallel photoregu-lation of a chloroplastic enzyme which is activated in the lightand inhibited in the dark.

Several compounds eliminated the effect of light on spinachchloroplastic NADH-NAAP. Enzymic activity was not inhibitedin the light when chloroplasts were incubated with 10 ,tm DCMU,a concentration which completely blocked photosynthesis. Theaddition of 25 ,UM FeSO4 to the chloroplast incubation mixturealso eliminated the light-mediated inhibition of NADH-NAAP.In a typical experiment with spinach chloroplasts, the rates ofpmol/mg Chl h ofNADH-NAAP were: chloroplasts in dark for10 min, 4.9; chloroplasts photosynthesizing for,10 min, 2.5; chlo-roplasts kept in dark for 10 min + 25 tLM FeSO4, 11.2; andchloroplasts photosynthesizing for 10 min + 25 !s? FeSO4, 12.9.

DISCUSSION

In every plant assayed, whether the test material was leaf, seed,chloroplast or four green and one red alga, both AAP and NAAPwere found together. The results of our study do not rule out thepossibility that NAAP is the same protein as AAP, or alternatively,a combination of AAP and some other protein. Thus, it is apossibility that we are measuring ascorbate peroxidase followedby a reoxidation of NAD(P)H with the resulting dehydroascor-bate. Several features of the NAAP enzyme may be mentionedwhich distinguished it from other peroxidase preparations whichhave been reported.

Beevers (6) has described a NADH oxidation system in cucum-ber extracts which is stimulated by ascorbate, extremely sensitiveto sulihydryl reagents and cyanide but lack of inhibition bycatalase discounts similarity to NAAP. Horseradish peroxidasehas been reported to oxidize reduced pyridine nucleotide in the

M

, 6

A DARK

o 0 - ~~~~~~~~~~~20'44 ~~~~~~~~~LIGHT-J ~~~~~~~~~OFF.<

LIGHT0FF 10 20 30 40

10 20 30 40 MINUTESMINUTES

FIG. 3. Effect of light on NADH-NAAP and AAP from spinach chlo-roplasts. Spinach chloroplasts, isolated by rapid mechanical isolation, wereincubated at 250C in a medium containing 50 mm Hepes-NaOH (pH 7.0),0.25 mm KH2PO4, 330 mM sorbitol, 5 mim NaHCO3, 2 mim disodiumEDTA, 1 mm MgCl2, and 1 mM MnCl2 in total volume of 1.0 ml. Thechloroplasts were 50 to 60% intact by ferricyanide assay and fixed CO2 ata rate of40 to 60 ,umol/mg Chl * h. Half of the chloroplasts were incubatedin saturating light for photosynthesis (0), while the other half wereincubated in complete darkness (A). Samples (0.1 ml) were taken at theindicated times, diluted with 0.9 ml 50 mm Hepes-NaOH (pH 7.0), andcentrifuged at 10,000g for 30 s with a Beckmann microfuge B. Enzymeactivity was assayed using 0.9 ml ofthe chloroplast extract. NADH-NAAP(A) and AAP (B) were measured with different chloroplast preparations.

presence of catalytic amount of certain phenols and Mn2' and thereaction is sensitive to cyanide and catalase (1). NAAP clearlydiffers from horseradish peroxidase since ascorbate and Fe2' donot substitute for phenol and Mn2' in the reaction described byAkazawa and Conn (1) and furthermore, their preparation wasinsensitive to pyrophosphate.Our preparation of spinach NAAP has properties very similar

to those reported for the spinach chloroplastic membrane-boundenzyme of Groden and Beck (11), and the soluble, cytoplasmic-located enzymes of Kelly and Latzko (15) and Skigeoka et al (21,22) obtained from peas and E. gracilis, respectively. For example,in addition to reasonably similar Km values for ascorbate andH202, NAAP mimics the spinach, pea and Euglena AAP withrespect to sensitivity to cyanide (11, 15, 21, 22), insensitivity tosulfhydryl inhibitors (21) and the role of Fe2+ (21). In contrast,the pH profiles differ inasmuch as the optimum for spinachNAAPis 6.8 which compares with 6.2 for Euglena (21) and 7.5 to 8.0 forspinach chloroplastic membrane bound peroxidase (11).NADH-NAAP is presumably a hemoprotein like other plant

peroxidases (18) and this is indicated by cyanide inhibition. Butloss of enzymic activity by the Fe2+ chelator, a,a-dipyridyl, andrestoration of activity by Fe2+ following dialysis indicates thatnoncovalently bound iron may also play a role. It is also possiblethat the inhibition of NADH-NAAP by DTT may, in part, beassociated with the heavy metal chelation property of the dithiols.The AAP purified from Euglena by Shigeoka et aL (21) has theinteresting property of extreme lability in the absence of Fe2 .This instability would appear to be associated with the loss ofFe2+during purification of their enzyme and is similar to the instabilitywe find for NADH-NAAP. The ferrous form of iron has beenimplicated in a number of plant enzymes, notably aconitase andraminolevulinate synthase but its cofactor or structural role inthose enzymes (19) and in NADH-NAAP remain to be deter-mined.A portion of the cells' NADH-NAAP is located in the chloro-

plasts ofspinach and S. praealtum (Table III, Fig. 4). Both NADH-NAAP and its AAP activity are lower following incubation of

10 2MINUTES

FIG. 4. Effect of light on NADH-NAAP and malate dehydrogenasefrom S. praealtum chloroplasts. S. praealtum chloroplasts, isolated fromprotoplasts, were incubated and sampled in a procedure identical to theone used for spinach chloroplasts in Figure 3, with the exception that thechloroplasts were incubated at 30 C. Chloroplast samples were assayed forNADH-NAAP (A) or malate dehydrogenase (B). Chloroplasts were in-cubated continuously in dark (A), or incubated in the light (0) and thendarkness (arrow).

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Plant Physiol. Vol. 69, 1982

spinach and S. praealtum chloroplasts under photosynthesizingconditions. An inhibition by light has been reported for glucose-6-P dehydrogenase from spinach chloroplasts (3) and pea leafphosphofructokinase, a cytoplasmic enzyme (13). The photo-regulation observed here for NADH-NAAP is consistent with thehypothesis advanced by others (2, 7) that light-generated reducingagents regulated metabolism by inhibiting and stimulating certainenzymic steps. Light-mediated activation or inactivation of en-

zyme activity appears to involve a thiol-disulfide reaction on themodulatable protein and can usually be duplicated in vitro byDTT. Thus, inactivated glucose-6-P dehydrogenase (3), phospho-fructokinase (13), and NADH-NAAP reported here are all in-hibited by DTT but at least with respect to NADH-NAAP,inhibition by the dithiol does not seem to be compatible with theineffectiveness of iodoacetamide, N-ethylmaleimide, and arsenite.

Light-mediated inhibition of NADH-NAAP is abolished whenthe photosynthesizing chloroplasts are incubated with 10 ptMDCMU. This finding is readily explained on the need of a

reductant generated during the photochemical act. On the otherhand, the apparent regulatory role of Fe2+ on NADH-NAAP isnot readily explained on the basis of current concepts of interac-tion between the photochemical act and enzymic activity.

Finally, inasmuch as H202 has been shown to inhibit photosyn-thetic activity in isolated chloroplasts (14, 20), then chloroplast-localized ascorbate peroxidase activity may be a protective mech-anism to counter H202 production which is not destroyed bydiffusion into other cellular compartments. Indeed, Groden andBeck (11) have proposed that the physiological role of chloroplas-tic peroxidatic activity is H202 detoxification which is producedin the chloroplast by photosynthetic 02 reduction. Our observa-tions that NADH-NAAP functions optimally at a pH (Fig. 2),similar to that of the stromal pH of darkened chloroplasts (12)and that the soluble chloroplastic peroxidase activity is inhibitedduring photosynthesis (Figs. 3 and 4) are consistent with thenotion that this enzyme functions primarily during dark metabo-lism in the chloroplast. We suggest that a possible role for NAAPin the chloroplast would be to recycle reduced pyridine nucleotidegenerated from such metabolic activity as starch breakdown eitherby glycolysis or the oxidative pentose-P cycle.

Acknowledgments-We thank Nancy O'Donoghue, Rene Gfeller, and DwightPeavey for growing the algal cultures and to Dr. J. A. Schiff for the culture of E.gracilis.

LITERATURE CITED

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2. ANDERSON LE 1979 Interaction between photochemistry and activity ofenzymes.In M Gibbs, E. Latzko, eds, Photosynthesis II: Photosynthetic Carbon Metab-olism and Related Processes. Springer-Verlag, New York, pp 271-281

3. ANDERSON LD, JX DUGGAN 1976 Light modulation of glucose-6-phosphatedehydrogenase. Partial characterization ofthe light inactivation system and itseffect on the properties of the chloroplastic and cytoplasmic forms of theenzyme. Plant Physiol 58: 135-139

4. ARNON DI 1949 Copper enzymes in isolated chloroplasts. Polyphenoloxidase inBeta vulgaris. Plant Physiol 24: 1-15

5. BRADFORD MM 1976 A rapid and sensitive method for the determination ofmicrogram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem 72: 248-254

6. BEEvERs H 1954 The oxidation of reduced diphosphopyridine nucleotide by anascorbate system from cucumber. Plant Physiol 29: 265-269

7. BUCHANAN BB 1980 Role of light in the regulation of chloroplast enzymes. AnnuRev Plant Physiol 31: 341-374

8. FOYER CH, B HALLIWELL 1976 The presence of glutathione and glutathionereductase in chloroplasts: a proposed role in ascorbic acid metabolism. Planta133: 21-25

9. GORMAN DS, LEVINE RP 1965 Cytochromefand plastocyanin: their sequence inthe photosynthetic electron transport chain of Chlamydomonas reinhardii ProcNatl Acad Sci USA 54: 1665-1669

10. GREENBLATT LL, JA SCHIFF 1959 A pheophytin-like pigment in dark-adaptedEuglena gracilis J Protozool 6: 23-28

11. GRODEN D, E BECK 1979 H202 destruction by ascorbate-dependent systems fromchloroplasts. Biochim Biophys Acta 546: 426-435

12. HELDT HW, K WERDAN, M MILOVANCEV, G GELLER 1973 Alkalization of thechloroplast stroma caused by light dependent protein flux into the thylakoidspace. Biochim Biophys Acta 314: 224-241

13. KACHRU RB, LE ANDERSON 1975 Inactivation of pea leaf phosphofructokinaseby light and dithiothreitol. Plant Physiol 55: 199-202

14. KAISER W 1976 The effect of hydrogen peroxide on CO2 fixation of isolatedintact chloroplasts. Biochim Biophys Acta 440: 476-482

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