Plant Physiol. (1982) 69, 72-76 0032-0889/82/69/0072/05/$00.50/0

Oxidation of Reduced Pyridine Nucleotide by a System Using Ascorbate 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 GIBBS Institutefor Photobiology of Cells and Organelles, Brandeis University, Waltham, Massachusetts 02254

ABSTRACT Ofthe soluble , the most characterized is the Euglena gracilis which is a hemoprotein (21) like many other plant A NAD(P)H oxiding system (NAAP) was detected and partially peroxidases (18). Euglena AAP is an unusual hemoprotein in that purife from leaves of spinh and Sedwn praeabu, seeds and leaves of it is stabilized by sucrose and by ferrous sulfate. In addition, the pea and cells of green and red algae which oxiized NAD(P)H in the enzyme has high affinities for ascorbate and H202, with Km values presence of ascorbate and H202. of410 and 56 ILK respectively (21). The insoluble spinach enzyme The partially-purified spinach system had substrate K. values of 5 has similar substrate affinities and also appears to be a hemopro- micromolar for NADH, 50 micromolar for H202, and 300 micromolar for tein. i-ascorbic acid at the pH opthnum of 6.8 NADH was a better electron We report here on a peroxidative activity similar to AAP in donor than NADPH. Among other electron donors, isoascorbic acid had peas, spinach, Sedum praealtum and algae. This enzyme system considerable activty but hydroquinone and resorcinol had only weak designated as NAAP uses NAD(P)H, ascorbate, and H202 as activities. The enzyme was inhbited by cyanide, a,a'-dipyridyl, an mono- substrates. Furthermore, this system can be solubilized from the and di-thol reagents. Inhibition by thiol-reagents was partially restored by chloroplast and is subject to photoregulation. Fe'+ as was enzyic actvity lost following dialysis against buffer. Subcelular locization studies with spinach and S. praeaiuu leaves indicated that a portion of the cei's NAAP was in the chloroplast fraction. MATERIALS AND METHODS Photosynthetic conditions resulted in a decrease in this actvity solubilized Plants and Algae. S. praealtum and peas (Pisum sativum var. from spinach and S. praealuw chrwoplasts. The presence of 3-(3,4-di- Progress No. 9) were grown in the greenhouse. Sedum and peas chlorophenyl)-1,1-dimethylurea or Fe2' in the incubation medium elimi- were grown in a vermiculite-soil mixture or in vermiculite, respec- nated the light-mediated inhibition of NAAP. tively. Spinach (Spinacia oleracea) was cultivated under controlled NAAP may function in the recycling ofNAD(P)H generated in the dark conditions (12 h light at 22°C and 12 h dark at 18°C) in a within the chloroplast. Inasmuch as all preparations of NAAP contained vermiculite-soil mixture. The algae with the exception of Chla- ascorbate activity, the data do not rule out the possibility that mydomonas reinhardii F-60 (9) and Euglena gracilis (10) were NAAP is the same protein as ascorbate peroxidase or, alternatively, a grown photoautotrophically with CO2 as the carbon source. combination of ascorbate peroxidase and some other enzyme. Cell-Free Preparations. About 2 kg of fully expanded spinach leaves 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) at 4°C. Pea seed which were imbibed in water overnight was also disintegrated in the blender. After the homogenate was filtered through cheesecloth, the homogenate was centrifuged at 10,000g Recently, ascorbate peroxidases have been solubilized from for 10 min to remove debris. The homogenate was then fraction- several higher plants (15) and algae (15, 22) and an insoluble form ated by solid (NH4)2SO4 precipitation. The portion ofthe homog- has been localized on the thylakoids of the spinach chloroplast enate insoluble in 50 to 80%1o (NH4)2SO4 was resuspended in 50 (11). The stoichiometry for the spinach chloroplast enzyme is I mM Hepes-NaOH (pH 7.0) and absorbed onto calcium phosphate mol each of H202 and ascorbate consumed for I mol of dehy- gel. After elution from the gel with 0.1 M K-phosphate, this droascorbate formed (11) and AAP4 has been envisioned as a solution free of was used as the source of higher plant mechanism of detoxifying H202 through the use of ascorbate, an ascorbate peroxidases. The result of treatment with (NH4)2SO4 abundant constituent in Chl-containing cells. In the chloroplast, and gel was an approximate 5-fold purification. Cell-free algal at least, the presence of reduced glutathione and glutathione preparations were made by grinding the organisms with an equal reductase (8) provides a potential means of recycling dehydroas- weight ofaluminum oxide for 5 min in a chilled mortar and pestle. corbate. About 5 to 10 ml of 50 mm Hepes-NaOH buffer (pH 7.0) was added and the slurry was centrifuged at 25,000g for 10 min to 1 Supported by National Science Foundation Grant PCM 79-22612 and remove the cellular debris. The clear supernatant fluid was used Department of Energy Grant EY-76-S-3231-14. for enzyme assays. 2 Prewnt address: Department of Biochemistry, University of Wiscon- Enzyme Activity. NAAP was assayed at 250C as the oxidation sin, Madison, WI 53706. of NAD(P)H at 340 um. The 1-ml assay volume contained 50 mM 3 Present address: Department of Agronomy and Soils, Washington Hepes-NaOH (pH 7.0), 0.2 mM NAD(P)H, and 5 mm ascorbate. State University, Pullman, WA 99164. After a baseline of absorbance change was established, a reading 4 Abbreviations: AAP, ascorbate peroxidase which catalyzes the reaction was taken 1 min after the addition of 0.18 mM H202. AAP was involving ascorbate and H202; NAAP, reoxidation of reduced pyridine assayed at 25°C as the oxidation of ascorbate at 265 nm (15). The nucleotide with a preparation involving ascorbate, H202 and NAD(P)H. 1-ml assay volume contained 50 mM Hepes-NaOH (pH 7.0) and Most experiments were carried out with NADH-NAAP. 0.1 mm ascorbate. The reaction was started by adding 0.18 mM 72 REDUCED PYRIDINE NUCLEOTIDE, ASCORBATE, H202 73 H202. Malate dehydrogenase and triose-P dehydrogenase were Table I. Activity ofNAAP and AAP From Dif7erent Sources measured as the oxidation of NADPH at 340 rm. Malate dehy- Pea, spinach, S. praealtum leaf, and pea seed preparations were partially drogenase assays contained, in a final 1.1 ml volume: 50 mm purified from crude homogenates by 50 to 80% (NH4)2SO4 precipitation, Hepes-NaOH (pH 7.0), 0.11 mM NADPH, and 0.22 mm oxalace- followed by elution from calcium phosphate gel. Spinach chloroplast tate to initiate the reaction. Triose-P dehydrogenase assays con- particles were prepared by rupturing intact chloroplasts in 50 mM Hepes- tained in a final 1.0 ml volume: 50 mm Hepes-NaOH (pH 7.6), 0.2 NaOH (pH 7.0). The particulate matter was spun down and washed once mm NADPH, 1 mm glycerate-3-P, 1 mm MgCl2, 1 mm DTT, and with the same buffer. To make cell-free algal preparations, the organisms 30 units glycerate-3-P kinase. Glucose-6-P dehydrogenase was were ground with an equal weight of A1203 for 5 min. Then 5 to 10 ml measured as the reduction of NADP at 340 nm. The l.0-ml assay Hepes-NaOH (pH 7.0) buffer was added and slurry was centrifuged at medium contained 50 mm Hepes-NaOH (pH 7.6), 1 mm MgCl2, 25,000g for 10 min to remove A1203 and cellular debris. The clear 2 mm glucose-6-P, and 0.5 mM NADP. Cytochrome oxidase was supematant fluid was used for enzyme essays. measured as the oxidation of reduced Cyt c at 550 nm (24). was monitored as the breakdown of at 240 am NAAP Catalase H202 (16). Plant Sources AAP Chloroplast Isolation. Spinach leaves were chopped into small NADH NADPH pieces and ground (1.5 s) with a VirTis model 45 in a ginding medium of 50 mm Hepes-NaOH (pH 6.8), 330 mm sorbitol, 2 mM Mmol NAD(P)H/mg umol ascor- disodium EDTA, 1 mm MgCl2, and 1 mm MnCl2. The homogenate protein *mpnbate/mg was filtered through two layers ofMiracloth (Chicopee Mills, Inc., protein Miltown, NJ) with the filtrate being centrifuged at 750g for 1 min. min The chloroplast pellet was resuspended in fresh grinding medium Pea leaf 2.95 2.01 4.32 and recentrifuged. The final washed chloroplast pellet was resus- Spinach leaf 1.59 0.64 2.51 pended in a minimal volume of grinding medium and used for S. praealtum leaf 2.13 0.90 5.32 chloroplast incubations. Chloroplasts were at least 50%7v intact as Pea seed 0.25 0.14 0.03 measured by ferricyanide-stimulated 02 evolution. Spinach chloroplast particles NDa ND 1.16 In some experiments, spinach chloroplasts were prepared from E. gracilis Z 0.15 0.04 0.32 protoplasts according to the procedure ofNishimura et aL (17). In Scenedesmus obliquus 0.23 0.14 0.16 this procedure, the protoplasts were disrupted by passage through Chlamydomonas reinhardii F-60 0.16 0.20 0.68 a syringe into the grinding medium used for the spinach leaves. Chlamydomonas reinhardii 1.00 0.16 1.77 S. praealtum leaves were sliced and digested enzymically to Chlorella vulgaris (dark) 0.19 0.15 0.09 yield protoplasts as described by Spalding and Edwards (23). The Chlorella vulgaris (light) 0.12 0.10 0.04 protoplasts were gently broken by passage through a syringe and Porphyridium cnrentum 1.01 0.71 2.17 the homogenate obtained was centrifuged in a flotation gradient a Not detected. to yield a chloroplast fraction. These chloroplasts were at least 90% intact as measured by ferricyanide-stimulated 02 evolution. Chemical Assays. Protein content was determined according to Bradford (5), using standards of BSA. Chl was determined ac- cording to Arnon (4). z RESULTS I- Comparison of NAAP and AAP Activties. NAAP and AAP (Table I) coexisted in roughly equal levels in the leaf and algal Q. preparations with the possible exception of pea seed, spinach z thylakoids, and C. vulgaris (light and dark grown). Only a trace of AAP was detected in pea seed while the particulate matter from z the spinach chloroplast was free of NAAP and with respect to -J Chlorella, the ratio of NAAP:AAP averaged about 2:1. NADH at 0 0.2 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 pea leaves could be concentrated but not separated by (NH4)2SO4 fractionation and treatment with calcium phosphate gel, indicating the two enzyme activities may be associated with the same protein. Further attempts to resolve the higher plant by alcohol precipitation or by DEAE chromatography resulted in a total loss of peroxidase activity. Attempts to determined stoichiometry for [mM NADH]r NAAP were unsuccessful due to the presence of AAP activity. FIG. 1. Lineweaver-Burk plot of spinach NADH-NAAP as a function Effect of Substrate Concentration on the Rate of Spinach ofascorbate, H202, and NADH concentrations. Reaction mixtures were as NAAP. The apparent Km values were 5 ,UM for NADH, 50 um for described in Figure 2 using Hepes-NaOH as a buffer. The concentration H202, and 300 um for ascorbate (Fig. 1). The V,,. for ascorbate of ascorbate (upper), H202 (middle), and NADH (lower) were varied as was 1.07 psmol/mg protein *min, a value which contrasts with 0.47, indicated. 0.17, and 0.06 when ascorbate was replaced by 5 mM isoascorbate, hydroquinone, or resorcinol. NAAP isolated from the spinach leaf or from the spinach chlo- pH Profile of Spinach NAAP. The pH optimum for enzyme roplast was unaffected by heating for 5 minat 40°C. Heating at activity was around 6.8, both for NADH (Fig. 2) and NADPH 60°C for 5mincompletely inactivated both preparations. On the (data not shown)-linked activity. At pH 8.0 enzyme activity was other hand, only 50% of the chloroplasts membrane-bound AAP less that 25% of the rate at pH 6.8. was destroyed after 5 minat 60°C. Effect of Temperature on the Stability of NAAP and AAP. Inhibition Studies with Spinach and Pea NADH-NAAP. Several 74 KOW ET AL. Plant Physiol. Vol. 69, 1982

2.0 1 mm NaN3. The Fe2+ chelator, a,a'-dipyridyl at 0.2 mm inhibited by 70%o Z spinach NAAP while the chelators of Fe3+ (Tiron) and of Cu2+ (diethyldithiocarbamate) were completely ineffective at 0.2 mm 1.5 w levels. The addition of 10 mm pyrophosphate inhibited the enzyme by 80%o. Effect of Fe2 on Spinach NADH-NAAP. Since inhibition by

0~ pyrophosphate and cyanide indicated participation of a metal ion and the finding with a,a-dipyridyl specified Fe2 , this property of NAAP was studied in some detail. Half of the NADH-linked peroxidase activity was lost after 1 h 0 I dialysis 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 was included in the dialyzing fluid. The activity ofthe dialyzed enzyme 6 7 8 9 was completely restored on addition of 10 to 25 ,UM Fe2+. Higher concentrations (50-100 ,iM) of Fe2+ severely inhibited. Bound iron in the form of ferredoxin or ferrocyanide was not active in FIG. 2. pH profile of spinach NADH-NAAP. Reaction was monitored restoring activity lost during dialysis. Other ions such as Zn2+ by following the oxidation of 0.2 mM NADH in the presence of 5 mm Mg2+, Co2+, Ni +, MoO42+ at 25 ,UM had either no effect or were ascorbate and 0.18 mm H202 in the appropriate buffer (100 mM) as inhibitory. indicated: (0), phosphate; (M), Hepes buffer, (A), Tricine buffer. Cellular Localization of NAAP and AAP. The data recorded in Table I indicated the presence of AAP but not to NAAP bound Table II. Inhibitors ofSpinach NADH-NAAP to the spinach chloroplastic thylakoids. On lysis of spinach pro- Spinach preparations were incubated in the presence of the indicated toplasts, about 20 to 30%o of the total NADH-linked ascorbate compounds for a period (usually 2-5 min) at 25°C prior to enzyme assay. peroxidase and AAP was found to be associated with a 650g pellet The control rate was 1.3 pmol/mg protein.min. which contained the bulk of the NADPH-glyceraldehyde-3-P dehydrogenase, a chloroplast marker (Table III). Another inter- Inhibitor Activity esting feature of Table III is the finding that repeated washing of this fraction in an isotonic medium did not change the specific Control 100 activity on a Chl basis of either NADPH-glyceraldehyde-3-P KCN, 0.1 mM 30 dehydrogenase or the ascorbate peroxidases while the specific 2,3-Dimercaptopropanol, 25 ,UM 30 activity of catalase, the microbody marker and Cyt c oxidase, the L-Cysteine, 0.2 mm 50 mitochondrial marker decreased several fold. Mercaptoethanol, 0.3 mM 30 Photoregulation of Ascorbate Peroxidases. The amount of DTT, 0.3 mM 30 NAAP and AAP solubilized from a chloroplast preparation was a,a'-Dipyridyl, 0.2 mM 30 Tiron, 0.2 mM 105 III. Enzyme Activities Associated with Particles Sedimenting at Diethyldithiocarbamate, 0.2 mM 99 Table Pyrophosphate, 10 mM 18 650 g Spinach protoplasts were prepared and then ruptured with a syringe to classes of compounds were tested as inhibitors with the 5-fold yield a cell homogenate in a volume of 1.5 to 2 ml. The homogenate was purified spinach NADH-NAAP (Table II). Cyanide at 0.1 mm centrifuged at 650g for 1 min and separated into a supernatant fraction inhibited enzyme activity by 70%o. Incubation with sulfhydryl and pellet. In experiment 2, a portion of the pellet was washed by two reagents such as 2,3-dimercaptoethanol, DTT, cysteine, and mer- resuspensions in fresh medium and recentrifuged at 650g prior to final captoethanol reduced enzyme activity but 2 mm reduced glutathi- resuspension. Samples were then frozen and thawed. In experiment 1, the one had no effect. The degree of inhibition by these -SH con- frozen and thawed materials were sonicated in a Branson cell disrupted taining compounds was dependent upon the order of substrate (model 200) for 20 s. The sonicated sample was centrifuged at 2,000g for addition. For example, 25 ,pm 2,3-dimercaptopropanol and 300 3 min to remove debris. The samples from the other experiment were p,m 2-mercaptoethanol inhibited the peroxidase by 70%o ifthe thiol assayed directly without sonication and centrifugation. The data are compound was incubated for 5 min with the enzyme preparation enzyme activities associated with the 650g pellet, with the value in paren- prior to the addition of 5 mM ascorbate. If the ascorbate was thesis representing percent of total protoplast enzyme activity associated added first, the inhibitory effect ofthe thiol reagents were reduced with the pellet. to about 10o. Activity in 650g Pellet Several oxidized compounds were tested to determine if they could reverse the dithiol inhibition of NADH-linked ascorbate Enzyme Experiment 2 peroxidase. Dehydroascorbate, oxidized glutathione, or oxidized Experiment 1 DTT at 1 mm had no effect on the peroxidase. When NAAP was Initial Washed incubated for 5 min with 0.1 mM DTT it was inhibited 60%o and munol/mg Chl.h the inhibition value was reduced to 30%o on addition of 1 mm dehydroascorbate but 1 mm oxidized DTT or 1 mm oxidized NADH-NAAP 8.8(20) 11.2(17) 22.1 glutathione was without effect. AAP 20.8(30) 18.7(32) 28.1 The pea leaf NADH-linked peroxidase showed similar re- Triose-P dehydrogenase sponses to the reagents listed in Table II. Furthermore, the pea (NADPH) 155.3(94) 123.2(65) 123.7 leafperoxidase was unaffected by inhibitors like arsenite (1.0 mM), Catalase 6911(17) 8092(8) 2038 iodoacetamide (5 mM), and N-ethylmaleimide (5 mM) indicating Cytochrome oxidase 0.07(1) 4.7(17) 0.5 that a sul&hydryl group is not required for enzyme activity. The Glucose-6-P dehydro- enzyme was inhibited 50Yo by a 5 mm NaN3 but little affected by genase 0 0 0 REDUCED PYRIDINE NUCLEOTIDE, ASCORBATE, H202 75 found to be dependent upon whether the chloroplasts were pho- presence of catalytic amount of certain phenols and Mn2' and the tosynthesizing or kept in the dark prior to organelle rupture. For reaction is sensitive to cyanide and catalase (1). NAAP clearly example, NADH-NAAP (Fig. 3A) and AAP (Fig. 3B) declined to differs from since ascorbate and Fe2' do 20 to 30% in photosynthesizing spinach chloroplasts when com- not substitute for phenol and Mn2' in the reaction described by pared to the enzymic activity in dark-kept chloroplasts. The Akazawa and Conn (1) and furthermore, their preparation was inhibitory effect of light was reversible inasmuch as NADH- insensitive to pyrophosphate. NAAP and AAP increased once the light was turned off. The Our preparation of spinach NAAP has properties very similar light-dark response was the same regardless ofwhether chloroplast to those reported for the spinach chloroplastic membrane-bound incubation and lysis was carried out at pH 7.1 or 8.1. enzyme of Groden and Beck (11), and the soluble, cytoplasmic- Light treatment also reduced the level of NADH-NAAP (Fig. located enzymes of Kelly and Latzko (15) and Skigeoka et al (21, 4A) and AAP (not shown) solubilized from S. praealtum chloro- 22) obtained from peas and E. gracilis, respectively. For example, plasts. NADPH-dependent malate dehydrogenase (Fig. 4B) was in addition to reasonably similar Km values for ascorbate and assayed as a control in order to demonstrate parallel photoregu- H202, NAAP mimics the spinach, pea and Euglena AAP with lation of a chloroplastic enzyme which is activated in the light respect to sensitivity to cyanide (11, 15, 21, 22), insensitivity to and inhibited in the dark. sulfhydryl inhibitors (21) and the role of Fe2+ (21). In contrast, Several compounds eliminated the effect of light on spinach the pH profiles differ inasmuch as the optimum for spinach NAAP chloroplastic NADH-NAAP. Enzymic activity was not inhibited is 6.8 which compares with 6.2 for Euglena (21) and 7.5 to 8.0 for in the light when chloroplasts were incubated with 10 ,tm DCMU, spinach chloroplastic membrane bound peroxidase (11). a concentration which completely blocked photosynthesis. The NADH-NAAP is presumably a hemoprotein like other plant addition of 25 ,UM FeSO4 to the chloroplast incubation mixture peroxidases (18) and this is indicated by cyanide inhibition. But also eliminated the light-mediated inhibition of NADH-NAAP. loss of enzymic activity by the Fe2+ chelator, a,a-dipyridyl, and In a typical experiment with spinach chloroplasts, the rates of restoration of activity by Fe2+ following dialysis indicates that pmol/mg Chl h of NADH-NAAP were: chloroplasts in dark for noncovalently bound iron may also play a role. It is also possible 10 min, 4.9; chloroplasts photosynthesizing for,10 min, 2.5; chlo- that the inhibition of NADH-NAAP by DTT may, in part, be roplasts kept in dark for 10 min + 25 tLM FeSO4, 11.2; and associated with the heavy metal chelation property ofthe dithiols. chloroplasts photosynthesizing for 10 min + 25 !s? FeSO4, 12.9. The AAP purified from Euglena by Shigeoka et aL (21) has the interesting property of extreme lability in the absence of Fe2 . DISCUSSION This instability would appear to be associated with the loss ofFe2+ during purification oftheir enzyme and is similar to the instability In every plant assayed, whether the test material was leaf, seed, we find for NADH-NAAP. The ferrous form of iron has been chloroplast or four green and one red alga, both AAP and NAAP implicated in a number of plant enzymes, notably aconitase and were found together. The results of our study do not rule out the raminolevulinate synthase but its or structural role in possibility that NAAP is the same protein as AAP, or alternatively, those enzymes (19) and in NADH-NAAP remain to be deter- a combination of AAP and some other protein. Thus, it is a mined. possibility that we are measuring ascorbate peroxidase followed A portion of the cells' NADH-NAAP is located in the chloro- by a reoxidation of NAD(P)H with the resulting dehydroascor- plasts ofspinach and S.praealtum (Table III, Fig. 4). Both NADH- bate. Several features of the NAAP enzyme may be mentioned NAAP and its AAP activity are lower following incubation of which distinguished it from other peroxidase preparations which have been reported. Beevers (6) has described a NADH oxidation system in cucum- ber extracts which is stimulated by ascorbate, extremely sensitive to sulihydryl reagents and cyanide but lack of inhibition by catalase discounts similarity to NAAP. Horseradish peroxidase has been reported to oxidize reduced pyridine nucleotide in the

A DARK

M

, 6 o44 0 ~~~~~~~~~LIGHT- ~~~~~~~~~~~20' -J ~~~~~~~~~OFF.< LIGHT 0FF 10 20 30 40 10 20 30 40 MINUTES MINUTES FIG. 3. Effect of light on NADH-NAAP and AAP from spinach chlo- roplasts. Spinach chloroplasts, isolated by rapid mechanical isolation, were incubated 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 disodium 10 2 EDTA, 1 mm MgCl2, and 1 mM MnCl2 in total volume of 1.0 ml. The MINUTES chloroplasts were 50 to 60% intact by ferricyanide assay and fixed CO2 at FIG. 4. Effect of light on NADH-NAAP and malate dehydrogenase a rate of40 to 60 ,umol/mg Chl * h. Half ofthe chloroplasts were incubated from S. praealtum chloroplasts. S. praealtum chloroplasts, isolated from in saturating light for photosynthesis (0), while the other half were protoplasts, were incubated and sampled in a procedure identical to the incubated in complete darkness (A). Samples (0.1 ml) were taken at the one used for spinach chloroplasts in Figure 3, with the exception that the indicated times, diluted with 0.9 ml 50 mm Hepes-NaOH (pH 7.0), and chloroplasts were incubated at 30 C. Chloroplast samples were assayed for centrifuged at 10,000g for 30 s with a Beckmann microfuge B. Enzyme NADH-NAAP (A) or malate dehydrogenase (B). Chloroplasts were in- activity was assayed using 0.9 ml ofthe chloroplast extract. NADH-NAAP cubated continuously in dark (A), or incubated in the light (0) and then (A) and AAP (B) were measured with different chloroplast preparations. darkness (arrow). 76 KOW ET AL. Plant Physiol. Vol. 69, 1982 spinach and S. praealtum chloroplasts under photosynthesizing 2. ANDERSON LE 1979 Interaction between photochemistry and activity ofenzymes. In M Gibbs, E. Latzko, eds, Photosynthesis II: Photosynthetic Carbon Metab- conditions. An inhibition by light has been reported for glucose- olism and Related Processes. Springer-Verlag, New York, pp 271-281 6-P dehydrogenase from spinach chloroplasts (3) and pea leaf 3. ANDERSON LD, JX DUGGAN 1976 Light modulation of glucose-6-phosphate phosphofructokinase, a cytoplasmic enzyme (13). The photo- dehydrogenase. Partial characterization ofthe light inactivation system and its regulation observed here for NADH-NAAP is consistent with the effect on the properties of the chloroplastic and cytoplasmic forms of the enzyme. Plant Physiol 58: 135-139 hypothesis advanced by others (2, 7) that light-generated reducing 4. ARNON DI 1949 Copper enzymes in isolated chloroplasts. Polyphenoloxidase in agents regulated metabolism by inhibiting and stimulating certain Beta vulgaris. Plant Physiol 24: 1-15 enzymic steps. Light-mediated activation or inactivation of en- 5. BRADFORD MM 1976 A rapid and sensitive method for the determination of zyme activity appears to involve a thiol-disulfide reaction on the microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254 modulatable protein and can usually be duplicated in vitro by 6. BEEvERs H 1954 The oxidation of reduced diphosphopyridine nucleotide by an DTT. Thus, inactivated glucose-6-P dehydrogenase (3), phospho- ascorbate system from cucumber. Plant Physiol 29: 265-269 fructokinase (13), and NADH-NAAP reported here are all in- 7. BUCHANAN BB 1980 Role oflight in the regulation ofchloroplast enzymes. Annu hibited by DTT but at least with respect to NADH-NAAP, Rev Plant Physiol 31: 341-374 8. FOYER CH, B HALLIWELL 1976 The presence of glutathione and glutathione inhibition by the dithiol does not seem to be compatible with the reductase in chloroplasts: a proposed role in ascorbic acid metabolism. Planta ineffectiveness of iodoacetamide, N-ethylmaleimide, and arsenite. 133: 21-25 Light-mediated inhibition of NADH-NAAP is abolished when 9. GORMAN DS, LEVINE RP 1965 Cytochromefand plastocyanin: their sequence in the photosynthesizing chloroplasts are incubated with 10 the photosynthetic electron transport chain of Chlamydomonas reinhardii Proc ptM Natl Acad Sci USA 54: 1665-1669 DCMU. This finding is readily explained on the need of a 10. GREENBLATT LL, JA SCHIFF 1959 A pheophytin-like pigment in dark-adapted reductant generated during the photochemical act. On the other Euglena gracilis J Protozool 6: 23-28 hand, the apparent regulatory role of Fe2+ on NADH-NAAP is 11. GRODEN D, E BECK 1979 H202 destruction by ascorbate-dependent systems from not readily explained on the basis of current concepts of interac- chloroplasts. Biochim Biophys Acta 546: 426-435 12. HELDT HW, K WERDAN, M MILOVANCEV, G GELLER 1973 Alkalization of the tion between the photochemical act and enzymic activity. chloroplast stroma caused by light dependent protein flux into the thylakoid Finally, inasmuch as H202 has been shown to inhibit photosyn- space. Biochim Biophys Acta 314: 224-241 thetic activity in isolated chloroplasts (14, 20), then chloroplast- 13. KACHRU RB, LE ANDERSON 1975 Inactivation of pea leaf phosphofructokinase localized ascorbate peroxidase activity may be a protective mech- by light and dithiothreitol. Plant Physiol 55: 199-202 14. KAISER W 1976 The effect of hydrogen peroxide on CO2 fixation of isolated anism to counter H202 production which is not destroyed by intact chloroplasts. Biochim Biophys Acta 440: 476-482 diffusion into other cellular compartments. Indeed, Groden and 15. KELLY GJ, E LATZKO 1979 Soluble ascorbate peroxidase detection in plants and Beck (11) have proposed that the physiological role of chloroplas- use in estimation. Naturwissenschaften 66: 617-618 tic peroxidatic activity is H202 detoxification which is produced 16. LUCK H 1965 Catalase In HU Bergmeyer, ed, Methods of Enzymatic Analysis. Elsevier, Amsterdam, pp 885-894 in the chloroplast by photosynthetic 02 reduction. Our observa- 17. NismsnmuR M, D GRAHAM, T AKAZAWA 1976 Isolation of intact chloroplasts tions that NADH-NAAP functions optimally at a pH (Fig. 2), and other cell organelles from spinach leaf protoplasts. Plant Physiol 58: 309- similar to that of the stromal pH of darkened chloroplasts (12) 314 and that the soluble chloroplastic peroxidase activity is inhibited 18. PAUL KG 1963 Peroxidases. In PD Boyer, H Lardy, K Myrback, eds, The Enzymes, Vol 8. Academic Press, New York, pp 227-274 during photosynthesis (Figs. 3 and 4) are consistent with the 19. RAINs DW 1976 Mineral Metabolism. In J Bonner, JE Varner, eds, Plant notion that this enzyme functions primarily during dark metabo- Biochemistry, 3rd Ed. Academic Press, New York, pp 561-597 lism in the chloroplast. We suggest that a possible role for NAAP 20. ROBINsoN JM, MG SMITH, M GIBBS 1980 Influence of hydrogen peroxide upon in the chloroplast would be to recycle reduced pyridine nucleotide carbon dioxide photoassimilation in the spinach chloroplast. 1. Hydrogen peroxide generated by broken chloroplasts in an "intact" chloroplast prepara- generated from such metabolic activity as starch breakdown either tion is a causal agent of the Warburg effect. Plant Physiol 65: 755-759 by glycolysis or the oxidative pentose-P cycle. 21. SHIGEOKA S, Y NAKANO, S KITAOKA 1980 Purification and some properties of L-ascorbic acid-specific peroxidase in Euglena gracilis Z. Arch Biochem Bio- Acknowledgments-We thank Nancy O'Donoghue, Rene Gfeller, and Dwight phys 201: 121-127 Peavey for growing the algal cultures and to Dr. J. A. Schiff for the culture of E. 22. SHIGEOKA S, Y NAKANo, S KITAOKA 1980 Metabolism of hydrogen peroxide in gracilis. Euglena gracilis Z by L-ascorbic acid peroxidase. Biochem J 186: 377-380 23. SPALDING MH, GE EDwARDs 1980 Photosynthesis in isolated chloroplasts ofthe LITERATURE CITED Crassulacean acid metabolism plant Sedumpraealtumn Plant Physiol 65: 1044- 1048 1. AKAZAWA T, EE CONN 1958 The oxidation of reduced pyridine nucleotides by 24. WHARTON DC, A TZAGOLOFF 1967 Cytochrome oxidase from beef heart mito- peroxidase. J Biol Chem 232: 403-415 chondria. Methods Enzymol 10: 245-250