Plant Physiol. (1988) 86, 098-103 0032-0889/88/86/0000/06/$01.00/0 Dihydroxyacetone Phosphate Reductase in Plants' Received for publication May 19, 1987 and in revised form September 10, 1987 ROBERT W. GEE, RICHARD U. BYERRUM*, DENNIS W. GERBER, AND N. E. TOLBERT Michigan State University, Department ofBiochemistry, East Lansing, Michigan 48824 ABSTRACr Two forms of dihydroxyacetone phosphate reductase are present in spinach, soybean, pea, and mesophyll cells of corn leaves. An improved homogenizing medium was developed to measure this activity. The en- zyme was detectable only after dialysis of the 35 to 70% saturated (NH4)2S04 fraction and the two forms were separated by chromatography on either DEAE cellulose or Sephacryl S-200. About 80% of the reduc- tase was one form in the chloroplast and the rest was a second form in the cytosol as determined by chromatography and by fractionation of subcellular organelles. The amount of activity detectable in the chloro- plast fraction was 10.7 micromoles of dihydroxyacetone phosphate re- ductase per hour per milligram chlorophyll from spinach leaves and 4.9 from pea leaves. The chloroplast form eluted first from DEAE cellulose and, being smaller, it eluted second from Sephacryl S-200. Activity of the chloroplast form was stimulated 3- to 5-fold by the addition of 1 millimolar dithiothreitol or 50 microgram reduced Escherichia coli thio- redoxin or 4 micrograms spinach thioredoxin to the assay mixture. This stimulation was not observed with monothiols. Activity of the cytosolic form was not affected by either reduced thioredoxin or dithiothreitol. In plant metabolism glycerol phosphate must be produced for the synthesis of various phospholipids, sulfolipids, galactolipids, and triglycerides. The metabolic reaction in leaf tissue of higher plants for glycerol phosphate synthesis is catalyzed by a DHAP2 reductase which has been partially purified from spinach leaves and castor bean endosperm, and which catalyzes the reduction of DHAP at pH 7.0 using NADH as the reductant (6, 17). In the reverse reaction the enzyme nomenclature lists it as a sn-glycerol phosphate:NAD oxidoreductase or dehydrogenase (EC 1.1.1.8). Because the activity as a dehydrogenase requires a pH of around 9.5 and very high substrate concentrations, no physiological significance is attached to the dehydrogenase activity and the enzyme will be referred to as DHAP reductase. DHAP is a central compound for several metabolic pathways. It can be converted in the chloroplast by the C3 photosynthetic carbon cycle to hexoses for starch synthesis or for regeneration of ribulose bisphosphate. DHAP is the transport component of the triose phosphate shuttle out of the chloroplast to the cytosol. In the cytosol it can be converted to hexoses, sucrose, and cell wall constituents or oxidized by glycolysis to pyruvate. In both the chloroplast and cytosol, DHAP can be reduced to glycerol phosphate by the two DHAP reductases described in this paper. Because of the number of reactions DHAP may undergo, its Supported by United States Department of Agriculture Grant 86- CRCR-1-2135 and published as Michigan Agricultural Experiment Sta- tion Report No. 12290. 2Abbreviations: DHAP, dihydroxyacetone phosphate; ME, mercap- toethanol; PVPP, polyvinylpolypyrrolidone. metabolism must be highly controlled, as is expected of com- pounds and enzymes at metabolic branch points. A number of free living algae produce glycerol as a major product of photosynthesis rather than accumulate starch or sucrose. Zooxanthellae, symbiotic, unicellular algae in the polyps of reef building corals and other marine invertebrates, excrete to their host up to 40% of their photosynthate as glycerol (14, 15). The halotolerant alga, Dunaliella tertiolecta (5, 20) and Chia- mydomonas (1 1) can synthesize and accumulate large amounts of glycerol as an osmoregulator. Haus and Wegmann (9) have partially purified a glycerol phosphate dehydrogenase (measured as DHAP reductase) in D. tertiolecta and Brown et al. (4) have shown that in this organism the dehydrogenase is in the chloro- plast. It is, therefore, possible for algae to synthesize glycerol as a major end product of photosynthesis. To date there is little information about the amounts of glycerol phosphate and free glycerol which exist in higher plants, even though the reaction pathway for their formation from the first triose phosphate formed in CO2 fixation in photosynthesis involves only DHAP reductase and glycerol phosphate phosphatase reactions. In ad- dition, essentially nothing is known about the regulation of DHAP metabolism to form glycerol phosphate and glycerol in higher plants. The purpose of the present study has been to look for the occurrence of DHAP reductase in a variety of plants and to determine its subcellular distribution and some aspects of its regulation. MATERIALS AND METHODS Plant Material. Plants used were corn (Zea mays L. cv Great Lakes 5922), castor bean (Ricinus communis L. cv 1778 Sangui- neus), soybean (Glycine max L. cv MSU 1066), pea (Pisum sativum L. cv Dwarf Progress 9), and spinach (Spinacia oleracea L. cv Longstanding Bloomsdale). Plants were grown in the greenhouse supplemented with high output, cool-white fluores- cent lights for 8 h/d at temperatures between 18 to 23°C except for spinach which was illuminated with 400 W high intensity discharge lamps (General Electric Corp. MVR 400/u). Chemicals. Chemicals unless otherwise indicated were pur- chased from Sigma Chemical Co. DHAP was obtained as the dimethylketal di-monocyclohexylamine salt, which was hydro- lyzed with Dowex 50 as described by Sigma. Sephacryl S-200 was obtained from Pharmacia Corp., DEAE cellulose was DE52 from Whatman Ltd., and ultrapure (NH4)2SO4 was from Swartz/ Mann Inc. E. coli thioredoxin was purchased from Chemical Dynamics Corp. and a mixture of spinach f and m thioredoxin was kindly provided by B. B. Buchanan. Enzyme Preparation. DHAP reductase preparations were ini- tially prepared by the procedure of Santora et al. (17) in which leaves were homogenized in a 10 mm phosphate buffer contain- ing 10 mm mercaptoethanol (Table I). Subsequently, more en- zyme activity was recovered by following a modified procedure which was used for most of the preparations in the present work. A given weight of leaf material was homogenized for about 1 to 2 min in an Oster blendor at 3°C in 2 volumes (w/v) of a medium 98 Downloaded from https://academic.oup.com/plphys/article/86/1/98/6083031 by guest on 23 February 2022
Dihydroxyacetone Phosphate Reductase in Plants'Received for publication May 19, 1987 and in revised form September 10, 1987
ROBERT W. GEE, RICHARD U. BYERRUM*, DENNIS W. GERBER, AND N. E. TOLBERTMichigan State University, Department ofBiochemistry, East Lansing, Michigan 48824
ABSTRACr
Two forms of dihydroxyacetone phosphate reductase are present inspinach, soybean, pea, and mesophyll cells of corn leaves. An improvedhomogenizing medium was developed to measure this activity. The en-
zyme was detectable only after dialysis of the 35 to 70% saturated(NH4)2S04 fraction and the two forms were separated by chromatographyon either DEAE cellulose or Sephacryl S-200. About 80% of the reduc-tase was one form in the chloroplast and the rest was a second form inthe cytosol as determined by chromatography and by fractionation ofsubcellular organelles. The amount of activity detectable in the chloro-plast fraction was 10.7 micromoles of dihydroxyacetone phosphate re-
ductase per hour per milligram chlorophyll from spinach leaves and 4.9from pea leaves. The chloroplast form eluted first from DEAE celluloseand, being smaller, it eluted second from Sephacryl S-200. Activity ofthe chloroplast form was stimulated 3- to 5-fold by the addition of 1millimolar dithiothreitol or 50 microgram reduced Escherichia coli thio-redoxin or 4 micrograms spinach thioredoxin to the assay mixture. Thisstimulation was not observed with monothiols. Activity of the cytosolicform was not affected by either reduced thioredoxin or dithiothreitol.
In plant metabolism glycerol phosphate must be produced forthe synthesis of various phospholipids, sulfolipids, galactolipids,and triglycerides. The metabolic reaction in leaf tissue of higherplants for glycerol phosphate synthesis is catalyzed by a DHAP2reductase which has been partially purified from spinach leavesand castor bean endosperm, and which catalyzes the reductionofDHAP at pH 7.0 using NADH as the reductant (6, 17). In thereverse reaction the enzyme nomenclature lists it as a sn-glycerolphosphate:NAD oxidoreductase or dehydrogenase (EC 1.1.1.8).Because the activity as a dehydrogenase requires a pH of around9.5 and very high substrate concentrations, no physiologicalsignificance is attached to the dehydrogenase activity and theenzyme will be referred to as DHAP reductase.DHAP is a central compound for several metabolic pathways.
It can be converted in the chloroplast by the C3 photosyntheticcarbon cycle to hexoses for starch synthesis or for regenerationof ribulose bisphosphate. DHAP is the transport component ofthe triose phosphate shuttle out of the chloroplast to the cytosol.In the cytosol it can be converted to hexoses, sucrose, and cellwall constituents or oxidized by glycolysis to pyruvate. In boththe chloroplast and cytosol, DHAP can be reduced to glycerolphosphate by the two DHAP reductases described in this paper.Because of the number of reactions DHAP may undergo, its
Supported by United States Department of Agriculture Grant 86-CRCR-1-2135 and published as Michigan Agricultural Experiment Sta-tion Report No. 12290.
metabolism must be highly controlled, as is expected of com-pounds and enzymes at metabolic branch points.A number of free living algae produce glycerol as a major
product of photosynthesis rather than accumulate starch orsucrose. Zooxanthellae, symbiotic, unicellular algae in the polypsof reef building corals and other marine invertebrates, excrete totheir host up to 40% of their photosynthate as glycerol (14, 15).The halotolerant alga, Dunaliella tertiolecta (5, 20) and Chia-mydomonas (1 1) can synthesize and accumulate large amountsof glycerol as an osmoregulator. Haus and Wegmann (9) havepartially purified a glycerol phosphate dehydrogenase (measuredas DHAP reductase) in D. tertiolecta and Brown et al. (4) haveshown that in this organism the dehydrogenase is in the chloro-plast. It is, therefore, possible for algae to synthesize glycerol asa major end product of photosynthesis. To date there is littleinformation about the amounts of glycerol phosphate and freeglycerol which exist in higher plants, even though the reactionpathway for their formation from the first triose phosphateformed in CO2 fixation in photosynthesis involves only DHAPreductase and glycerol phosphate phosphatase reactions. In ad-dition, essentially nothing is known about the regulation ofDHAP metabolism to form glycerol phosphate and glycerol inhigher plants. The purpose of the present study has been to lookfor the occurrence ofDHAP reductase in a variety of plants andto determine its subcellular distribution and some aspects of itsregulation.
MATERIALS AND METHODS
Plant Material. Plants used were corn (Zea mays L. cv GreatLakes 5922), castor bean (Ricinus communis L. cv 1778 Sangui-neus), soybean (Glycine max L. cv MSU 1066), pea (Pisumsativum L. cv Dwarf Progress 9), and spinach (Spinacia oleraceaL. cv Longstanding Bloomsdale). Plants were grown in thegreenhouse supplemented with high output, cool-white fluores-cent lights for 8 h/d at temperatures between 18 to 23°C exceptfor spinach which was illuminated with 400 W high intensitydischarge lamps (General Electric Corp. MVR 400/u).
Chemicals. Chemicals unless otherwise indicated were pur-chased from Sigma Chemical Co. DHAP was obtained as thedimethylketal di-monocyclohexylamine salt, which was hydro-lyzed with Dowex 50 as described by Sigma. Sephacryl S-200was obtained from Pharmacia Corp., DEAE cellulose was DE52from Whatman Ltd., and ultrapure (NH4)2SO4 was from Swartz/Mann Inc. E. coli thioredoxin was purchased from ChemicalDynamics Corp. and a mixture of spinach f and m thioredoxinwas kindly provided by B. B. Buchanan.Enzyme Preparation. DHAP reductase preparations were ini-
tially prepared by the procedure of Santora et al. (17) in whichleaves were homogenized in a 10 mm phosphate buffer contain-ing 10 mm mercaptoethanol (Table I). Subsequently, more en-zyme activity was recovered by following a modified procedurewhich was used for most of the preparations in the present work.A given weight of leaf material was homogenized for about 1 to2 min in an Oster blendor at 3°C in 2 volumes (w/v) of a medium
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containing 100 mM Tris at pH 7.0, 25 mM ascorbate, 10 mMmercaptoethanol, and 5 g of PVPP per 100 g of leaves. Thehomogenate was filtered through eight layers of cheesecloth andthe filtrate centrifuged at 20,000g for 30 min. The supernatantwas then made 35% saturated with 266 g of (NH4)2SO4/L andstirred for 30 min. The precipitate was discarded after centrifu-gation at about 15,000g for 30 min. The supernatant was thenmade 70% saturated with an additional 266 g/L of (NH4)2SO4and again stirred for 30 min. The enzyme in the precipitate wascollected by centrifugation and resuspended in the smallest pos-sible volume of a Tris/ME buffer containing 10 mm Tris at pH7.0 and 10 mM mercaptoethanol. This preparation was stable fora few months at - I5°C. Ten ml of resuspended protein then wasdialyzed overnight against three changes of 1 L of 10 mm Tris/ME buffer to remove residual (NH4)2SO4. The DHAP reductaseactivity in the dialyzed solution was stable for less than 24 h.The proteins in the 35 to 70% saturated ammonium sulfate
preparation were further separated by one or the other of twocolumn chromatographic procedures. The solution was appliedto the top of either a 2.5 x 90 cm column of Sephacryl S-200(Superfine), which was preequilibrated and eluted with the same50 mM Tris 10 mm ME buffer (pH 7.0), or after dialysis to thetop of a 2.5 x 40 cm DE52 cellulose column that had beenequilibrated with the 10 mM Tris/ME buffer. The DE52 columnwas subsequently washed with 200 ml of 10 mM Tris/ME bufferat pH 7 and then eluted with a linear gradient of 0 to 0.5 M KCIin the same buffer. Eight ml fractions were collected and moni-tored for protein content by measuring absorbance at 280 nmand were assayed for DHAP reductase activity. Peaks of enzymeactivity from the columns were pooled and concentrated byprecipitation with 70% saturation with (NH4)2S04 and the pre-cipitate resuspended in a small volume of the Tris/ME bufferand stored at -15°C. The enzyme could also be stabilized bystorage of the pooled fractions in 2 M glycerol at -15C. Unlessthe enzyme fractions were stored in either of these two ways,activity was stable for less than 24 h. Storage of enzyme at 4°Cor the addition of 1 mM DTT to the enzyme fractions did notprevent loss of activity. When the activity from the stored frac-tions were prepared for assay, they were again dialyzed againstthree 1 L changes of 10 mM Tris/ME buffer.
Subcellular Distribution of Enzyme Activity. Following theprocedure of Walker (19) a whole chloroplast fraction was iso-lated from about 50 g of spinach or pea leaves by rapid homog-enization and a very short centrifugation. The supernatant fromthe whole chloroplast fraction was then further fractioned bydifferential centrifugation. A pellet obtained by centrifugation at1,500g for 10 min was labeled thylakoids. The supernatant wasthen centrifuged at 10,OOOg for 30 min and the pellet obtainedwas labeled mitochondria plus peroxisomes. Finally, the super-natant was centrifuged at I00,OOOg for 1 h to obtain the micro-some fraction and the supernatant was considered to be thecytosol. All ofthe subcellular fractions were resuspended in dilutehomogenizing medium as described by Walker (19) to break theorganelles, and the solutions were then treated with (NH4)2S04to obtain the 35 to 70% saturated (NH4)2SO4 fraction whichcontained the DHAP reductase activity. This fraction was usedin all cases for enzyme assay after dialysis overnight against three1 L changes of 10 mm Tris/ME buffer.Enzyme Assay. DHAP reductase activity was measured by the
reduction ofDHAP with NADH, as monitored at 340 nm usinga Gilford Response spectrophotometer. The 1 ml assay mixturecontained 100 mM Hepes buffer (pH 6.9), 0.2 mm NADH, and0.35 mm DHAP. Tris buffer was not used in the assay becauseat 100 mm it would have been inhibitory as previously observed(9). DTT, when added, was 1 mM. When thioredoxin was addedto the assay mixture, either 50 gg of E. coli thioredoxin or 4 Agof a mixture of spinach f and m thioredoxins, as recommended
by Wolosiak et al. (22), were incubated with 1 mm DTT for 10min at room temperature and then centrifuged through SephadexG 25 in a Biorad econo-column containing 3 ml of resin. Thethioredoxin, separated from DTT by this rapid method, wasimmediately incubated with the enzyme for 10 min beforeassaying for DHAP reductase. Sodium dithionite (1 mM) couldalso be used to reduce thioredoxin but glutathione, cysteine, andmercaptoethanol could not serve as reducing agents. The endog-enous reaction rate was observed for 2 min before DHAP wasadded and the reaction rate was then recorded for 8 min. Amodified Lowry method (2) was used to determine proteinconcentration with BSA used as a standard. Chl was measuredas described by Arnon (1).
RESULTSSurvey and Isolation Procedure. As reported previously (17)
significant levels of DHAP reductase activity could not be de-tected in leafhomogenates or in undialyzed (NH4)2SO4 fractions.However, DHAP reductase activity could be measured in thedialyzed fraction obtained after precipitation between 35 to 70%saturated (NH4)2S04 from leaf homogenates of the plants tested(Table I). The necessity for dialysis was due in part to (NH4)2SO4inhibition of the enzyme and to other dialyzable inhibitorymaterials which precipitated with the enzyme but which havenot been characterized. Part of the differences in the specificactivity shown in Table I is related to different amounts of otherprotein present in this fraction. The Tris-ascorbate homogenizingmedium with mercaptoethanol and PVPP usually gave a muchmore active enzyme preparation than did the phosphate bufferpreviously used (17) (Table I). Therefore, the Tris-ascorbatebuffer was adopted as the homogenizing buffer for further exper-iments. Using the isolation procedure outlined in "Materials andMethods" the enzyme was partially purified by a 35 to 70%saturated ammonium sulfate fractionation followed by chroma-tography on either a Sephacryl S-200 column or a DE52 anionexchange column. This procedure resulted in the isolation oftwoDHAP reductases which had not been achieved before (4, 6, 9,17).The enzyme elution profile based on ionic charge differences
during DEAE cellulose chromatography of the dialyzed 35 to70% (NH4)2S04 fraction from spinach leaves had two peaks ofDHAP reductase activity designated peak I and peak II (Fig. 1).
Table I. A SurveyforDHAP ReductaseThe leaves (10 g) were homogenized in 50 ml of a buffer of 100 mm
phosphate (pH 7) and 10 mM mercaptoethanol (17) or in a buffer of 100mM Tris (pH 7.0), 20 mM ascorbate, 10 mm mercaptoethanol, and 5 gPVPP/100 g leaf. Activity was assayed on the 35 to 70% saturatedammonium sulfate fraction after dialysis of a 10 ml sample overnightagainst three changes of 1 L each of a 10 mm Tris and a 10 mm MEbuffer (pH 7). Assays were run in the presence or absence of 1 mim DTTand results expressed as specific activity in the ammonium sulfate frac-tion.
Homoge HomogenizingHomogenizng MeimoMedium of Medium of
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0
Is
200 400 1Volume (ml)
A nearly identical enzyme activity profile was obtained by DEAEchromatography ofthe dialyzed fraction obtained between 35 to70% (NH4)2S04 saturation from soybean leaves (data not shown).Further results to be presented subsequently proved that peak Iis the form ofDHAP reductase in chloroplasts and peak II is thecytosolic form. The elution profile in Figure 1 was obtained usinga 10 mm Tris/ME buffer with a 0 to 0.5 M KCI linear gradient.Tris/ME at 10 mm concentration was chosen for elution of theDEAE cellulose, since higher concentrations permitted someprotein and enzyme activity to come through the column withthe void volume.The elution profile based on size during Sephacryl S-200
chromatography ofthe undialyzed 35 to 70% (NH4)2S04 fractionfrom spinach leaves also had a large and small peak of activityas shown in Figure 2. To be consistent with peak numberingfrom the DEAE column, the large peak with the smaller mol wtis designated as I (from the chloroplasts) and the small peak witha larger mol wt is form II (from the cytosol). Chromatographyofthe fraction obtained between 35 to 70% (NH4)2S04 saturationfrom pea, soybean, and corn all gave elution profiles fromSephacryl S-200 similar to the spinach profile shown in Figure2.
041
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Volume (ml)
FIG. 2. Separation of dihydroxyacetone phosphate reductases fromspinach leaves on Sephacryl S-200. The same 35 to 70% (NH4)2SO4fractionation described in the legend for Figure 1 was applied to a 2.5 x90 cm column of Sephacryl S-200 and eluted with 50 mM Tris and 10mM mercaptoethanol.
FIG. 1. Separation of spinach dihydroxyacetone phosphosphatereductases on DEAE cellulose. The fraction precipitated between35 and 70% (NH4)2SO4 saturation ofthe homogenate was dialyzedagainst three 1 L changes of a 10 mm Tris and 10 mM ME bufferat pH 7.0 and applied to a 2.5 x 40 cm DEAE cellulose (WhatmanDE52) column. The column was washed with 200 ml of the samebuffer and then eluted with a gradient of 0 to 0.5 M KCI in thebuffer.
Effect of pH on Reductase and Dehydrogenase Activities ofthe Chloroplastic and Cytosolic Enzymes. The chloroplasticDHAP reductase activity had a pH optimum at about 6.9whereas the cytosolic form exhibited a double peak betweenabout 6.8 to 7.2 (Fig. 3). The reason for the double peak isunknown but was replicable. The reductase activity was meas-ured with 0.2 mM DHAP as substrate, which is close to thephysiological concentration of 0.1 mm DHAP calculated from
0.15- Chloroplost DHAP Reductose
@ \ ~~~~~~~~~~Glycerol- P
01
a. Dehydrogenose
E AE
E iCytos 0
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6.0 7.0 pH 8.0 9.0 10.0
FIG. 3. The effect of pH on dihydroxyacetone phosphate reductaseand glycerol phosphate dehydrogenase activities of the chloroplastic andcytosolic enzymes. The DHAP reductase assay (0) was run with 0.2 mMDHAP and 0.1 mm NADH in a mixture of 33.3 mm each of MES,Hepes, and Bicine adjusted to the indicated pH with KOH. The portionof the pH curve between pH 9 and 10 was determined with 100 mmglycine as buffer. The dehydrogenase activity was measured with 2 mMglycerol phosphate and 2 mM NAD (A) as well as with 20 mM glycerolphosphate and 2.5 mM NAD (0). All assays contained 1 mM DTT. Barsrepresent 1 SD.
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DIHYDROXYACETONE PHOSPHATE REDUCTASE
the data of Stitt et al. (18). Both enzymes had optimum dehy-drogenase activity at about pH 9.5. Two curves are shown fordehydrogenase activity, one using 20 mm glycerol phosphate anda second using 2 mm glycerol phosphate as substrate. At asubstrate concentration of 2 mM there was no detectable dehy-drogenase activity with the chloroplast enzyme and only a slightactivity with the cytosol enzyme. Some dehydrogenase activitycould be measured with 20 mm glycerol phosphate as substrate.This substrate concentration as well as the high optimum pHseem to be well above physiological values. Even with the highsubstrate concentration for the dehydrogenase activity the ratioof reductase to dehydrogenase activities is 10 to 1.
Subcellular Location of Dihydroxyacetone Phosphate Forms.Because DHAP reductase could not be assayed until inhibitorshad been removed by dialysis of the 35 to 70% saturated(NH4)2SO4 fraction, larger samples were required than normallywould be used for isopycnic sucrose gradients. Consequently, theWalker procedure (19) was used to isolate whole chloroplastsfrom spinach and pea leaf homogenates by rapid centrifugation.This procedure was followed by differential centrifugation toyield thylakoids (broken chloroplasts and membranes), mito-chondria plus peroxisomes, microsomes, and cytosol. No detect-able DHAP activity could be found in any of these cellularcomponents until they were solubilized, fractionated using(NH4)2SO4, and then dialyzed. DHAP reductase activity was
Table II. Distribution ofDHAP Reductase in Cellular FractionsThe homogenate was separated into subcellular fractions as described
in "Materials and Methods" and a dialyzed 35 to 70% saturated(NH4)2SO4 fraction was prepared from each. Assays were run in thepresence of 1 mm DTr. Specific activities were calculated from proteinor Chl analyses done on subcellular fractions before (NH4)2SO4 fraction-ation. The specific activity ofthe whole chloroplast enzyme is equivalentto 10.7 ,umol/h mg Chl from spinach leaves and 4.9 gmol/h-mg Chlfrom pea leaves. No activity was detected in the fraction containingmitochondria and peroxisomes or in the microsome fraction.
Whole Tyaod yooChloroplasts Thylakoids Cytosol
70 g spinach leavesTotal nmol/min 1560 950 3771nmol/min mg protein 103 49 46nmol/min.mg Chl 179 53
41 g pea leavesTotal nmol/min 505 331 1344nmol/min mg protein 12 7 6nmol/min mg Chl 81 23
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found only in the chloroplastic and cytosolic fractions (Table II).No enzyme activity was observed in the mitochondria plusperoxisome fraction or in the microsomes. From Chl analyses ofboth pea and spinach it was determined that about one-third ofthe chloroplasts were isolated in the whole chloroplast fraction.To estimate total DHAP reductase in the chloroplasts, if all ofthem had been intact, the activity in the chloroplast fractionfrom spinach (1560) was multiplied by 100/33 or a total of 4727nmol/min. The amount (3771 nmol/min) measured in the cy-tosol included DHAP reductase from two-thirds of the chloro-plasts (3167 nmol/min) which had been broken minus theactivity still entrapped in the broken chloroplasts or thylakoids(950 nmol/min). Thus, the cytosolic form of DHAP reductasewas 1554 nmol/min. These calculations indicated that the dis-tribution of total DHAP reductase activity in the chloroplastfraction was 75% for spinach and 70% for pea leaves, while inthe cytosol there was 25% for spinach and 30% for peas of thetotal activity. From chromatographic analyses it was observedthat peak I (chloroplastic form) contained 83% ofthe total DHAPreductase activity from a DEAE column and 78% from theSephacryl S-200 column. The remainder was in peak II (cytosolicform). An approximate average value from these analysis indi-cates that the total DHAP reductase was distributed about 75 to80% in the chloroplastic form and 20 to 25% in the cytosolicform. A similar distribution was found for DHAP reductase inyoung and mature leaves from both spinach and peas. Exactenzyme specific activity per g of leaf, mg Chl, or mg proteincould not be measured directly because the activity could onlybe detected after dialysis of the fraction obtained between 35 to70% (NH4)2SO4 saturation of the leaf homogenate. Loss ofactivity during fractionation and the presence of inhibitors re-sulted in underestimation of total enzyme activity. However, forspinach or pea leaves the activity detectable in a whole chloro-plast fraction after addition of 1 mM DTT to the above dialyzedfraction was 10.7 Mmol of DHAP reductase/h-mg Chl and forpea leaves 4.9 Mmol ofDHAP reductase/h * mg Chl.To establish that peak I, which had the larger amount of
DHAP reductase activity, was the chloroplastic form, intactchloroplasts isolated by the method ofWalker (19) were disruptedby osmotic shock and the solubilized protein was fractionatedby saturation between 35 and 70% with (NH4)2SO4. This chlo-roplast protein fraction was then dialyzed overnight against threechanges of 1 L of 10 mM Tris/ME buffer and the dialysatechromatographed on DEAE cellulose (Fig. 4). Only peak I waspresent; there was none of the cytosolic peak II.The two DHAP reductases had different stabilities during
isolation and storage. When the fraction obtained by saturation
N
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00
I
FIG. 4. DEAE cellulose separation of dihydroxyacetone phos-phate reductase from isolated spinach chloroplast. Intact isolatedchloroplasts were broken by resuspension in one-tenth dilution ofthe homogenizing buffer described by Walker (19). After centrif-ugation, the broken chloroplasts were washed with the same dilutebuffer, both solutions were combined, and DHAP reductase con-centrated by fractionation with 35 to 70% (NH4)2SO4 saturation.The dialyzed sample was chromatographed on DEAE cellulose asdescribed in Figure 1.
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between 35 and 70% with (NH4)SO4 was chromatographed onSephacryl S-200 and stored in 2 M glycerol at -15C, 90% of theoriginal chloroplast enzyme was still active after 6 months andthe cytosolic form showed 90% of the original activity after 4months at -15C. When the same ammonium sulfate fractionwas taken up in a minimum volume of Tris/ME buffer andstored at - 15C for 3 weeks before chromatography on SephacrylS-200, only 30% of the original chloroplast enzyme was activewhereas 80% of the cytosolic enzyme activity remained. Storageof the fraction obtained between 35 and 70% (NH4)2SO4 satu-ration either before or after chromatography on Sephacryl S-200changed the ratio ofchloroplastic and cytosolic enzyme activities.
Stimulation ofChloroplastic Dihydroxyacetone Phosphate Re-ductase Activity by Dithiothreitol or Thioredoxin. The additionof 1 mm DTT or thioredoxin in the assay enhanced DHAPreductase activity 3- to 10-fold in the dialyzed 35 to 70% satu-rated (NH4)2SO4 fraction from leaf homogenates, a whole chlo-roplast fraction or from peak I from either chromatographicprocedure. Enhancement of activity with increasing DTT con-centration is shown in Table III. DTT stimulation occurred eventhough the enzyme had been prepared throughout in solutionscontaining 10 mm mercaptoethanol (Table I). Other thio com-pounds did not enhance DHAP reductase activity (Table III).Essentially, no DTT or thioredoxin stimulation was observedwith the cytosolic form of the enzyme from either spinach orsoybean leaves.
Oxidized thioredoxin did not stimulate the chloroplasticDHAP reductase, and when DTT was added to thioredoxin toreduce it, the mixture did not enhance enzyme activity abovethat observed with DTT alone. To show thioredoxin stimulationof DHAP reductase, it was necessary first to reduce the thiore-doxin with DTT or sodium dithionite. Reduced thioredoxinstimulated the spinach chloroplast enzyme about 5-fold and thesoybean chloroplast enzyme 3-fold (Table IV). When stimulationby a mixture of spinach f and m thioredoxin was compared to
Table III. Stimulation ofthe Chloroplastic DHAP ReductasefromSpinach Leaves by Mercapto Compounds
The enzyme was the dialyzed 35 to 70% saturated (NH4SO4 fractionfrom peak I, after DEAE cellulose separation of the two reductase formsfrom the 35 to 70% saturated (NH4)2S04 fraction from spinach leafhomogenates (Fig. 1). Different control activities with no added mercaptocompound is due to different enzyme preparations.
stimulation by E. coli thioredoxin, the stimulation by the spinachpreparation was 3-fold greater than with the E. coli preparation(data not shown).
DISCUSSION
The properties ofthe chloroplast and cytosolic forms ofDHAPreductase are summarized in Table V. The chloroplast form hada pH optimum at about 6.9 and the cytoplasmic form hadmaximal activity between pH 6.8 to 7.2. The Km (NADH) forthe chloroplast form was 62 gM and for the cytosolic form 23 ,M(data not shown). The chloroplast form of the enzyme repre-sented about 80% of the total in the leaf and it was stimulated3- to 5-fold by DTT or reduced thioredoxin. This stimulationwas not additive. Both forms of the reductase used NADH andno activity was observed with NADPH. Both forms were char-acteristically inhibited by NADH at concentrations greater than150 to 200 mm (data not shown). The requirement for NADHby the chloroplastic DHAP reductase is different from mostother chloroplast dehydrogenases associated with photosyntheticcarbon metabolism that use NADPH. The concentration ofNADH in the chloroplast may be an important regulator ofenzyme activity, as well as the unusually low pH optimum of6.9.
In our first investigation of DHAP reductase from spinachleaves (17) only one form of the enzyme was noted after chro-matography of the dialyzed 35 to 70% saturated (NH4)2SO4fraction. This fraction had the elution profile of the chloroplastform. However, with an improved homogenizing medium andmore total activity, a smaller component, the cytosolic form, hasbeen found in significant amounts from all the leaves examined.In general, about 20 to 25% of the total DHAP reductase was inthis cytosolic form and the rest in the chloroplast. The chloroplastform has a smaller mol wt (elutes secondly from Sephacryl S-200) but is more charged (elutes at higher ionic strength fromDEAE column) than the cytosolic form. DTT and reducedthioredoxin stimulate the chloroplastic form but not the cytosolicform. DTT and thioredoxin regulation of enzymes in chloro-plasts is well recognized (22) whereas different mechanisms maybe involved in regulation of cytosolic enzymes. It seems reason-able to presume that the glycerol phosphate produced from theDHAP reductase reaction is so essential for lipid synthesis thatboth a chloroplastic pool and a cytosolic pool are produced bythe two DHAP reductases. Consequently, control over glycerolphosphate production in the cell, other than the chloroplast,would depend in part on the triose phosphate shuttle and glycol-ysis rather than on a glycerol phosphate transporter from thechloroplast. Other factors are also involved in the differentialregulation ofthese two DHAP reductase forms (our unpublisheddata).Only one form of DHAP reductase has been reported from
Table IV. Stimulation ofPartially PurifiedDHAP Reductases by ThioredoxinE. coli thioredoxin had been reduced by incubation with DTT or sodium dithionites as described in
"Materials and Methods." Approximately 50 ,g ofE. coli thioredoxin was required for maximum enhancement(data not shown). The low specific activities in the preparation used with the Sephacryl S-200 columns is dueto the loss of activity in the 35 to 70% saturated (NH4hS04 fraction during storage for 2 weeks.
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DIHYDROXYACETONE PHOSPHATE REDUCTASE
Table V. Properties ofthe Two DHAP Reductase Forms in Leaves
Form I Form IIChloroplast Cytosolic
pH optimum 6.9 6.8-7.2Km(NADH) 62 uM 23 gMDTT stimulation 3- to 5-fold 0-30%Thioredoxin stimulation 3- to 5-fold NoneDistribution of total activity 75-80% 20-25%NADH substrate inhibition Over 200 Mm Over 150 AM
the green halotolerant alga Dunaliella tertiolecta (4, 9) and it hasKm values for substrate and NADH and a pH optimum similarto the chloroplast enzyme in higher plants. Furthermore, Brownet al. (4) showed that the algal enzyme is associated with chlo-roplasts and it is possible that upon closer examination some ofthe cytosolic enzyme will also be found. Whether the higherplant chloroplast enzyme is different from the algal reductase ispresently uncertain.
Multiple forms of NADH linked DHAP reductase or NADlinked glycerol phosphate dehydrogenase have been seen inanimals. Chicken liver and muscle (21), mammalian liver (16),insects (3), and E. coli. (12) contained multiple forms of thisenzyme as well. In both rat and mouse liver one form was shownto be in the peroxisomes (7).According to enzyme nomenclature rules, the enzymes we
have examined in this study have been numbered as NAD linkedglycerol phosphate oxidoreductases or dehydrogenases (EC1.1.1.8). However, the dehydrogenase activity was nearly non-existent at pH 9.5 (the optimum) with 10 times higher substrateconcentrations than required for the reductase activity. Thereductase assay was run with 0.2 mM DHAP and 0.1 mM NADH.The reverse reaction or dehydrogenase activity could only bemeasured at pH 9.5 with extremely high concentrations of 20mM glycerol phosphate and 2.5 mm NAD. These conditions donot occur physiologically. There are no reports on the glycerolphosphate pool size in leaves, but many chromatographic ex-aminations of 14C labeled products of leaves have never found asignificant amount of this ester to be present. The DHAP poolsize is about 100 ,AM (18), but the glycerol phosphate must bemuch less. Thus, there is no evidence that there is sufficientglycerol phosphate in leaves forDHAP reductase to ever functionas a dehydrogenase. Even at near V. the ratio of 10 to 1 forthe reductase to dehydrogenase activity is unusually high (Fig.3). This ratio for other dehydrogenases such as malate, lactate,or glycerate dehydrogenases is generally about 4 to 1. It seemslikely that this enzyme physiologically can only catalyze thereduction of DHAP and for these reasons we have called it aDHAP reductase. Under such circumstances the reverse or de-hydrogenase reaction can be expected to be catalyzed by differentenzymes in different cellular locations. The mitochondrial flavinlinked glycerol phosphate dehydrogenase (EC 1.1.99.5) frommicroorganisms or animal tissue transfers electrons to the cyto-chrome system for generating a sufficient H+ gradient to synthe-size two ATP. This dehydrogenase in plant tissues has not beencharacterized. It was detected in (bean) endosperm tissue, but inthe same investigation the DHAP reductase was not noted inleaf homogenates probably because of inhibitors (10). Glycerol
phosphate may also be oxidized to DHAP by an oxidase locatedin plant peroxisomes (8). In Dunaliella tertiolecta, which pro-duces much glyercol as an osmoticum, the reversal is apparentlyaccomplished by a different pathway with a glycerol dehydrogen-ase plus a dihydroxyacetone kinase (4, 9).
Randall and his colleagues have recently reported (13) on aglyoxylate reductase in spinach leaves which has some of theproperties of the DHAP reductase reported in the present study.DEAE cellulose chromatographic separates the glyoxylate reduc-tase activity from both DHAP reductases reported here (data notshown). These are, therefore, completely different enzymes.
LITERATURE CITED
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