Plant Physiol. (1980) 65, 939-9430032-0889/80/65/0939/05/$00.50/0

Synthesis of Nitrate Reductase in ChlorellaI. EVIDENCE FOR AN INACTIVE PROTEIN PRECURSOR'

Received for publication July 16, 1979 and in revised form December 22. 1979

EDWARD A. FUNKHOUSERDepartment of Plant Sciences, Texas Agricultural Experiment Station, Texas A&M University, College Station,Texas 77843TEH-CHIEN SHEN2, AND RENATE ACKERMANNForschungsstelle Vennesland der Max-Planck-Gesellschaft, Harnackstrasse 23, D-1000 Berlin 33 (Dahlem)Federal Republic of Germany

ABSTRACT

Synthesis of nitrate reductase (EC 1.6.6.1) in Chlorelka vulgaris wasstudied under inducing conditions, ie. with cells grown on ammonia andthen transferred to nitrate medium. Cycloheximide (but not chloramphen-icol) completely inhibited synthesis of the enzyme, but only if it was addedat the start (i.e. at the time of nitrate addition) of the induction period.Cycloheximide inhibition became less effective as induction by nitrateproceeded. Enzyme from small quantities of culture (I to 3 milliliters ofpacked cells) was purified to homogeneity with the aid of blue dextran-Sepharose chromatography. Incorporation of radioactivity from labeledarginine into nitrate reductase was measured in the presence and absenceof cycloheximide. Conditions were found under which the inhibitor com-pletely blocked the incorporation of labeled amino acid, but only slightlydecreased the increase in nitrate reductase activity. The results indicatethat synthesis of nitrate reductase from amino acids proceeds by way of aprotein precursor which is inactive enzymically.

Bacteria, fungi, green algae, and higher plants which are sup-plied with and use nitrate as the sole source of nitrogen, containhigh levels of nitrate reductase. When the nitrogen source isswitched from nitrate to ammonia, the nitrate reductase in theseorganisms usually decreases to extremely low levels (1). Becauseof this behavior, nitrate is often described as an inducer andammonia a repressor of synthesis of nitrate reductase even thoughthe molecular mechanism of this "induction" and "repression" arelargely unknown (9).

Nitrate and ammonia are not the only substrates which cause"induction" or "repression" (24). Environmental changes will alsoaffect the level of enzyme. Nitrogen starvation (10) and light havebeen used to induce synthesis of nitrate reductase in Chlorella. Intobacco callus tissue (26) the incorporation of labeled amino acidsinto active enzyme was used as evidence of induction and de novosynthesis. Further support for de novo synthesis after inductioncomes from the observations that cycloheximide inhibits the early,rapid rise when light triggers "induction" of nitrate reductase in

' Supported in part by a grant to Dr. B. Vennesland from the DeutscheForschungsgemeinschaft and a contribution of the Texas AgriculturalExperiment Station.

2 Recipient of Alexander von Humboldt-Stiftung fellowship. Permanentaddress: Department of Botany, University of Malaya, Kuala Lumpur,Malaysia.

synchronous cultures ofAnkistrodesmus (3) and Chlorella (20, 21).Cycloheximide alos inhibits the appearance of enzyme activity inthese two algae when induction is initiated by nitrogen starvation(10).When Chlorella vulgaris is grown photoautotrophically with

nitrate under high levels of CO2, it produces amounts of nitratereductase, reaching about 0.1% of the soluble protein (18). Theenzyme from this source is easily purified ( 17). This system permitsthe rapid isolation and analysis of enzyme synthesized in thepresence of labeled amino acids. We have investigated some ofthe events which occur during the synthesis of nitrate reductasewhen Chlorella cultures grown on ammonia and containing littleenzyme, are "induced" with nitrate addition. The results showthat the process is more complex than a simple de novo synthesisof active enzyme. A preliminary account of this research hasappeared (4).

MATERIALS AND METHODS

The '4C-amino acids were uniformly labeled and were obtainedfrom Amersham Buchler (Braunschweig). The specific radioactiv-ity of [U-'4C]arginine used in these experiments was 318-324mCi/mmol. Blue Dextran 2000 and Sepharose 4B were obtainedfrom Pharmacia; ,B-NADH and FAD from Boehringer; Bio-GelA-1.5m (200-400 mesh) from Bio-Rad Laboratories; BSA, cyclo-heximide, and dithioerythritol from Serva; and all other chemicalsfrom Merck.

Growth of Algae. Cultures of C. vulgaris were maintained aspreviously described (13, 19). Cells were grown autotrophically incontinuous white light (16,000 to 20,000 lux supplies by metalfilament lamps; 150 w, cool-flood, Philips) in a stream of 5% (v/v)CO2 in air, at 21 to 22 C. The composition of nitrogen-free, nitrate,and ammonium media is given in Table I. In the experimentsreported here, cells which had been maintained on ammoniamedium for at least five transfers were used as an inoculum. Atthe start of each experiment, the initial cell density was 0.24 ,ulcells/ml culture. Nitrate reductase activity was low (less than 0.05units/ml extract). Cells grown continuously in nitrate mediumunder otherwise identical conditions normally contain between 2and 3 units/ml of extract. After growth in fresh ammonia mediumfor one day, the cells were harvested by centrifugation, washedonce with nitrogen-free medium, and resuspended in fresh nitratemedium. Cell densities after I day of growth ranged from 2 to 3td cells/ml. Labeled amino acids or inhibitors were added asindicated in each experiment. Label was added without the addi-tion of carrier. Preliminary experiments showed that unlabeledamino acids, at concentrations 100 times that of the label, addedat the time of application or as a chase, did not alter the uptake

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FUNKHOUSER, SHEN, AND ACKERMANN

Table I. Composition of Culture Media

Nitrogen- Nitrate Me- Ammonia Me-Component free Medium dium (pH 4.3) dium (pH 6.3)

(pH 4.3)mM

MgSO4 20 20 20KH2PO4 18 18 18Na2HPO4 10NaCl 34 34 34CaCl2 2 2Ca(N03)2 2KNO3 20NH4Cl 20

Microelement solutions (ml/250ml culture)

Solution 1 2Solution la 2Solution II I ISolution III 0.1 0.1

Composition of microelement solutions. Solution I: 500 mg FeSO4.7H20; 1,000 mg Fe(NO3)3.9H20; 300 mg H3BO:3; 200 mg MnSO4. H20;22 mg ZnSO4.7H20; 8 mg CuS04.5H20; 2 mg (NH4)6Mo7,0244H20; perliter 0.5 mm H2S04. Solution Ia: As solution I, except Fe(NO:):i -9H20 and(NH4)6Mo7024.4H20 were omitted and substituted with I g NH4Fe(S04)2.12H20. Solution 11: 5 mg CoSO4.7H20; 5 mg NiSO4.7H20; 5 mg Na2WO4;5 mg KCr(S04)2.12H20; 5 mg VOS04.2H20; per liter 0.5 mm H2S04.Solution III: 500 mg NaVO3.4H20 per liter H20.

pattern. Uptake of these tracer amounts of the amino acids wasessentially complete in 15-20 min. Nitrate reductase synthesizedunder these inducing conditions was completely in the active form,i.e. there was no further increase in activity after activation withferricyanide (I 1, 23).

Preparation of Crude Extracts. Cells were harvested by centrif-ugation, washed once with 10 mm K-phosphate (pH 7.6) andresuspended to give a cell suspension which contained 250 ,uIcells/ml buffer (13). The cell suspension was disrupted in a Frenchpressure cell (Aminco) at 10,000 psi. The extract was centrifugedat 30,000g in an SS-34 rotor in a Sorvall RC 2-B centrifuge. Thesupernatant fraction was then tested for nitrate reductase activityand stored at -20 C. Essentially all of the activity was recoveredin the supematant fraction. All manipulations after the cells werewashed were carried out at 1-4 C.

Purification of Nitrate Reductase. After thawing, the superna-tant fraction from French Press extract was again clarified bycentrifugation for 15 min at 30,000g. To insure that all the enzymewas in the active form, and to oxidize any NADH in the extract(which prevents binding to blue dextran-Sepharose) ferricyanide(5 mM) was added to the extract (I l). Enzyme from 500 to 1,000ml culture (1-3 ml packed cell volume) was purified to homoge-neity by chromatography on blue dextran-Sepharose as previouslydescribed (17) with minor variations. Blue dextran-Sepharose wasprepared by the method of Ryan and Vestling (14). The finalproduct contained about 12 mg blue dextran/ml Sepharose 4B.The extract was applied to a column which contained 4 ml gel,

which had previously been equilibrated with 80 mm sodium-potassium phosphate (pH 6.9), 0.1 ims EDTA, and 0.1 mM dithi-oerythritol (buffer A). Essentially all of the enzyme was bound tothe column. In experiments with radioactively labeled aminoacids, a wash of 80-120 ml equilibration buffer A was necessaryin order to lower the radioactivity of a 0.5-ml sample of theeffluent to less than two times background. Nitrate reductaseactivity was then specifically eluted with a series of 2-ml portions200 ,UM NADH in buffer A. About two-thirds of the activity whichwas applied was recovered in the second and third fractions.

Another 15-25% was recovered when the column was washedwith 3 M KCI. After this treatment, the column was ready to beequilibrated again with buffer A and to be reused for anotherpassage of crude extract. The ratio between radioactivity andenzyme activity was not constant in the fractions which wereeluted with NADH, so that another purification step was requiredin order to obtain a homogeneous protein preparation.The material which was eluted with NADH was concentrated

by precipitation with 50% saturated ammonium sulfate, and re-suspended in buffer A to which FAD (16 mg/I) had been added.The enzyme was then chromatographed on a Bio-Gel A-.1Smcolumn (I x 36 cm). The enzyme was eluted with buffer A whichalso contained FAD. The presence of FAD increased the yield ofenzyme. The peak of eluted enzyme activity was identical to theradioactivity peak, so that the ratio of radioactivity to enzymeactivity was constant across the peak. This material also gave asingle band when subjected to electrophoresis both in the presenceand absence of SDS. The proportion of radioactivity in the secondpeak varied with the amino acid used as the source of label.

Assay of Nitrate Reductase Activity. In the experiment de-scribed in Figure 4, nitrate reductase activity was assayed bydetermining the rate of nitrite formation as described by Pistoriuset al. (13). In the other experiments activity was assayed bymeasuring the rate of nitrate-dependent NADH oxidation at 334nm, as previously described (25). One unit of enzyme is thatamount which catalyzes the reduction by NADH of I [tmol ofnitrate to nitrite under standard conditions.

Electrophoresis. Polyacrylamide gel electrophoresis both in thepresence and absence of SDS was done as previously described(18). Protein was measured by a modified method of Lowry aspreviously described (16) with BSA as the standard.

Radioactivity Measurements. Radioactivity measurements weremade with a Nuclear Chicago ISOCAP/300 scintillation counter.Counting efficiencies were determined by the channels ratiomethod. Up to 0.5 ml aqueous samples were added to 15 mlscintillation fluid (4 g 2,5-diphenyloxazole [PPO] in 400 ml ethanoland 600 ml toluene).

RESULTS

Purification. Interpretation of the data obtained from theseexperiments is based on the assumption that we have successfullyisolated a homogeneous protein from relatively small volumes ofculture. Preliminary experiments indicated that nitrate reductasefrom cells which had been incubated with radioactively labeledamino acids, and which had been eluted from blue dextran-Sepharose with NADH was not homogeneous. Namely, the peakof radioactivity was skewed from the peak of the enzyme activity.Our method of purification differed from that of Solomonson (17)in that the initial ammonium sulfate fractionation was omitted.Also, enzyme was eluted from the column with a constant concen-tration of NADH instead of a gradient.The material which was eluted by NADH from blue dextran-

Sepharose contained a substantial amount of radioactivity whichwas not associated with nitrate reductase. When this material wasconcentrated by ultrafiltration (UM-2 membrane Amicon Co.),most of the radioactivity was retained along with nitrate reductaseactivity. Polyacrylamide gel electrophoresis of this sample showedseveral proteins to be present (not shown). When the sample wasconcentrated by precipitation with 50%Yo saturated ammoniumsulfate, most of the contaminating radioactivity remained soluble.Polyacrylamide gel electrophoresis of the material which precipi-tated showed what seemed to be a single protein band which alsohad diaphorase activity (not shown). Apparently the radioactivitythat remained in the soluble fraction was associated with the fastermigrating proteins. However, even after this step there was stillsome contaminating material associated with nitrate reductase.The last traces of the contaminating radioactivity was removed

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NITRATE REDUCTASE PRECURSOR

by chromatography on an agarose column. When a relativelylarge amount of material (about 70 units) was chromatographedon a Bio-Gel A-1.5m column a small amount of non-nitratereductase protein was detected (Fig. 1). When the nitrate reductasepeak was analyzed by polyacrylamide gel electrophoresis, againonly a single broad protein band, which had diaphorase activity,was detected (not shown). However, when the material from thesmall A280 peak was similarly analyzed, two proteins, with nearlythe same electrophoretic mobility were detected. Only the fastermoving material had diaphorase activity and was presumed to benitrate reductase since activity had trailed into this region (notshown). Although the contaminant represented only a small per-centage of the total nitrate reductase protein, the effective removalof this contaminant was important because there were someexperiments in which the contaminant contained most of theradioactivity which had been applied to the Bio-Gel column(Table II).

Nitrate reductase obtained from the Bio-Gel column step wasjudged to be homogeneous by the following criteria: (a) a constantratio between radioactivity and enzyme activity (Fig. 2); and (b)

0.1

v0 5 10 15

wLI)

15 -D0W _

10 C Ew n

5 --<0ccr D3

z

O

FRACTION NUMBER

FIG. 1. Purification of nitrate reductase. Nitrate reductase (70 units)which was eluted with NADH from blue dextran-Sepharose was concen-

trated by precipitation with 50%N saturated ammonium sulfate, resuspendedin buffer A and chromatographed on a Bio-Gel A-I.5m column. Elutionwas with buffer A which contained FAD (16 mg/l). The 90-drop fractionswere assayed for nitrate reductase activity. Note the small non-nitratereductase contaminant. This small amount of protein contained varyingproportions of the radioactivity which was applied to the column (TableII). ( ): A at 280 nm; (O-O): nitrate reductase activity.

one protein band on polyacrylamide gel electrophoresis (notshown). This band also contained disphorase activity. (c) Oneprotein band on SDS-polyacrylamide gels (not shown).

Choice of Amino Precursor. Preliminary experiments with dif-ferent radioactively labeled amino acids showed that some aminoacids labeled nitrate reductase better than others. Table II showsthe effectiveness of the various amino acids which were tested.Arginine resulted in enzyme with the greatest specific radioactiv-ity. The percentage of label in the contaminant was also minimalwith arginine. The high rate of incorporation of arginine intonitrate reductase was not due to an N-terminal modification ofpreexisting protein by arginyl-tRNA transferase (15). While thisactivity has been detected in cereal embryos (12), N-terminalanalysis by the dansylchloride method (7) of purified nitratereductase showed the N-terminal amino acid to be either lysine ortyrosine (not shown).We are at a loss to explain the effectiveness of arginine in

labeling nitrate reductase. There are 33 arginine residues persubunit of nitrate reductase (6) which is not very different fromthe number for the other amino acids tested. Therefore the effec-tiveness of arginine as precursor can not be explained on the basisof high arginine content.

Effect of Cycloheximide on Induction. When cells grown onammonia medium are transferred to nitrate medium, there is, aftera variable lag period, a rapid increase in nitrate reductase activity.In other systems, this has been shown to be due to de novosynthesis (26) and not due to activation. If cycloheximide (10 ,tg/ml) is added at the start of induction, there is no increase inenzyme activity (Fig. 3). Other reports have also shown thatincrease in nitrate reductase activity is sensitive to cycloheximide(3, 8, 10, 20, 21). If however, cycloheximide is added after induc-tion, it only partially inhibits continued synthesis (Fig. 3). Itbecomes less effective as an inhibitor as synthesis continues.Puromycin and chloramphenicol were without effect, and emetinewas only slightly inhibitory. Incorporation of radioactivity from["4Clarginine into soluble protein was inhibited 60% by chloram-phenicol while puromycin and emetine had little effect (notshown). A possible hypothesis to explain the effect of cyclohexi-mide is that the synthesis of nitrate reductase occurs in at leasttwo distinct steps. The final step is not sensitive to cycloheximide,but is dependent on a protein synthesized after nitrate addition.The synthesis of this protein is cycloheximide-sensitive.

Table II. Incorporation of U-'4C-Amino Acids into Nitrate ReductaseUniformly labeled amino acids of similar specific radioactivity were added to standard cultures of Chlorella or

to cultures under inducing conditions. After incubation, the cells were harvested and broken. Nitrate reductasewas recovered from blue-dextran Sepharose and concentrated by precipitation with ammonium sulfate. Thismaterial, which also contained a radioactively labeled impurity was chromatographed on a Bio-Gel A-1.5mcolumn. The specific radioactivity of the purified nitrate reductase was then determined.

Activity Incubation Specific Ra-

Amino Acid Applied to Induction with 4C tivityin Ex- dioactivity of ity in Impu-Each 500- with Nitrate Amino tract Nitrate Re- ityml Flask Acid tract ductase rity

,iCi h units/ml dpm/unit % of sampleapplied to Bio-

GelLysine 2 a 1 2.0 4 91Phenylalanine 10 a 1 2.1 36 89Phenylalanine 10 a 24 2.3 20 86Tyrosine 10 25 25 1.6 9 93Isoleucine 8 4 1 0.8 53 71Isoleucine 8 24 20 2.2 11 83Arginine 10 5 1 0.8 1300 42Arginine 8 4 1 1.8 700 39Arginine 8 28 25 1.6 870 10

'Grown continuously on nitrate.

,0s

I

* .0~~~~~~

0.2_

n,,-

Plant Physiol. Vol. 65, 1980 941

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FUNKHOUSER, SHEN, AND ACKERMANN

--~ ~ ~in 15 5 10 15 2

E

choaorpe anBoGlanFgr .The frcin er sae

0

<r05

-0 o

5 10 15 20

FRACTION NUMBER

FIG. 2. Purification of nitrate reductase. Radioactively labeled nitrate

reductase (11I units from the last arginine experiment in Table II) was

chromatographed on Bio-Gel as in Figure 1. The fractions were assayed

for nitrate reductase activity (- ), and for radioactivity (O-O).The ratio between radioactivity and enzyme activity (X X) was con-

stant across the nitrate reductase peak.

3.0c-E

' 2Cw

U)

0

w 1.Cw

cr

Z

0.

2 6 8

HOURS OF INDUCTION

FIG. 3. Effect of cycloheximide on the synthesis of nitrate reductase.One-day-old ammonia grown cells were transferred to nitrate medium(inducing condition). Aliquots were harvested periodically. Cells were

resuspended (250 ul/ml) and broken in a French pressure cell. Afteraddition of ferricyanide to ensure that all enzyme was in the active form.nitrate reductase activity in the extracts was determined. (O-O): Noadditions; (X X): cycloheximide (10 ug/ml) added at the start ofinduction; (@-4): cycloheximide (10 ,tg/ml) added after 2 h of induc-tion.

Effect of Cycloheximide on Incorporation of 11'ClArginine. Twoexperiments were designed to test this hypothesis. First a series ofpreliminary experiments established standard conditions for in-duction and incorporation of label into nitrate reductase. Therewas no difference in the uptake or in the labeling pattern betweencells which were given a small amount of label and cells whichwere given label followed by a cold chase with 100-fold moreunlabeled amino acids. Essentially the two experiments tested theeffect of cycloheximide on the incorporation of labeled aminoacid when each was added, at various times, to 1-day-old ammo-nia-grown cells which were transferred to nitrate medium. Underthese conditions the enzyme which accumulated was fully active.Nitrate reductase was purified and was tested for radioactivity.These experiments were repeated several times with modifications.Each substantiated the results of the others. Only the two mostrepresentative are reported here.

In Experiment I (Table III), cycloheximide was added 15 minbefore the labeled arginine after the cells had been under inducingconditions for 2 h. Under these conditions, the amount of activeenzyme is increasing, but the inhibitor completely prevents incor-poration of label into the nitrate reductase protein. Uptake of thelabel was the same with and without cycloheximide. There wassome incorporation of label into other soluble proteins in thepresence of cycloheximide at a level of about 10%o of the unin-

Table III. Incorporation of Radioactivity into Nitrate ReductasefromArginine in the Presence of Cycloheximide

Ammonia-grown cells were transferred to nitrate medium and incubatedfor 2 h. The first sample (A) was harvested. Cycloheximide (CH) (10 ,ug/ml) was added 15 min prior to the addition of [U-'4Clarginine (5 ,uCi; 324mCi/mmol) to the second sample (B). The third sample (C) had only theamino acid applied. An hour later the second and third samples wereharvested by centrifugation, cells were resuspended to 250 ,ul/ml andbroken in a French pressure cell. Nitrate reductase activity was determinedin the extracts, the enzyme was purified, and the specific radioactivitydetermined.

Nitrate Reductase Specific Radioactiv-Treatment Activity in Crude ity of Purified En-

Extract zyme

units/ml dpm/unitA 0.33B (+ CH) 0.63 0C (- CH) 0.72 1,500

Table IV. Incorporation of Radioactivity into Nitrate ReductasefromArginine in the Presence of Cycloheximide

Ammonia-grown cells were transferred to nitrate medium and[U-'4Clarginine (5 ,uCi; 324 mCi/mmol) was added to both at the start ofinduction. After 2 h one culture (A) was harvested and cycloheximide(CH) (10 jLg/ml) was added to the second culture (B). After an additional2 h induction this second culture was also harvested. After collecting thecells by centrifugation, they were resuspended to 250 ll/ml and broken ina French pressure cell. Nitrate reductase activity was determined in thesecrude extracts, the enzyme was purified, and the specific radioactivitydetermined.

Nitrate Reductase Ac- Specific Radioac-Treatment tivity in Crude Extract tivity of Purified

Enzyme

units/ml dpm/unitA 0.43 1,500B (+ CH) 1.08 1,400

hibited control. If incorporation into nitrate reductase had oc-curred even at this level, it would have been easily detected. Fromthis experiment we conclude that active nitrate reductase is beingmade from a precursor protein which is assembled from aminoacids by cycloheximide-sensitive translation. Even ifnew synthesisof precursor protein is blocked by cycloheximide, the process ofconversion of precursor to active enzyme can continue, at least fora while, in the presence of cycloheximide.

In Experiment 2 (Table IV), labeled arginine was added at thestart of the induction period. Two hours later, one sample washarvested and cycloheximide was added to the other. After anadditional 2 h of induction, the second sample was harvested.Even though the level of active enzyme increased more than 2-fold during the second 2 h, the specific radioactivity of the totalenzyme remained the same. Since the first experiment showedthat in the presence of cycloheximide there was no incorporationof label into active nitrate reductase from free amino acids, thecontinued increase in enzyme activity must have been from preex-isting proteins or peptides. In fact, whereas 4,000 dpm/flaskappeared in nitrate reductase during the first 2 h, 6,000 dpmappeared during the second 2 h in experiment 2, even in thepresence of cycloheximide.

DISCUSSION

We must account for the fact that cycloheximide preventsincrease in nitrate reductase activity if added at the beginning ofthe induction period, though not when added later. The simplestway of explaining this is to assume that the synthesis of active

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NITRATE REDUCTASE PRECURSOR

nitrate reductase from precursor protein requires another enzymeor substrate which is lacking in ammonia-grown cells. We canrefer to this as an "NR activator," without implying exactly whatit is and how it functions. The synthesis of "NR activator" mustbe sensitive to cycloheximide, but once the activator is formed, itcan "act" in the presence of cycloheximide. Thus removal ofammonia or addition of nitrate is not directly "inducing" the NRitself, but is inducing the "NR activator", which is essential forassembly of the active nitrate reductase molecule. The latter islarge and complex, and contains bound heme, FAD, and Mo (6,18). The NR activator may be responsible for assembling activeenzyme from protein of peptide components or precursors. How-ever, because cycloheximide inhibits processes other than proteinsynthesis (2) it is premature to draw any conclusions on the natureof cycloheximide inhibition.When ammonia-grown cells were treated with cycloheximide

for one or 3 h prior to transfer to inducing conditions (the transferinvolves washing out ammonia and cycloheximide, as well asaddition of nitrate), then the cycloheximide treatment had theeffect of prolonging the lag period before the increase in nitratereductase activity (not shown). The longer the cycloheximidepretreatment, the greater the effect. Such experiments suggest thatcycloheximide-sensitive nitrate reductase-precursor-protein maybe synthesized in ammonia-grown cells. Support for this hypoth-esis comes from the observation that there is material in extractsfrom ammonia-grown cells which reacts with purified antibodiesto nitrate reductase (5). In fact the amount of material whichreacts with the antibodies is similar in extracts from both nitrate-and ammonia-grown cells.Whatever the details of the interpretation, our present experi-

ments show that nitrate reductase is not simply released frompolysomes in active form, but that inactive precursor protein(s) isfirst synthesized and then assembled or activated in a separatestep. Tischner and Huttermann (21) have reached a similar con-clusion regarding the rapid and large increase in nitrate reductaseactivity which occurs in synchronized Chlorella at the beginningof the light period. This is a different type of "induction". Thesynchronized Chlorella cells are exposed to nitrate all the time,but show large fluctuations in nitrate reductase activity. Theenzyme activity is very low at the end of the dark period and risesrapidly when the cells are exposed to light. Because this rise wasinhibited by cycloheximide, it was first concluded that it was dueto synthesis of new enzyme from amino acids (20), but later studieswith deuterium-labeled amino acids showed that the newly ap-pearing enzyme did not contain label in sufficient amount toaccount for de novo synthesis (21). Thus the active enzyme formedon illumination must have been formed from protein or poly-peptide precursors, under these conditions also.

Hipkin and Syrett (10) have studied the rise in nitrate reductasethat occurs when ammonia-grown cells are deprived of a sourceof nitrogen. They used Ankistrodesmus, Dunaliella, and a Chlorellaspecies different from ours. In many respects their experimentaldesign resembles ours. They noted that the rise in enzyme activity,which occurred after a lag period, was inhibited by cycloheximide,but found also that cycloheximide abruptly stopped appearanceofmore enzyme when added after the rise had commenced. Unlessthere are species differences, the difference between their resultsand ours must be due to the fact that we used nitrate whichpermits growth and they used nitrogen starvation as the "inducer".Tischner and Lorenzen (22) have shown that the presence ofnitrate (rather than lack of ammonia) is required for the formationof active enzyme.

Hipkin and Syrett (10) also showed that 6-methylpurine did notinhibit the "induction" of nitrate reductase in their system. Fromthis they concluded that the mRNA for nitrate reductase is present

in ammonia-grown cells. Thus, the control of the enzyme levelwas posttranscriptional. Gupta et al. (8), working with a com-pletely different system, also reached the conclusion that nitrate"induction" was controlled posttranscriptionally. Our results in-dicate that the control is postranslational as well. We suggest thatit is the synthesis of "NR activator" which is inhibited by ametabolite ofammonia, not the translation of the nitrate reductasemessenger. This implies that messenger RNA is present, and isbeing translated in ammonia-grown cells. But the protein pieces,in the absence of the nitrate reductase activator or processingenzyme cofactor, can not be assembled into active enzyme andare probably "turning over" rapidly, as shown by antibody pre-cipitation studies (5).

Acknowledgment-The authors are grateful to Professor Birgit Vennesland for hercontinued support and encouragement.

LITERATURE CITED

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2. ELLIs RJ 1977 Protein synthesis by isolated chloroplasts. Biochim Biophys Acta463: 185-215

3. FISCHER S. W SIMONIS 1979 Diurnal periodicity and light-induced increase ofnitrate reductase activity in synchronized cultures ofAnkistrodesmus braunii. ZPflanzenphysiol 92: 143-152

4. FUNKHOUSER EA, TC SHEN, R ACKERMAN 1976 Precursor(s) of nitrate reductasein Chlorella. Plant Physiol 57: S-69

5. FUNKHOUSER EA, CS RAMADOSS 1980 Synthesis of nitrate reductase in ChlorellaII. Evidence for synthesis in ammonia-grown cells. Plant Physiol 65: 944-948

6. GIRI L. CS RAMADOSS 1979 Physical studies on assimilatory nitrate reductasefrom Chlorella vulgaris. J Biol Chem 254: 11703-11712

7. GRAY WR 1972 End-group analysis using dansyl chloride. Methods Enzymol 25:121-143

8. GuPrA A. SK SOPORY, S GUHA-MUKHERJEE 1979 The presence of a noninduciblephase for nitrate reductase during early phase of germination in barley em-bryos. Z Pflanzenphysiol 92: 249-254

9. HEWITT EJ 1975 Assimilatory nitrate-nitrite reduction. Annu Rev Plant Physiol26: 73-100

10. HIPKIN CR, PJ SYRErT 1977 Post-transcriptional control of nitrate reductaseformation in green algae. J Exp Bot 28: 1270-1277

1 1. JETSCHMANN K. LP SOLOMONSON, B VENNESLAND 1972 Activation of nitratereductase by oxidation. Biochim Biophys Acta 275: 276-278

12. MANAHAN CO, AA APP 1973 An arginyl-transfer ribonucleic acid proteintransferase from cereal embryos. Plant Physiol 52: 13-16

13. PisroRIus EK HS GEWITz, H Voss, B VENNESLAND 1976 Reversible inactivationof nitrate reductase in Chlorella vulgaris in vivo. Planta 128: 73-80

14. RYAN LD, CS VESTLING 1974 Rapid purification of lactate dehydrogenase fromrat liver and hepatoma: a new approach. Arch Biochem Biophys 160: 279-284

15. SOFFER RL 1970 Enzymatic modification of proteins. II. Purification and prop-erties of the arginyl transfer ribonucleic acid-protein transferase from rabbitliver cytoplasm. J Biol Chem 245: 731-737

16. SOLOMONSON LP 1974 Regulation of nitrate reductase activity by NADH andcyanide. Biochim Biophys Acta 334: 297-308

17. SOLOMONSON LP 1975 Purification of NADH-nitrate reductase by affinity chro-matography. Plant Physiol 56: 853-855

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943Plant Physiol. Vol. 65, 1980

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