Plant & Cell Physiol. 21(8): 1461-1474 (1980)

Isolation and characterization of NADH-glutam.ate synthase from. pea (Pisum sativum L.) Toru Matoh l, Shoji Ida s and Eiichi Takahashi!

(Received August 14, 1980)

Both ferredoxin-glutamate synthase (EC 1.4.7.1) and NADH-glutamate synthase (EC 1.4.1.14) were isolated separately on DEAE-cellulose chromatography from etiolated pea shoots. The latter enzyme was purified 1,400-fold by ammonium sulfate fractionation and column chromatographies of DEAE-cellulose, Sephadex G-200 and blue-Sepharose. The enzyme had a molecular weight of 220,000 and an isoelectric point of 4.3. The optimum pH was 7.6. Apparent Km values for L-glutamine, 2-oxoglutarate and NADH were 400, 37 and 4 pM, respectively. The enzyme had its absorption maxima at 275, 375 and 440 nm, suggesting that pea NADH-glutamate synthase is a flavoprotein. It showed NADH-diaphorase activity toward ferricyanide and 2,6-dichlorophenol indophenol as the electron acceptor. Sulfhydryl reagents, metal-chelating reagents, phthalein acids and azaserine were strong inhibitors. Ammonium and phosphate ions enhanced the enzyme activity. Key words: Blue-Sepharose Etiolated pea shoot Ferredoxin-glutamate synthase - NADH-glutamate synthase - Pisum sativum.

The glutamine synthetase/glutamate synthase (GS/GOGAT) pathway plays a major role in inorganic nitrogen assimilation in a wide range of the higher plants, green algae and nitrogen-fixing organisms (19). Recently glutamate synthase has been implicated in the amino acid metabolism active in seed development (3, 24). Despite accumulated information on the physiological functions in these diverse aspects of nitrogen metabolism, we have scanty knowledge about the enzymatic characteristics of the glutamate synthase from higher plants because investigations of the enzyme have been largely restricted to unpurified preparations. Originally, the glutamate synthase in higher plants was found as a pyridine nucleotide-dependent enzyme in nonchlorophyllous tissues (7, 9). Subsequently Lea and Miflin (14) reported the presence of a different enzyme active only with Fd in pea chloroplasts. The Fd-linked enzyme recently was prepared in purified form from plant leaves and some of its properties have been determined (16, 28), whereas the enzymatic nature of pyridine nucleotide-glutamate synthase remains largely unknown. We here describe the presence of both Fd-glutamate synthase Abbreviations: DCIP, 2,6-dichlorophenol indophenol; Fd, ferredoxin; ME, 2-mercaptoethanol;

MV, methyl viologen; DEAE, diethylaminoethyl; Tricine, N-tris(hydroxymethyl) methylglycine. 1 2

Permanent address: Department of Agricultural Chemistry, Kyoto University, Kyoto 606, Japan. To whom inquiries should be addressed. 1461

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The Research Institute for Food Science, Kyoto University, Uji, Kyoto 611, Japan

1462

T. Matoh, S. Ida and E. Takahashi

(EC 1.4.7.1) and NADH-glutamate synthase (EC 1.4.1.14) in etiolated pea shoots. The NADH-linked enzyme was isolated and purified to characterize its enzymatic properties. Materials and rnerhods

Plant materials

Chemicals The following chemicals were used: Benzyl viologen from BDH, Poole, U.K.; DEAE-cellulose from Brown Co., New York, U.S.A.; bovine serum albumin, glutamate dehydrogenase, yeast alcohol dehydrogenase and Reactive Blue 2 from Sigma Chemical Co., St. Louis, U.S.A.; rabbit muscle aldolase and cow milk xanthine oxidase from Boehringer, Mannheim, Germany; carrier ampholytes, Ampholine from LKB-Produkter AB, Stockholm, Sweden; and azaserine from P-L Biochemicals, Milwaukee, U.S.A. Other chemicals were of reagent grade. Commercial L-glutamine was purified as described previously (15). Fd was prepared in purified form from spinach (12). Blue-Sepharose was prepared according to the procedure of Hyens and De Moore (I I) using Reactive Blue 2 and Sepharose 6B.

Enzyme purification A 1,490 g batch of etiolated pea shoots was ground in a motor-driven mortar with 1,490 ml of 200 mu phosphate buffer, pH 7.5, containing 100 mu KCI, 0.20/0 ME, 2 mM EDTA, 0.2 mM diisopropylfluorophosphate (DFP) and 0.1 % Triton X-IOO as well as appropriate amounts of sea sand. [Phosphate buffer is potassium

salt thronghont the present study.] Maceration and subsequent operations were The homogenate was filtered through 4 layers of carried out below 5°C. gauze, after which the filtrate was fractionated with ammonium sulfate. The precipitate formed between 25 and 55% saturation was used for further purification. It was dialyzed against 50 msr phosphate buffer, pH 7.5, containing 0.1 % ME, 2 mM 2-oxoglutarate, 0.5 rmr EDTA and 10% glycerol; henceforth, referred to as the buffer. This dialyzed solution was charged on a DEAE-cellulose column (5.5 X 39 em) equilibrated with the buffer, then the column was washed with the buffer enriched with 100 mu KCI until the absorbance of the eluate was that of the applied buffer. During this development Fd-glutamate synthase was eluted, whereas NADH-linked enzyme was recovered when the buffer with 200 mu KCI was applied. Both enzymes were separately pooled and used for subsequent purification. The latter enzyme solution, NADH-linked glutamate synthase, was concentrated with a membrane (Amicon PM 30) to 2 mg protein ml-1, then it was precipitated with ammonium sulfate (70% saturation). The precipitate was dissolved in a minimal volume of the buffer and the solution was subjected to gel filtration chromatography on Sephadex G-200. The column (4.5 X 90 em) was equilibrated with the buffer supplemented with 200 mn KCI and developed with

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Pea seeds (Pisum sativum L. cv. Alaska) were soaked in running tap water overnight then germinated in vermiculite at 22°C in the dark. After 8 days, shoots about 10 ern from the top were harvested immediately before use.

NADH-glutamate synthase from Pisum

1463

Assay ofenzyme activity The reaction mixture for NADH-glutamate synthase contained: 50 mM phosphate buffer, pH 7.5, 10 mM L-glutamine, 10 mM 2-oxoglutarate, 0.6 mx NADH and enzyme up to 20 flu nits in a final volume of 1.5 ml. The reaction was started by adding NADH and the mixture was incubated at 30aC for 20 min. Glutamate and glutamine in the reaction mixture were determined as described previously (16). Consumption of NADH was monitored by following the decrease in absorbance at 340 nm using the same reaction mixture, but the concentration of NADH was reduced to 0.15 msr. Fd- and MV-linked activity was assayed as reported previously (15), except that the pH of the assay medium was 7.5. One unit of enzyme activity is defined as the amount of enzyme catalyzing the formation of 1 flmole glutamate min-I. Specific activity is expressed as units·mg proteirr". Protein was determined by the procedure of Bensadoun and Weinstein (4) with bovine serum albumin as the standard.

Gel electrophoresis and isoelectric focusing Polyacrylamide gel electrophoresis was performed according to the method of Gabriel (10) on 7.5% acrylamide gel with a stacking gel of 2.50/0 acrylamide and Tris-glycine buffer, pH 8.3. Both gels contained 25% glycerol. Gels were stained for proteins with Coomassie brilliant blue R250. To locate the enzyme, gels were stained by the method of Tarmy and Kaplan (26) with a slight modification. After electrophoresis, gels were immersed for 30 min in 200 mu phosphate buffer, pH 7.5, then incubated for 30 min in 4 ml of a reaction mixture containing 4 mg NADH, 15 mM 2-oxoglutarate and 15 mM L-glutamine in 100 rmr phosphate buffer, pH 7.5, at 30aC. After the reaction mixture had been removed by aspiration, the staining reagent containing 40 mg of nitroblue tetrazolium and 2.4 mg of phenazine methosulfate in 4 ml of 50 mM Tris-HCI buffer, pH 8.5, was added, and it was incubated at 30°C for 1 hr. Diaphorase activity on the gel was located as follows: Gels were immersed in 200 mM phosphate buffer, pH 7.5, containing 1 mx DCIP for 30 min, then they were incubated at 30°C in 50 mM phosphate buffer, pH 7.5, containing 1 mg NADH ml-I. During this incubation the dye was reduced at each point on the gel where NADH-diaphorase activity was localized.

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the same buffer. Active fractions were pooled and concentrated with the membrane. After a 4-fold dilution with the buffer, the enzyme solution was loaded onto a blue-Sepharose column (1.4 X 30 cm). The column was eluted with a linear gradient of KCI concentration formed with 250 ml of the buffer and 250 ml of the same buffer enriched with 1.5 M KCl. The enzyme were eluted between 250 mn and 300 mM KCl. The enzyme solution was concentrated with the membrane then diluted 5-fold with the buffer before rechromatography on blue-Sepharose. This column (1 X 25 em) was equilibrated with the buffer, and elution was carried out with a 100 mllinear gradient of 0 to 0.5'mM NADH in the buffer. The enzyme fractions were immediately pooled and concentrated with the membrane. Potassium ferricyanide was added to the concentrated enzyme solution at a final concentration of 50 flM and left for 1 hr, after which it was dialyzed against the buffer supplemented with 200 msr KCl. The dialyzed enzyme was made 500~ glycerol and stored in a freezer at -20°C as the purified enzyme.

1464

T. Matoh, S. Ida and E. Takahashi

The isoelectric point of the enzyme was determined by the gel electrofocusing technique (30) using glass tubes (0.5 X 8 ern) filled with 5% polyacrylamide gel containing 2% Ampholine, pH range 3-6 and 10% glycerol. A cathodic lower chamber contained 0.1 M NaOH and 0.1 M H3P04 in an anodic upper chamber. The run was carried out in a cold room (4°C) for 5 hr with 200 V across the electrode. To locate the enzyme was stained the gel for enzyme activity as described above. The pH gradient was determined as follows: After electrophoresis the gel was sliced into 5 mm segments, and the pH of a 0.5 ml of a water extract from each section was measured with a Hitachi pH meter F-7 at O°C.

ofabsorbance and absorption spectra

Absorbance and absorption spectra were recorded with a Shimadzu recording spectrophotometer MPS-5000. Results

Presence

of two kinds of enzyme

The crude extract of etiolated pea shoots had both Fd- and NADH-dependent glutamate synthase activities. The presence of two forms of glutamate synthase enzyme in developing pea cotyledons has been reported (17), and we confirmed this in etiolated pea shoots (Fig. 1). Each of the two forms showed strict specificity toward the electron donor as in the enzymes from developing pea cotyledons. Another feature that differed between two forms was revealed on DEAE-cellulose

".

T 2.0

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100

Tube number (3.8ml) Fig. 1. Elutionprofile ofglutamate synthase enzymesfrometiolatedpea shoots ona Sephadex G-200 column. The column (2.5 X 95 em) was equilibrated and eluted with the buffer containing 200 mM KCl. The applied sample (69 mg in 2.8 ml) was from the 25-55% ammonium sulfate fraction. Procedures for the preparation and the enzyme assay are given in the text. 0, NADH-dependent activity; e, Fddependent activity.

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Measurements

1465

NADH-glutamate synthase from Pisum

4.0

400

T I

~

u

~ 3.0 ~

o

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ex:> N

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100

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Fig. 2. Elutionprofile ofglutamate synthase enzymes on DEAE-cellulose chromatography. The 200-ml sample containing 5.5 g protein, from the ammonium sulfate fraction, was applied on a DEAE-cellulose column (5.5 X 39 em), equilibrated with the buffer. The column was eluted with the buffer enriched with concentrations of KCI as indicated by the arrows. Symbols and the enzyme assay are the same as in Fig. 1.

chromatography (Fig. 2). Fd-linked enzyme was recovered in the 100 mx KCl elution, whereas NADH-dependent activity appeared in the eluate with 200 mM KCl. This is evidence that two different species of glutamate synthase enzymes exist in etiolated pea shoots, each having a distinct molecular weight, ionic charge and specifity for the electron donor.

Extraction andpurification

of NADH-linked

enzyme

When etiolated pea shoots were homogenized in a Waring blender without Triton X-IOO, little enzyme was obtained in the supernatant fraction (10,000 xg for 30 min). The sedimented fraction contained the enzyme activity which was detected only when 0.1 % Triton X-IOO was added. Without detergent, substantial activity was recovered in the soluble fraction after it was ground in a mortar with sea sand. This may be because of the membrane bound nature of the enzyme in chloroplasts (14) and proplastids (29). The final two steps of purification involved affinity chromatography with blue-Sepharose. Although considerable purification took place when the first column was eluted with the KCI gradient, a second chromatography was required with a different elution system (Fig. 3). In this rechromatography, NADH was used for the elution, but the NADH-containing enzyme solution was unstable and was oxidized immediately by ferricyanide which then was removed by dialysis. Table 1 summarizes the purification procedure for NADH-glutamate synthase from 1,490 g of etiolated pea shoots. The enzyme was purified I,400-fold over the crude extract with an overall yield of 9°~.

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C1J

/'

E

.......

E o o

E

1466

T. Matoh, S. Ida and E. Takahashi

~

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700 600

0

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400

1

0.4

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20

40

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Tube number (1.95ml) Fig. 3. Elutionprofile of NADH-glutamate synthase on a blue-Sepharose column. The column (1 X 25 em) was equilibrated with the buffer. Sixty milliliters of enzyme solution containing 15.6 units (specific activity 1.93) was applied to the column, which was washed with the buffer up to tube number 40, then eluted with a linear gradient of NADH (0-0.5 mx) in a total volume of 100 ml of the buffer. e, absorbance at 280 nm; 0, enzyme activity, the broken line shows the concentration ofNADH.

The concentrated enzyme was relatively stable when kept under the specified conditions. The presence of sulfhydryl-reducing reagents (such as ME and dithiothreitol) was essential at all times, as noted in previous studies (5, 16). 2-0xoglutarate stabilized the enzyme, as first reported for the bacterial enzyme (21).

An addition of glycerol (100/0) was not always effective for stabilization in the early stages of purification, but did act as stabilizer when the diluted enzyme solution was used as in affinity chromatography.

Purity and absorption spectrum When 20 flg of the purified enzyme was put through disc gel electrophoresis, Table 1 Summary of thepurification of NADH-glutamate synthasefrom etiolatedpeashoots Purification step

Protein (mg)

Crude extract Ammonium sulfate (25-55%) DEAE-cellulose Sephadex G-200 1st blue-Sepharose 2nd blue-Sepharose

8630 5500 245 53.3 8. 10 0.552

Activity (units)

Specific activity (unitsomg ) proteinr!

Yield (%)

Purification (factor)

94.9 68.8

0.011 0.013

100 72

1

40.5 22.2 15.6 8.6

O. 165 0.417 1. 93 15.6

43 23 16 9

1.2 15.0 37.9 175 1420

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3

0.2 300 ...., ~ .~ ...., 200 u

Isolation and characterization of NADH-glutamate synthase from pea (Pisum sativum L.).

Both ferredoxin-glutamate synthase (EC 1.4.7.1) and NADH-glutamate synthase (EC 1.4.1.14) were isolated separately on DEAE-cellulose chromatography fr...
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