10
Purification and Properties of Ornithine Carbamoyl Transferase 1 from Alnus glutinosa Root Nodules 1) Laboratoire de Microbiologie Forestiere, Institut National de la Recherche Agronomique- C.N.R.F. Champenoux, F-S4280 Seichamps, France 2) Laboratoire de Physiologie Vegetale Metabolique ERA CNRS nO 799, Universite de Paris- Sud, Centre d'Orsay, Bat 430 F-9140S, Orsay O!dex, France Received December 3,1982 . Accepted June 8, 1983 Summary The main isoform of ornithine carbamoyl transferase (carbamoyl phosphate: L-ornithine car- bamoyl transferase; EC 2.1.3.3) from Alnus glutinosa root nodules, called OCT h has been purified to electrophoretic homogeneity. The homogeneous enzyme has a specific activity of 93 nkat per milligram of protein. The molecular mass of the native protein as determined from polyacrylamide gradient gel electrophoresis is 2oo,000± 10,000. The ornithine carbamoyl trans- ferase 1 has a Km value for ornithine of 10.0mM and displays apparent inhibition with respect to ornithine. Km values for carbamoyl phosphate are found in the range 1.1 to 2.8 mM depend- ing of the ornithine concentration. Key words: Alnus glutinosa, black alder, symbiose, nodules, citrulline biosynthesis, ornithine car· bamoyl transferase. Introduction It has been reported (Miettienen and Virtanen, 1952; Leaf et al., 1958; Schubert et al., 1981; Blom et al., 1981) that citrulline is the most prominent free amino acid in root nodules of Alnus glutinosa. This is the main product of N2 fixation (Bond et al., 1958) and is the main nitrogen compound tanslocated from the nodules and the roots to the shoots (Blom et al., 1981; Schubert et al., 1981). Ornithine carbamoyl transferase (carbamoyl phosphate: L-ornithine carbamoyl transferase, EC 2.1.3.3) (OCT) catalyses the synthesis of citrulline from ornithine and carbamoyl-Po This enzyme has been measured in many higher plants but is poorly known (for a review see Thompson, 1980). Recently, OCT activity has been demonstrated in cell-free extracts of A. glutinosa nodules (Blom et al., 1981; Martin et al., 1982 a), roots and leaves (Martin et al., 1982 a). By investigating the tissular and intracellular localisation of OCT in A. glutinosa nodules, it has been shown (Blom et 3) To whom correspondence should be sent. Abbreviations: carbamoyl-P: carbamoyl phosphate, EDTA: ethylene diamine tetraacetic acid, 2-ME: 2-mercaptoethanol, OCT: ornithine carbamoyl transferase, PAGE: polyacryl- amide gel electrophoresis, PVP: polyvinylpyrollidone. Z. Pjlanzenphysiol. Bd. 111. s. 413-422.1983.

Purification and Properties of Ornithine Carbamoyl Transferase 1 from Alnus glutinosa Root Nodules

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Page 1: Purification and Properties of Ornithine Carbamoyl Transferase 1 from Alnus glutinosa Root Nodules

Purification and Properties of Ornithine Carbamoyl Transferase 1 from Alnus glutinosa Root Nodules

1) Laboratoire de Microbiologie Forestiere, Institut National de la Recherche Agronomique­C.N.R.F. Champenoux, F-S4280 Seichamps, France

2) Laboratoire de Physiologie Vegetale Metabolique ERA CNRS nO 799, Universite de Paris­Sud, Centre d'Orsay, Bat 430 F-9140S, Orsay O!dex, France

Received December 3,1982 . Accepted June 8, 1983

Summary

The main isoform of ornithine carbamoyl transferase (carbamoyl phosphate: L-ornithine car­bamoyl transferase; EC 2.1.3.3) from Alnus glutinosa root nodules, called OCT h has been purified to electrophoretic homogeneity. The homogeneous enzyme has a specific activity of 93 nkat per milligram of protein. The molecular mass of the native protein as determined from polyacrylamide gradient gel electrophoresis is 2oo,000± 10,000. The ornithine carbamoyl trans­ferase 1 has a Km value for ornithine of 10.0mM and displays apparent inhibition with respect to ornithine. Km values for carbamoyl phosphate are found in the range 1.1 to 2.8 mM depend­ing of the ornithine concentration.

Key words: Alnus glutinosa, black alder, symbiose, nodules, citrulline biosynthesis, ornithine car· bamoyl transferase.

Introduction

It has been reported (Miettienen and Virtanen, 1952; Leaf et al., 1958; Schubert et al., 1981; Blom et al., 1981) that citrulline is the most prominent free amino acid in root nodules of Alnus glutinosa. This is the main product of N2 fixation (Bond et al., 1958) and is the main nitrogen compound tanslocated from the nodules and the roots to the shoots (Blom et al., 1981; Schubert et al., 1981).

Ornithine carbamoyl transferase (carbamoyl phosphate: L-ornithine carbamoyl transferase, EC 2.1.3.3) (OCT) catalyses the synthesis of citrulline from ornithine and carbamoyl-Po This enzyme has been measured in many higher plants but is poorly known (for a review see Thompson, 1980). Recently, OCT activity has been demonstrated in cell-free extracts of A. glutinosa nodules (Blom et al., 1981; Martin et al., 1982 a), roots and leaves (Martin et al., 1982 a). By investigating the tissular and intracellular localisation of OCT in A. glutinosa nodules, it has been shown (Blom et

3) To whom correspondence should be sent.

Abbreviations: carbamoyl-P: carbamoyl phosphate, EDTA: ethylene diamine tetraacetic acid, 2-ME: 2-mercaptoethanol, OCT: ornithine carbamoyl transferase, PAGE: polyacryl­amide gel electrophoresis, PVP: polyvinylpyrollidone.

Z. Pjlanzenphysiol. Bd. 111. s. 413-422.1983.

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414 F. MARTIN, B. HIREL and P. GADAL

aI., 1981; Scott et aI., 1981) that in this symbiotic association the OCT is mainly active in host-tissue mitochondria.

In a previous paper, we have reported the occurrence of two isoforms of ornithine carbamoyl transferase, called OCT 1 and OCT 2 (Martin et aI., 1982 a). The present investigation reports the purification and properties of the main isoform, named OCT 1, which represents about 85 % of the total OCT activity of black alder nodules.

Material and Methods

Plant material. Black alder [Alnus glutinosa (L.) Gaertn] was grown from seed (Versepuy, France) in pots containing vermiculite. The nutrient solution was a Quispel's salt solution which included 1 mM KN03 (Pizelle, 1978).

Young plants (40 day-oldd) were inoculated with nodule brei (200 p.g nodule fresh weight· I-I N-free nutrient solution). After nodules were developed, alders were irrigated with N-free nutrient solution. Plants were grown in a growth chamber at 21°C in the light and 18 °C in the dark. The daylength was 14 h and the light intensity approximately 2000 W . m -2 (Gro-Lux, Sylvania, USA). The relative humidity fluctuated between 70 and 80 % saturation.

Nodules were collected from 0.5 year-old plants. About 5 g fresh weight of nodules were obtained from each plant and samples were assayed for nitrogen fixing activity (acetylene reduc­ing. activit1)' and those which pro.duced 300 to 2000 nmoles C2fu' g-I nodule fresh weIght· h - were used for OCT extractIOn.

Preparation of the crude extract

All operations were carried out at 4 0c. Buffers were adjusted to the indicated pH at 22°C. 30 g of fresh root nodules were ground with a Polytron PT 10 mixer at maximum speed for

4 xIS s in 100 ml 50 mM Tris buffer (pH 8.0) containing 2 mM EDTA, 50 mM 2-ME and 2 % soluble PVP 44,000 and 5 g of insoluble PVP (Polyclar AT). The brei was filtered through two layers of cheesecloth and centrifuged at 30,000 g for 30 min.

Purification of OCT

Ornithine carbamoyl transferase was purified by chromatography through DEAE-Sephacel and hydroxylapatite, and by preparative PAGE.

DEAE-Sephacel chromatography

The supernatant was diluted twice with cold distilled water and layered on the top of a column (20 x 2 cm) of DEAE-Sephacel preequilibrated in 25 mM Tris buffer (pH 7.6) contain­ing 2mM EDTA, 14mM 2-ME and 1 % soluble PVP 44,000. The column was washed with 200 ml of the same buffer. Proteins were eluted from the column by a linear gradient of increas­ing concentration of NaCI (0 to 400 mM) in 200 ml25 mM Tris buffer containing 14 mM 2-ME and 2 mM EDT A. Four ml fractions were collected and flow-rate was adjusted to 20 mI· h -1.

Hydroxylapatite chromatography

The fractions containing OCT activity were dialysed for 16 h against 25 mM acetate buffer (pH 6.0) containing 14mM 2-ME and 2mM EDTA. The dialysed sample was applied to a column (20 x 2 cm) of hydroxylapatite previously equilibrated with the dialysing buffer. The enzyme was found to adsorb more strongly to the hydroxylapatite at pH 6.0 than at higher pH values. The column was washed with 100 ml of the dialysing acetate buffer and then eluted with a linear gradient of increasing K-phosphate concentration (0 to 400 mM) in a total volume of

Z. Pjlanzenphysiol. Bd. 111. S. 413-422.1983.

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Ornithine carbamoyl transferase from alder root nodules 415

100 ml of the acetate buffer. Two ml fractions were collected and the flow rate was adjusted to 10ml·h-1•

Preparative PAGE

High specific activity OCT fractions were pooled and dialysed against 25 mM Tris-glycine buffer (PH 8.3) containing 14mM 2-ME and 2mM EDTA. The dialysed sample was then sub­mitted to preparative PAGE in 6 % acrylamide disc gel (7 x 2.5 cm). The electrophoresis was performed in 25 mM Tris-glycine buffer (pH 8.3) during 4 h (20 rnA, 300 V). After the elec­trophoresis, the gel was incubated 15 min in OCT assay medium and the Pi produced by enzymatic hydrolysis of carbamoyl-P was localized in the gel by precipitation with 100 ml CaCh as described by Hirel and Gadal (1980). The Pi precipitating band which corresponds to the OCT activity was cut out with a razor blade and was finely ground. OCT was eluted from the brei gel by electrophoresis. The apparatus consists of two separate chambers the upper of which contains a bed support, the lower (elution chamber) is covered with a dialysis membrane. OCT emerging from the gel brei enters the elution chamber. The electrophoresis was con­ducted during 4 h (5 rnA, 200 V). The protein solution was recovered and stored at -20°C.

Estimation of molecular mass and analytical PAGE

The molecular mass of OCT was estimated by using polyacrylamide gradient gel PAA 4-30 according to the manufacturer (Pharmacia Fine Chemicals, Uppsala, Sweden). OCT was locat­ed by Coomassie Blue staining and citrulline formation activity.

Analytical PAGE was performed according to Hirel and Gadal (1980).

OCT assay

OCT was assayed by measuring citrulline formation. The assay medium contained in a total of 0.5 ml: 50/Lmol Tris buffer (pH 7.8), 5/Lmol L-ornithine, 5/Lmol carbamoyl-P prepared just before use and enzyme extract. The incubation was carried out for 20 min at 30 0C. In our con­ditions, citrulline formation was linear for 30 min. All components except enzyme preparation were mixed together and preincubated at 30°C for 5 min before initiating the reaction with enzyme. The reaction was stopped by addition of 0.2 ml of 12.5 % (w/v) trichloroacetic acid. The precipitated proteins were removed by centrifugation in a clinical centrifuge for 5 min. Ali­quot of the supernatant was used to determine the amount of synthesized citrulline. A blank in which the ornithine was omitted served as control.

Citrulline was routinely determined colorimetrically at 530 nm by the diacetylmonoxime method of Boyde and Rahmatullah (1981). Ornithine utilization and stochiometrical formation of citrulline were also determined on representative samples by reverse phase high performance liquid chromatography of aliquot of the supernatant. Elution and detection at 340 nm of o-phthaldialdehyde derivatives of amino acids were performed according to Martin et al. (1982 b).

Protein determination

Protein concentration was determined by the method of Lowry et al. (1951) with bovine serum albumin as standard. Nodule extracts are rich in phenolic compounds and the determina­tion of protein content is greatly distorted if the method is used without modifications. We found that a combination of a 12.5 % trichloroacetic acid precipitation and the protective agents (PVP and 2-ME) occurring in extraction buffer was necessary in order to avoid interferences in the determination of the protein content (see also Agusti and Pio Beltran, 1982).

Z. Pjlanzenphysiol. Bd. 111. S. 413-422.1983.

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416 F. MARTIN, B. HlREL and P. GADAL

Results

Purification of the enzyme

Table 1 summarizes the purification of OCTl from alder root nodules. Because of the presence in the crude extract of high amount of phenolic compounds, the use of protective agents as PVP and 2-ME was necessary to improve the enzyme stability. The purification protocol yielded final preparations with specific activities of about 100 nkat per mg of protein. The enzyme was purified to homogeneity approximately 460-fold with a yield of 5 %. Disc gel electrophoresis indicated the presence of only one band when the gel was stained with Coomassie Blue (Fig. 1), which corresponded

e

<i)

~OCT

<II front dye

Fig. 1: Analytical PAGE of purified ornithine carbamoyl transferase 1 (20 J.Lg of protein) from black alder root nodules. Electrophoresis was conducted on 7 % gel and protein was stained with Coomassie Blue.

with the OCT activity (data not shown). Based on the specific activity of 93 nkat per mg of protein of the homogeneous enzyme, it is calculated that 1 g of fresh alder root nodule contains about 33 p.g of OCT 1 and that about 0.21 % of the soluble protein in the crude extract is consisted of the enzyme which represents a catalytic activity of 3 nkat· g-l of nodule fresh matter.

Molecular mass of OCT.

The molecular mass of OCT 1 as determined by polyacrylamide gradient gel was 200,000 ± 10,000 (n = 3) (Fig.2). This is in close agreement with the 224,000 molecu­lar mass value reported for the partially purified OCT from sugarcane (Glenn and Maretzki, 1977).

Z. P/lanzenphysiol. Bd. 111. s. 413-422.1983.

Page 5: Purification and Properties of Ornithine Carbamoyl Transferase 1 from Alnus glutinosa Root Nodules

I-:I: £! w ~

0:: « ...I :::l U W ...I 0 ::E

c: ...I

Ornithine carbamoyl transferase from alder root nodules 417

Thyroglobulin 669000

13

440000

12.5

• Catalase 232 000

\ ,OCT 200 000 ± 10000

\ Aldolase 158000 4%_----'A..:,;C:..:.R:..:.Y.=:LA..:,;M..:.;I.::,D:;"E _________ 30 %

12~ __ ~ __ ~~ __ ~ __ ~ ____ ~ __ ~ __ ~

2 4 6

MIGRATION em

Fig. 2: Determination of ornithine carbamoyl transferase 1 molecular mass by electrophoresis on polyacrylamide gradient gel PAA 4-30. The value is the mean of three separate experi­ments±SE.

Kinetic and regulatory properties of OCT

The pH-dependent enzyme activity was examined, and the pH optimum was found to be close to 7.8 (Fig.3), which is similar to the value found with sugarcane and pea enzymes (Glenn and Maretzki, 1977; Eid et aI., 1974). Kleczkowski and Cohen (1964), working with a highly purified pea OCT, reported an optimal pH of 8.45. The effect of temperature on the rate of reaction was studied over the range 20 to 60°C. The temperature optimum was found in the range 35 to 45 °C (Fig. 4).

Because OCT has two substrates, steady-state parameters were calculated from rates of reaction at a variety of concentrations of one of the substrate for each concen­tration of the second. Double-reciprocal plots of initial velocity versus ornithine con­centrations at a series of fixed concentrations of carbamoyl-P (Fig.5) resulted in a family of intersecting lines that deviated from linearity.

Parameters were calculated according to Cleland (1970). The OCT 1 affinity with respect to ornithine was found to be 10.0 mM. The Ki values for ornithine were found to be 1.7 mM at 2.0 mM carbamoyl-P and 3.6 mM at 10.0 mM carbamoyl-Po

Z. Pjlanzenphysiol. Bd. 111. S. 413-422.1983.

Page 6: Purification and Properties of Ornithine Carbamoyl Transferase 1 from Alnus glutinosa Root Nodules

418 F. MARTIN, B. HIREL and P. GADAL

.2'Ul ~

~ g § f;J)

~ 0

~ 0

a 6 7 B

pH 9

o 1oJ....----'-----"--'----L--' 20 30 40 so 60

empera UN! o(

Fig. 3 Fig. 4

Fig. 3: Dependence of ornithine carbamoyl transferase 1 activity on pH. All results derive from triplicate determinations and are expressed as per cent of maximum activity (0.1 nkat).

Fig. 4: Dependence of ornithine carbamoyl transferase 1 activity on temperature of the assay medium. All results derive from triplicate determinations with a maximum SE of 5 % and are expressed as per cent of maximum activity (0.1 nkat).

2

II 5 6

~ c ~ " 6 ~ > ...... !: ~ .!l! 4 >

......

2

o 1 2 Cl5 10

11 [ornithine) (mMr' (!::arbc'lrrQyLP )(1111·1)·1

Fig. 5 Fig. 6

Fig. 5: Double-reciprocal plots of the ornithine carbamoyl transferase 1 velocity against the con­centration of ornithine at a series of fixed concentrations of carbamoyl-Po The carbamoyl-P concentrations (mM) are indicated near the lines. Assay medium contained 11'g of enzyme.

Fig. 6: Double-reciprocal plots of the ornithine carbamoyl transferase 1 velocity against the con­centration of carbamoyl-P at a series of fixed concentrations of ornithine. The ornithine con­centrations (mM) are indicated near the lines. Assay medium contained 11'g of enzyme.

Thus, it appears that the increase of carbamoyl-P concentration decreased the inhib­itory effect of ornithine (Fig. 5).

Z. Pjlanzenphysiol. Bd. 111. S. 413-422. 1983.

Page 7: Purification and Properties of Ornithine Carbamoyl Transferase 1 from Alnus glutinosa Root Nodules

Ornithine carbamoyl transferase from alder root nodules 419

When carbamoyl-P was the varied substrate at a series of fixed concentrations of ornithine, a series of lines with untypically intersects was observed (Fig. 6). It appears that the Km value with respect to carbamoyl-P increased as the concentration of ornithine increased suggesting that the binding of ornithine decreased the affinity of OCT1 for carbamoyl-Po The enzyme affinity to carbamoyl-P decreased from 1.1 mM at 1.25 mM ornithine to 2.8 mM at 10.0 mM ornithine. The plots obtained with the highest ornithine concentrations (5 and 10 mM) had a greater slope, which may be due to substrate (ornithine) inhibition (Cleland, 1970).

Discussion

Ornithine carbamoyl transferase is widely distributed in higher plants (Kleckowski and Cohen, 1964; Thompson, 1980). However, in comparison with microorganisms and livers from ureotelic animals, the enzyme occurs in relatively low concentrations in plants (De Martinis et al., 1981). Because of this, few studies on purification and properties of plant OCT have been carried out (Kleckowski and Cohen, 1964; De Martinis et al., 1981) and until now OCT has been purified to homogeneity only from pea seedlings (De Martinis et al., 1981). The enzyme from pea was purified by affinity chromatography about 750-fold but the properties were not reported.

OCT from pea seedlings was also highly purified (2000-fold) by Kleckowski and Cohen (1964) but the procedure included 11 steps with a final yield of 6 %.

In the present study, the final purification of alder nodule OCT 1 involves a 463-fold increase in specific activity over that of the initial extract. Despite the occurrence of high amounts of phenolic compounds in nodule extract, the procedure described herein allowed a 5 % final recovery of the enzyme activity. After three steps of puri­fication, the homogeneous enzyme has a specific activity of 93 nkat· mg-1 of protein. Thus, in alder nodule, OCT 1 comprises about 0.2 % of the soluble protein. This high level indicates the importance of citrulline synthesis in this tissue. From the specific activity of the final preparation (Table 1) a catalytic activity of 18 S-l can be calculated assuming a 200,000 molecular mass.

Steady-state kinetics of the enzyme at pH 7.8 show low affinities of OCT 1 with respect to ornithine and carbamoyl-Po Moreover, our results suggest that the binding of ornithine decreased the affinity for carbamoyl-P and that ornithine concentrations

Table 1: Purification of ornithine carbamoyl transferase 1 from 30 grams of fresh black alder root nodules.

Purification Step Activity Protein Specific Recovery Purification Activity

(nkat) (mg) (nkat·mg-1) (%) (fold)

Crude extract 94 468 0.2 100 1 DEAE-Sephacel 55 9.6 5.7 58 28 Hydroxylapatite 18 0.3 60.0 19 305 Preparative PAGE 5 0.054 92.6 5 463

Z. Pjlanzenplrysiol. Ed. 111. S. 413-422. 1983.

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420 F. MARTIN, B. HrREL and P. GADAL

higher than 1.25 mM inhibited the citrulline formation. Ornithine inhibition has been also reported for enzyme preparation from pea (Kleckowski and Cohen, 1964), sugarcane (Glenn and Maretzki, 1977), Vicia (Eid et aI., 1974), microorganisms, and animals (Hoogenraad et al., 1980; Marshall and Cohen, 1972).

Affinity of OCT 1 with respect to carbamoyl-P is close to the value reported for

crude extract of Phaseo/us aureus (1.58 mM) (Ong and Jackson, 1972) but lower than the Km found for sugarcane OCT (0.11 mM) (Glenn and Maretzki, 1977).

In higher plants, carbamoyl-P is a substrate for two important pathways, one leading

to pyrimidine nucleotide synthesis promoted by aspartate carbamoyl transferase, and the other to arginine by action of OCT. The low Km for carbamoyl-P and the kinetics properties displayed by aspartate carbamoyl transferase from Phaseo/us aureus lead Ong and Jackson (1972) to suggest that at low carbamoyl-P concentrations the pyrimidine pathway takes precedence over the arginine pathway in the utilization of carbamoyl-Po At higher carbamoyl-P concentrations, there could be considerably more spill-over into the citrulline biosynthesis and then the arginine pathway.

In young black alders, McClure et al. (1983) have shown that nodule cells possess a high CO2 fixation activity. The net incorporation of CO2 in this tissue was catalyzed by phosphoenolpyruvate carboxylase and by carbamoyl-P synthetase. The latter accounts for approximately 30 to 40 % of the CO2 fixed and provides carbamoyl-P for the synthesis of citrulline. The major labeled compounds in extracts of alder nod­ules exposed to l4C02 were citrulline, glutamate, aspartate and malate. Thus, it appears that in N2-fixing alder nodules, citrulline biosynthesis takes precedence over the pyrimidine pathway.

The kinetic properties presented in this paper are also consistent with a role of car­bamoyl-P on the regulation of citrulline biosynthesis in alder nodule. Indeed, high amounts of carbamoyl-P decreased the ornithine inhibition demonstrated by OCT 1 (Fig.5) though the physiological significance cannot be speculated upon until there are reliable estimates of the carbamoyl-P concentrations in the nodule.

At an estimated ornithine nodule concentration of about 0.3 mM (Blom et al., 1981), it is apparent that OCTl could only be partially saturated. However, levels of OCT 1 are high in nodule and its also possible that concentrations of ornithine could be somewhat different that what overall nodule concentration suggest, due to com­partmentalization in the nodule tissues and cells.

The present study is the second to be devoted to kinetic and physicochemical pro­perties of nitrogen metabolism enzymes from alder nodules (Hirel et al., 1981); fur­ther studies should be carried out with enzymes of ammonia assimilation pathway and ornithine cycle to establish the route of the ammonia derived from N2 fixation.

Acknowledgements

The authors are indebted to J. P. Jacquot, A. Suzuki and G. Pizelle for helpful suggestions and to S. Martin for typing the manuscript. This investigation was supported by a gant from Commissariat 11 l'Energie Solaire (ATP nO 1047).

Z. Pjlanzenphysiol. Bd. 111. S. 413-422. 1983.

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Ornithine carbamoyl transferase from alder root nodules 421

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