characterization ornithine transcarbamylase pea l

Plant Physiol. (1991) 96, 262-2680032-0889/91/96/0262/07/$O1 .00/0

Received for publication November 27, 1990Accepted January 18, 1991

Purification and Characterization of OrnithineTranscarbamylase from Pea (Pisum sativum L.I

Robert D. Slocum* and David P. RichardsonDepartment of Biology (R.D.S.) and Department of Chemistry (D.P.R.), Williams College,

Williamstown, Massachusetts 01267

ABSTRACT

Pea (Pisum sativum) omithine transcarbamylase (OTC) waspurified to homogeneity from leaf homogenates in a single-stepprocedure, using 5-N-(phosphonacetyl)-L-omithine-Sepharose 6Baffinity chromatography. The 1581-fold purified OTC enzyme ex-hibited a specific activity of 139 micromoles citrulline per minuteper milligram of protein at 370C, pH 8.5. Pea OTC representsapproximately 0.05% of the total soluble protein in the leaf. Themolecular weight of the native enzyme was approximately108,200, as estimated by Sephacryl S-200 gel filtration chroma-tography. The purified protein ran as a single molecular weightband of 36,500 in sodium dodecyl sulfate-polyacrylamide gelelectrophoresis. These results suggest that the pea OTC is atrimer of identical subunits. The overall amino acid compositionof pea OTC is similar to that found in other eukaryotic andprokaryotic OTCs, but the number of arginine residues is approx-imately twofold higher. The increased number of arginine resi-dues probably accounts for the observed isoelectric point of 7.6for the pea enzyme, which is considerably more basic thanisoelectric point values that have been reported for other OTCs.

which have been used to investigate the distribution and levelsof OTC protein in various plant tissues (28). These studies,and others in progress, should provide us with a better under-standing of the cellular and molecular mechanisms regulatingOTC activity in plants.

MATERIALS AND METHODS

Chemicals

L-Ornithine, phosphonacetic acid, ninhydrin, Hepes, DTT,L-glutamate, and EDTA were purchased from Sigma Chemi-cal Co. Basic copper carbonate, trifluoroacetic acid, and tri-fluoroacetic anhydride were from Aldrich. Formic acid, am-monium formate, sodium acetate, and sodium borate wereobtained from Baker. The bicinchoninic acid reagent wassupplied by Pierce. Chelex 100 chelating and BioRex MSZ- Ianion exchange resins and all SDS-PAGE reagents were pur-chased from Bio-Rad. Epoxy-activated Sepharose 6B wasobtained from Pharmacia. Ethanolamine (2-aminoethanol)was from Kodak. PALO, a transition-state analog inhibitorofOCT (27), was synthesized in our laboratory.

OTC2 (carbamyl phosphate: L-ornithine carbamyltransfer-ase, EC 2.1.3.3) catalyzes the formation of L-citrulline fromL-ornithine and CP. This reaction represents the first com-mitted step in de novo arginine biosynthesis in higher plants(31) and, in this context, changes in OTC activity couldsignificantly impact nitrogen and carbon allocation to thearginine pathway and related pathways involved in pyrimi-dine and polyamine biosynthesis. Meaningful investigationsof mechanisms regulating OTC activity will require (a) de-tailed knowledge of the physical and biochemical propertiesof plant OTCs and (b) the development of OTC-specificreagents, such as antibodies and DNA clones, which arepresently unavailable.

In the present communication, we describe the purificationof OTC from pea and report its general properties. We havealso produced antisera against the pea enzyme which recog-nize OTCs from phylogenetically diverse groups ofplants and

' This research was supported by grants from the Research Cor-poration (C-252 1) and the National Science Foundation (DCB-8806240) to R. D. S.

2 Abbreviations used: OTC, ornithine transcarbamylase; ATC, as-partate transcarbamylase; CP, carbamoyl phosphate; PALO, b-N-(phosphonacetyl)-L-ornithine; dH20, distilled, deionized water; pl,isoelectric point.

Plants

Four-week-old pea seedlings (Pisum sativum L. cv"Wando") were grown under standard greenhouse conditionsin potting soil supplemented with fertilizer.

Purification of OTC from Pea Seedlings

Leaf and stem tissue, 50 g, were homogenized at 0.2 g freshweight tissue/mL of homogenization buffer (100 mM Hepes,5 mM EDTA, 1 mm DTT, 1 mM L-glutamate, 1 mm PMSF,pH 7.5) at 4°C using a Polytron homogenizer (Brinkmann).The homogenate was centrifuged at 16,000g for 20 min, 4TC.The supernatant was adjusted to pH 8.5, mixed with PALO-Sepharose 6B, and stirred for 30 min at 25°C to permit OTCto bind to the immobilized ligand. Following this bindingstep, the gel was removed from the homogenate by collectiononto a scintered glass filter under gentle vacuum. The gel wasthen briefly washed, 5 min each, with three 30-mL changesof wash buffer (50 mM Hepes, 1 mm EDTA, 1 mm DTT, pH8.5), followed by 20 mL of the same buffer + 10 mM KCl,then 20 mL of buffer only. OTC was finally eluted from thegel in 20 mL of buffer + 5 mM CP, which was dissolved inthe buffer just before use. All wash solutions were maintainedat 40C. OTC activity and protein content in the solutions were

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CHARACTERIZATION OF PLANT ORNITHINE TRANSCARBAMYLASE

determined as described below. The final CP eluate containingOTC was dialyzed overnight against 1 mm ammonium ace-tate, pH 7.2, lyophilized to dryness, then dissolved in sterileaqueous 30% glycerol, and stored frozen at -80'C. Beforereuse, the PALO-Sepharose 6B resin was washed with 50 mLof 0.3 M sodium acetate buffer, pH 5.2 and then washedexhaustively with distilled H20. The resin was stored at 4°Cin 0.02% sodium azide.

OCT Assay

OTC activity was measured according to the method ofBoyde and Rahmatullah (4), based on the colorimetric detec-tion of citrulline with diacetylmonoxime. The reaction mix-ture contained 200 mm Hepes, 10 mM L-ornithine, 10 mmCP (dilithium salt), pH 8.5, and varying amounts of enzymein a final volume of 0.1 mL. The reaction was carried out at370C for 15 min and then stopped by addition of 0.1 mL of10% perchloric acid. Half of the acidified reaction mixture(0.1 mL) was combined with 0.9 mL ofdH20 and 4.5 mL ofchromogenic reagent (20 mM antipyrene 2,3-dimethyl- 1-phenyl-3-pyrazolin-5-one, 5 mm ferric ammonium sulfate in2.2 N H3PO4, 9 N H2SO4). Citrulline formation was estimatedfrom A464 readings using a citrulline standard curve, whichwas linear between 0 and 0.25 ,umol. OTC-catalyzed citrullinesynthesis in nondialyzed, crude extracts was estimated byrunning the reaction both with and without added CP. Oneunit ofOTC activity was defined as 1 ,mol citrulline/min at37°C, pH 8.5.

Protein Determination

Sample protein contents were determined either by the dye-binding method of Bradford (5) or by using the bicinchoninicacid assay of Smith et al. (29).

SDS-PAGE

The degree of homogeneity of the PALO-Sepharose 6Bpurified OTC protein sample was determined by SDS-PAGE(22). Samples were electrophoresed through 6% stacking and12% resolving gels at 20 mA constant current. Size estimationfor the OTC monomer was based on the mobility of enzymeprotein relative to mol wt standards (Pharmacia; phosphoryl-ase b, 94,000; BSA, 67,000; ovalbumin 43,000; carbonicanhydrase, 30,000; soybean trypsin inhibitor, 20,100; a-lac-talbumin, 14,400).

Gel Filtration Chromatography

The mol wt for native OTC was determined by gel filtrationchromatography using Sephacryl S-200 (Pharmacia). Approx-imately 5 ug of purified OTC protein was eluted from a 41.5-x 1-cm column in 20 mM Hepes, 1 mM DTT, 1 mM EDTA,pH 7.5, at a flow rate of 0.3 mL/min at 4°C. The column wascalibrated with low mol wt markers (Pharmacia; ribonucleaseA, 13,700; chymotrypsinogen A, 25,000; ovalbumin, 43,000;BSA, 67,000) supplemented with muscle enolase (82,000) andbovine y-globulins (158,000). The mol wt for pea OTC wasestimated from its partition coefficient, relative to markerproteins.

Determination of OTC pl

The pIs for both the monomeric and trimeric forms ofpurified pea OTC were determined using two separate meth-ods. The apparent pI for the OTC monomer was estimatedby isoelectric focusing using a modification of the method ofMiner and Heston (26). OTC protein (1 to 2 ,g) was focusedthrough 0.4- x 10-cm tube gels consisting of 7% acrylamide,0.25% Triton X-100, 5 M urea, and 2.5% carrier ampholines6 to 8 (LKB). Samples were applied at the anode beneath a2.5% ampholine/5% sucrose solution. The anodic buffer was40 mM DL-aspartic acid, pH 3.5, and the cathodic buffer was50 mM NaOH. Focusing was carried out at 500 V/tube for 6to 8 h at 250C, and then gels were stained with CoomassieBlue to visualize the position of the OTC protein. Replicategels, used to determine the pH gradient, were cut into 1-cmsections. Each section was incubated in 0.5 mL of dH20 at4°C overnight in sealed tubes and, after warming the tubes to25°C, the apparent pH values were used to estimate theapparent pI for the OTC monomer, as described by Gelsemaet al. (13).The pI for native OTC was estimated, based upon its pH-

dependent binding to the strong cation exchanger SP-Sepha-dex C50 (Pharmacia), using the method of Yang and Langer(32). After a 10-min incubation, OTC binding to the ionexchange beads in 20 mm Na-phosphate buffers between pH6.6 and 8.2 was determined by monitoring A280 of the super-natant after removal of the beads by centrifugation. OTCbinding was plotted as a function of pH and the midpoint ofthe inflection area of the plot, between adsorbed and nonad-sorbed phases, was taken as the approximate pI for the protein.

OTC Amino Acid Analysis

Amino acid analyses for purified pea OTC were carried outin duplicate by Dr. David Speicher at the Protein Microchem-istry Laboratory of the Wistar Institute, Philadelphia.

Synthesis and Purification of PALO

The synthesis and purification of PALO were carriedout using modifications of methods described previously byHoogenraad (15).

Copper-L-ornithine was prepared by dissolving 16.9 g (100mmol) of L-ornithine HCl in 300 mL of boiling water andthen slowly adding 30 g (136 mmol) ofbasic copper carbonate.The mixture was then cooled to room temperature and ablue-green precipitate was allowed to settle out of solution.The remaining dark blue solution was further clarified bycentrifugation at 10,000g for 10 min, and the supernatantwas made to 90% ethanol and stirred at 40C overnight. Thelight blue Cu24-L-ornithine precipitate was collected on What-man No. 1 filter paper, air dried, and stored at room temper-ature until further use.The mixed trifluoroacetic anhydride derivative of phos-

phonacetic acid was prepared by suspending phosphonaceticacid (15.4 g, 110 mmol) in trifluoroacetic acid (30.4 mL) andtrifluoroacetic anhydride (12.8 mL) at 25°C in a 500-mLround-bottom flask with an attached reflux condenser fittedwith a CaCl2 drying tube. The reaction mixture was heated toa gentle boil and refluxed for 10 min, whereupon the solid

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SLOCUM AND RICHARDSON

white phosphonacetic acid dissolved, giving a colorless solu-tion. The solution was allowed to cool to 25°C and stirred for2 h. The solvents were then removed using a rotaryevaporator.PALO was prepared by dissolving copper-L-ornithine (9.6

g, 29 mmol) in 40 mL of dH20 in a 250-mL round-bottomflask fitted with an addition funnel, a submersed glass pHelectrode, and a submerged thermometer. The deep bluesolution was adjusted to pH 10 using 2.6 mL of 10 M NaOH.The final solution was diluted to 50 mL with dH20, cooledto 0°C, and vigorously stirred. The trifluoroacetic acid-mixedanhydride of phosphonacetic acid (above) was added slowlyin 2- to 3-g portions, while the solution temperature was

maintained at 0 to 5°C. The reaction mixture was also main-tained at pH 9 to 10 throughout the addition ofthe anhydrideby admitting small volumes of 10 M NaOH through theaddition funnel. Before the purification ofPALO, the reactionmixture was adjusted to 0.5 M HCl with 4.2 mL of 6 N HCl,and dH20 was added for a final volume of 100 mL (pH 5.1).The reaction mixture containing PALO was applied to a

Chelex 100 column (23 cm x 1.8 cm2; Na+ form) equilibratedwith dH20 and eluted with dH20 into 200 x l-mL fractions.(The blue Cu2+ ion remains bound to the column and PALOwhile unreacted L-ornithine and phosphonacetic acid elute.)A 5-IAL aliquot of each fraction was spotted onto Whatman 3MM chromatography paper and sprayed with 0.25% ninhy-drin in absolute ethanol to visualize amino acid-containingfractions, which were pooled, adjusted to pH 8.0 with 10 N

NaOH, and concentrated by lyophilization to approximately75 mL.The concentrated Chelex 100 eluate was applied to a

BioRex MSZ-1 anion exchange column (78 cm x 1.8 cm2;HCOO- form) equilibrated with 0.1 M ammonium formatebuffer, pH 8. (We were unable to observe binding of PALOto the resin in the Cl- form, as was reported by Hoogenraad[15]. Given the relative selectivities of this resin for variousanions, i.e. number of bed volumes of 1 N NaOH required toconvert the resin to the OH- form [in brackets] as Cl- [22] >HPO4- [5.0] > HCOO- [4.6] > OH- [ 1.0], published by themanufacturer [Bio-Rad], this is not surprising.) The columnwas then washed with 200 mL of the equilibration buffer,removing nonbound L-ornithine (zwitterionic form). PALOand unreacted phosphonacetic acid were eluted from thecolumn with 1.0 M formic acid and collected in 5-mL frac-tions. The ninhydrin-positive, PALO-containing fractionswere pooled and concentrated to dryness by lyophilization.We eluted PALO from the MSZ-l column with 1.0 M

formic acid, rather than salt, because this acid is volatile andis completely removed from the sample during lyophilization.Furthermore, chromatography on MSZ- 1 in the HCOO- formdoes not require prior desalting of Cl- from the sample (15),because the Cl- ion is efficiently bound to the resin becauseof its high selectivity value relative to that of formate. Thus,PALO can be purified from the reaction mixture by a simpletwo-step procedure omitting desalting steps before and afteranion exchange chromatography.The chemical structure of purified PALO was confirmed

by 'H-NMR spectroscopy. Proton NMR spectra were meas-

ured on an IBM/Bruker WP 200SY spectrometer in D20.Peaks are reported on the scale, relative to the residual HDO

signal at 4.63 ppm and are listed in the order: chemical shift(multiplicity, number of protons, coupling constant in Hz).For comparison, IH-NMR spectra of authentic phosphona-cetic acid and L-Ornithine were also measured. The followingdata are representative.

Phosphonacetic acid. 0 2.73 (d, 2H, J = 20.8).L-Ornithine. 0 1.60 (t, 2H, J = 7.9); 1.73 (m, 2H); 2.85

(t, 2H, J = 7.9); 3.60 (t, IH, J = 5.8).Purified PALO. 0 1.50 (m, 2H); 1.79 (m, 2H); 2.55 (d, 2H,

J = 19.9); 3.08 (br. t, 2H, J = 5.8); 3.84 (br. t, IH, J = 5.0);8.02 (s, 1H).'H-NMR spectra of 1:1 mixtures of purified PALO with

either phosphonacetic acid or L-ornithine showed the char-acteristic peaks listed above as clearly resolved signals.The entire synthesis yielded approximately 400 mg of pure

PALO. For the free acid of PALO (formula weight = 254),this represents 1.6 mmol of PALO and a 6% yield, based oncopper-L-ornithine (formula weight = 328). Although this issomewhat lower than the 15 to 30% yield of PALO reportedby deMartinis et al. (9), we collected only peak fractions withrelatively high PALO content and made no attempt to recoverPALO quantitatively.

Coupling of PALO to Epoxy-Activated Sepharose 68

This step was carried out according to the method ofHoogenraad (15) and the instruction manual provided byPharmacia. Epoxy-activated Sepharose 6B resin, 2 g, washydrated in 15 mL of 0.1 N NaOH. The solution was thenadjusted to pH 12 with 6 N NaOH. The resin was degassedunder vacuum, then mixed with approximately 200 ,umol (50mg) of PALO, and dissolved in 3 mL of 0.1 N NaOH in acapped 50-mL plastic centrifuge tube. The final pH of thecoupling solution was adjusted to pH 12 with NaOH and thecoupling reaction was carried out at 37°C for 2 d. The resinwas collected by gentle centrifugation and washed in 2 X 50mL of 0.1 M acetate, 0.5 M NaCl buffer, pH 4.0, followed by2 x 50 mL of 0.1 M borate, 0.5 M NaCl buffer, pH 8.0. ThePALO-Sepharose 6B gel was further washed with 50 mL of 1M ethanolamine for 2 d at 25°C to block unreacted epoxygroups on the resin. No attempt was made to determine theefficiency of PALO coupling to the resin, but deMartinis etal. (9) reported that this method binds approximately 50 to60 ,umol PALO/g of suction-dried resin.

RESULTS AND DISCUSSION

Of the various procedures that have been used to purifyOTC, affinity purification of the enzyme using PALO-Seph-arose 6B is clearly the method of choice. Using this method,deMartinis et al. (9) reported that milligram quantities ofhomogeneous OTC protein could be purified from animaltissue homogenates in a few hours. They also described con-ditions for the purification of pea OTC using this method,noting the weaker PALO-binding characteristics of the plantenzyme, relative to animal OTCs. Building upon these earlierstudies, we have confirmed the usefulness of this method forthe routine purification of OTCs from plants and have char-acterized the highly purified pea enzyme.

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PALO-Sepharose 65 Affinity Purification of Plant OTC

Transition-state analogs, such as PALO, bind tightly andwith a high degree of specificity to enzymes (12). PALOimmobilized on Sepharose 6B via its a-amino group provedto be a useful affinity matrix for the routine purification ofpea OTC. A summary of a typical small-scale purification isgiven in Table I. In scale-up purifications, we have routinelyobtained 0.5- to 1.0-mg yields of purified OTC protein. Thepea OTC protein was purified 1581-fold with a 22% yield,exhibiting a specific activity of 139 units/mg protein. Thiscompares with a 750-fold purification ofthe pea enzyme witha 48% recovery and specific activity of 340 units/mg reportedby previous workers (9). Both OTC preparations are highlypurified, and differences in final recovery can be explained interms of activity eluted from the PALO-Sepharose 6B resinby the salt wash in our purification procedure.

In previous studies with animal OTCases, nonspecificallybound proteins were washed from the PALO affinity matrixwith buffers containing 100 mM KCl (15, 16, 19). deMartiniset al. (9) noted that pea OTC bound to PALO less tightlythan the animal enzyme and was eluted from the column by100 mM KC1. For this reason, they washed the column withbuffer containing only 50 mm KCl before elution of OTC.However, as can be seen in Table I, approximately 55% ofthe OTC activity bound to PALO was released by a 50 mmKC1 buffer wash in our hands. The reason for this is not clear,but we have found that a 10 mm KCl buffer wash releases<2% of the pea OTC activity from the PALO, whereas thepurity of the eluted OTC protein is not significantly differentfrom that seen after the 50 mM KCl wash. For this reason, wehave adopted this modified washing procedure.

deMartinis et al. (9) also reported that the pea enzymebound PALO-Sepharose 6B better at pH 6.5 than at pH 7.5,the pH used in the purification of the animal OTCs. Again,we did not observe this. The recovery of our pea OTC fromPALO-Sepharose 6B at pH 6.5 was only 73% ofthat recoveredat pH 7.5 which, in turn, was considerably lower than at pH8.5 (Table II). Furthermore, OTC activity in the homogenatewas rapidly lost at pH 6.5 throughout a period of 1 to 2 h,even in the presence of L-glutamate, which has been reportedto stabilize this enzyme (20); decreased OTC recovery mayreflect a greater instability ofthe enzyme at this pH. Similarly,we found that binding of corn leaf OTC is maximal near pH8.5 (Table II). For this reason, we now routinely adjust thepH ofplant homogenates to 8.5 just before the PALO-bindingstep.We estimate that 1 g of PALO-Sepharose 6B resin binds

approximately 100 to 150 ,ug of pea OTC protein under the

Table II. Recovery of Plant OTC Activity from PALO-Sepharose 6BAffinity Support as a Function ofpH

Percentage of activity recovered at pH 7.5 is shown in parentheses.pH OTC Activity

punol citrulline/hPea 6.5 334 (73)

7.5 456 (100)8.5 647 (142)

Corn 6.5 2.2 (19)7.5 11.5 (100)8.5 17.6 (153)9.0 8.8 (76)9.8 6.0 (52)

experimental conditions described, in contrast to the 3.5 mgof OTC/g of resin-binding capacity reported for the rat en-zyme (15). Ifwe assume that similar PALO-coupling efficien-cies were achieved in both studies, this suggests that the peaenzyme binds to the inhibitor with a considerably loweraffinity than does its mammalian counterpart, which exhibitsa Ki for PALO in the range of 4.7 x l0- M (27) to 2.7 x 10-7M (1 5). Indeed, Acaster et al. (1) reported Ki values for PALObetween 1.7 and 3.5 x 10-4 M for OTCs from eight differentplant species. We recently obtained partial amino acid se-

quence data for a pea OTC peptide which includes the con-sensus sequence for the CP-binding site (Slocum RD, unpub-lished data). The primary structure for the pea enzyme CPsite differs considerably from that of other OTCs for whichsequence data are available (17). This may explain, in part,why the plant enzyme binds PALO with a reduced affinity,because PALO is known to bind to the CP site of OTC (27).

Based on the purification data in Table I, and from similarexperiments with other plants, it appears that OTC is a low-abundance protein, representing between 0.03 and 0.07% ofthe total soluble protein in leaf homogenates. In hepatocytes,this enzyme constitutes about 0.01% of the total solubleprotein (18), although it represents 3 to 4% of the totalmitochondrial protein (7).

Yields of OTC were not significantly increased by increas-ing the time in which the PALO affinity adsorbent was

incubated with the homogenate. In fact, prolonged bindingtimes of several hours to overnight at 40C resulted in lowerrecoveries and greatly decreased OTC activities, presumablydue to proteolytic degradation of the enzyme (see below).

Immobilized PALO does not appear to be significantlymodified after incubation with plant homogenates. Carrotsuspension cultures continuously grown in the presence of a

Table I. PALO-Sepharose 6B Affinity Purification of OTC from PeaTotal Total Specific Fold %

Fraction Activity Protein Activity Purification Yield

units mg units/mgCrude homogenate 41.6 470.8 0.088 1 100Buffer + 50 mm KCI wash 8.5 5.2 5.8 66 20Buffer + 5 mm carbamoyl 9.3 0.07 139 1581 22phosphate eluate

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w

V qgmp*W

Figure 1. A, SDS-PAGE analysis of pea leaf OTC purification. Leafhomogenate (lane 1: 200 ;g of total protein) and PALO-Sepharose6B-purified OTC (lanes 2 to 4: 5, 10, and 15 Mug, respectively). B,OTC doublett" (10 ug; undegraded 36,500 subunit and 34,800 de-graded subunit protein) profile resulting from prolonged incubation ofleaf homogenate with the PALO ligand in absence of protease inhib-itor PMSF. Positions of mol wt standards (in thousands) are shown.

structurally related inhibitor (6-N-(phosphonacetyl)-aspartate;PALA) detoxify it through an, as yet, unknown mechanism(8), but a similar PALO-detoxifying activity may not occurin plant homogenates. We have now carried out more than20 purifications of OTC from pea and other plants using asingle batch of PALO-Sepharose 6B.

Characterization of Pea OTC

OTC Holoenzyme and Subunit Size

Purified pea OTC ran as a homogeneous protein withapparently identical subunit Mr = 36,500 on SDS-PAGE gels(Fig. 1A); a very minor M, 52,000 contaminant was alsoobserved in some enzyme preparations. In one experiment,in which the PALO-Sepharose 6B resin was incubated withthe pea leaf homogenate for 1 h at 25°C and then for severalhours at 4°C in an effort to maximize OTC binding to theligand, we observed the purified OTC protein to run as a"doublet" of 36,500 and 34,800 bands (Fig. 1B). We believethat the lower mol wt band is a proteolytic degradationproduct ofOTC, because it is not seen after standard (30 min)incubations or when the seine protease inhibitor PMSF isincluded in the homogenation buffer. Pea OTC antisera alsorecognize both bands on SDS-PAGE immunoblots (data notshown). deMartinis et al. (9) reported that PALO-purified pea

OTC ran as a major 37,000 band with a minor (ca. 35,000)contaminating band on SDS-PAGE gels, and it seems likelythat the lower mol wt band in their preparation is the prote-olytic degradation product. Pea OTC has also been purifiedby two other groups (10, 20), but size estimates for the OTCmonomer were not given.

Purified pea OTC eluted from a Sephacryl S-200 columnwith an Mr = 108,200 (Fig. 2). Based on the 36,500 monomersize, the native enzyme appears to be a trimer of identicalsubunits, as has been reported for carbamoyltransferases fromother organisms. It is interesting to note, however, that non-trimeric structures have been reported for other plant OTCs.For example, deRuiter and Kolloffel (10) concluded thatpartially purified pea OTC was a dimer (Mr = 77,600).Recently, Acaster et al. (1) reported that the mean Mr valuefor OTCs from several plant species was 158,000 and thesame value was obtained for the carrot enzyme (2), suggestingthat the enzyme was most likely a tetramer. Other mol wtsfor plant OTCs have also been reported and it is not clear towhat extent differences in the apparent mol wts of theseenzymes may be due to different methods for their prepara-tion and analysis.

OTC Amino Acid Composition and pi

The amino acid composition for pea OTC, normalized tothe 36,500 monomer size observed in SDS-PAGE gels, isshown in Table III and is compared with amino acid com-positions reported for several mammalian, yeast, and bacterialOTCs. Amino acid compositions obtained by chemical anal-yses may differ substantially from those obtained by translat-ing DNA sequences encoding the OTC genes (see rat andyeast comparisons, Table III) and some residues, such as Cysand Trp, are quantitatively lost during chemical analyses andcannot be accurately determined. It seems likely that the plantenzyme contains at least a single Cys, located within the L-ornithine-binding site consensus sequence, since several work-ers have shown that this Cys residue is essential for OTCactivity (21).

x

._.

(1)

1B4

0.0 0.2 0.4 0.6 0.8 1.0

Kav

Figure 2. Sephacryl S-200 gel filtration chromatography of nativepea OTC. mol wt marker proteins: A, 158,000; B, 82,000; C, 67,000;D, 43,000; E, 25,000; F, 13,000.


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Table Ill. Amino Acid Composition of Pea OTC and Comparison with Other Eukaryotic and ProkaryoticOTCs

No. of Residues/SubunitAminoAcdd Pea' Rath Humanc Cowd Saccharomycese Staphylococcus Escherichia

faecafisf'iAsx 29 28 (31) 30 28 45 (45) 36 27GIx 36 35 (39) 37 36 32 (30) 41 43Ser 20 22 (25) 21 22 29 (18) 15 15Gly 27 21 (24) 28 22 14 (15) 26 27His 11 7 (8) 9 7 9 (10) 8 9Arg 27 13(14) 12 11 11 (11) 13 12Thr 21 24 (26) 21 25 19 (17) 20 22Ala 27 22 (24) 26 22 25 (25) 26 36Pro 15 14 (24) 17 16 8 (8) 9 9Tyr 8 11 (12) 9 11 4(4) 10 11Val 21 16 (17) 16 16 21 (22) 26 20Met 8 11 (11) 8 11 9(2) 13 13Cys h 1 (2) 3 3 7 (1) 2 4lIe 19 16 (17) 16 16 28 (26) 18 12Leu 18 42 (50) 44 45 26 (27) 31 34Phe 17 11 (13) 12 11 24(24) 18 13Lys 22 23 (25) 27 27 26 (25) 23 23Trp h 5 (4) 6 5 1 (1) 3 4Total 327' 322 (357) 342 334 338 (311) 340 334

a Chemical analysis, present study. bTranslation of DNA sequence, ref. 30, chemical analysis(parentheses) ref. 24. c Chemical analysis, ref. 18. d Chemical analysis, ref. 18. e Translationof DNA sequence, ref. 17; chemical analysis (parentheses), ref. 11. 'Chemical analysis, ref.25. 9 Translation of DNA sequence (arg I gene), ref. 3. h Trp and Cys residues quantitatively lostduring chemical analysis. 'Minimum number of residues (minus Cys, Trp) normalized for subunitmol wt = 36,500.

The overall amino acid composition of pea OTC is similarto that of other OTCs, although there are significant differ-ences. For example, the number of Leu residues in the peaenzyme is 50 to 70% lower than for other OTCs, althoughthe percentage of total nonpolar residues is comparable. Thenumber ofArg residues in the plant enzyme is approximatelytwofold higher than in any of the other species, whereasnumbers of His and Lys residues are similar. Limited peptidesequence data available to us indicate that Glu/Asp make upapproximately one-half of the Glx/Asx pool in the pea en-zyme (data not shown), as for other OTCs. For these reasons,we expected pea OTC to be a more basic protein than theanimal enzyme, which exhibits a pI = 6.8 (18). Indeed, afterelectrophoresis in pH 8.8 native gels (6), the pea enzymemigrates toward the cathode with an Rf = 0.58 versus 0.32for rat OTC, indicating that pea OTC is a considerably morebasic protein than the rat enzyme. The native pea enzymeexhibited a pI of 7.6 in the pH-dependent binding assay andthe OTC monomer focused as a single band in tube gels withan apparent pI of 7.7.We attempted to identify the N-terminal amino acid residue

of the OTC monomer using standard protocols involvingprotein dansylation, hydrolysis, and identification of dansyl-amino acids by two-dimensional TLC (14). These experi-ments failed to identify the N-terminal residue and attemptsto obtain N-terminal sequence data for the purified proteinby the Edman degradation method were also unsuccessful(Speicher D, personal communication). We conclude that theN terminus is probably "blocked."

OTC Primary Structure

Cyanogen bromide cleavage of pea OTC produced peptidefragments whose number and size are consistent with boththe number of Met residues found in this protein and therelative positions ofMet within other OTC proteins for whichthe primary structure is known (17). These data suggest thatthe overall primary structure of the plant enzyme is probablysimilar to that of other proteins in this family. Recentlyobtained peptide sequence data for the pea OTC enzyme (notshown) should further enable us to design OTC-specific oli-gonucleotide primers for use in polymerase chain reaction-mediated cloning of the pea gene (23). These studies are inprogress.

In summary, we have described the purification of peaOTC from leaf homogenates using a single-step PALO-Seph-arose 6B affinity purification protocol, modified from earlierstudies. We have characterized several structural and chemicalfeatures of OTC from pea and compared them to those ofOTCs from other organisms. We have also used this methodto purify OTC from corn, Arabidopsis, and zucchini tissuesin our laboratory and it should be generally applicable to thepurification of OTCs from other plant species, as well.

ACKNOWLEDGMENTS

We thank Harry Nichols and Eleanor Mendoza for their experttechnical assistance with this work.

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characterization ornithine transcarbamylase pea l

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