Plant Physiol. (1988) 88, 46-510032-0889/88/87/0046/06/$0 1.00/0

Elicitor-Induced L-Tyrosine Decarboxylase from Plant CellSuspension Cultures1I. INDUCTION AND PURIFICATION

Received for publication October 5, 1987 and in revised form March 28, 1988

IVANO A. MARQUES AND PETER E. BRODELIUS*Institute ofBiotechnology, ETH Hbnggerberg, CH-8093 Zurich, Switzerland

ABSTRACT

L-Tyrosine decarboxylase (EC 4.1.1.25) activity was induced in cellsuspension cultures of Thalictrum rugosum Ait. and Eschscholtzia cali-fornica Cham. with a yeast polysaccharide preparation (elicitor). Thehighest L-tyrosine decarboxylase activity in extracts from 7-day-old cellcultures of E. californica was observed 5 hours after addition of 30 to 40micrograms elicitor per gram cell fresh weight. The enzyme extractedfrom cells of E. californica was purified 1540-fold to a specific activityof 2.6 micromoles CO2 produced per minute per millim protein at pH8A and 30°C. Purified enzyme from T. rugosum showed a specific activityof 0.18 micromoles per minute per milligram protein. The purificationprocedure involved ammonium sulfate fractionation, anion-exchange fastprotein liquid chromatography, ultrafiltration, and hydrophobic interac-tion chromatography. Sodium dodecyl sulfate-polyacrylamide gel electro-phoresis showed that the enzyme from the two plant cell cultures hadsubunits of identical molecular weight (56,300 1 300 daltons.

Aromatic amino acids are important precursors of varioussecondary products in higher plants (e.g. alkaloids, flavonoids,and coumarins). Studies with plant cell cultures have shown thatkey enzymes (e.g. phenylalanine ammonia lyase [7] and trypto-phan decarboxylase [11]) linking primary and secondary metab-olisms are often inducible. These regulatory enzymes are ofparticular interest in investigations on the regulation ofsecondarymetabolism in plants.Secondary product formation in plant cell suspension cultures

may be induced by fungal elicitors (4, 9, 15). Studies from thislaboratory have shown a good correlation between the inductionof enhanced berberine production in suspension cultures ofThalictrum rugosum Ait. and TDC2 activity within the cells afterelicitation with a yeast carbohydrate preparation (4,6). A possiblerole ofTDC as a key regulatory enzyme in isoquinoline alkaloidbiosynthesis has been suggested (6).

In order to determine the possible regulatory function ofTDCin isoquinoline alkaloid biosynthesis, it is first necessary toinvestigate its molecular and catalytic properties. For these typesof investigations, a purified enzyme is required. For conclusiveproofthat the elicitor-induced increase ofTDC activity observedin various alkaloid producing cell cultures is based on transcrip-tional regulation, the application of immunological techniques

' Supported by research grants from the Swiss National Science Foun-dation (3.318-0.86) and the Swiss Federal Institute of Technology.

2Abbreviations: TDC, L-tyrosine decarboxylase; PLP, pyridoxal-5-phosphate; PVPP, polyvinylpolypyrolidone.

(e.g. in vitro translation ofTDC mRNA) is necessary. Therefore,a pure enzyme will also be required for the production ofantibodies against TDC.

In the first part of these studies, we report on the extractionand purification of TDC from elicitor-induced cell suspensioncultures of T. rugosum and Eschscholtzia californica Cham.Since the enzyme is relatively unstable, a procedure has beendeveloped that allows the purification ofTDC to near homoge-neity within one and a half working days.

MATERIALS AND METHODS

Chemicals. MS- and B5-media were obtained from Flow Lab-oratories. Kinetin and PVPP were from Sigma and 2,4-D fromMerck. Silver nitrate, bis-Tris, EDTA, 2-mercaptoethanol, PLP,L-tyrosine, and DTT were supplied by Fluka. L-[1-'4C]tyrosine(53.8 mCi/mmol) and Protosol were purchased from New Eng-land Nuclear. All chemicals used for electrophoresis were fromBio-Rad. All other chemicals were obtained from commercialsources.

Cell Culture. Suspension cultures of Eschscholtzia californicawere established in 1984 and have been cultivated since then onB5 medium (5) supplemented with 4.5 gM 2,4-D, and 0.46 Mmkinetin on a gyratory shaker (120 rpm) in the dark at 26°C.Subculturing was carried out every 7 d by transferring 26 g ofcells (wet weight) into 300 ml of medium in 1 L Erlenmeyerflasks.

Cultures of Thalictrum rugosum were cultivated as describedpreviously (4).

Induction with Elicitor. The elicitor was prepared from yeastextract (8), and the applied solution had a carbohydrate concen-tration of 6.1 mg/ml. The conditions for induction of TDC incells of T. rugosum have been determined previously (6). Formaximal TDC activity within cells of E. californica, the condi-tions for induction were optimized in the following manner:

Culture Age. An appropriate number of 50 ml Erlenmeyerflasks, each containing 20 ml ofgrowth medium, were inoculatedwith 1.5 g cells (fresh weight). Each day (d 1-8), one flask wastaken for the determination of fresh weight, dry weight, andamount of sucrose remaining in the medium. Elicitor (100 MAgcarbohydrate/g fresh weight) was added to one additional flask.Seven h after addition of elicitor, the cells from the induced andfrom one noninduced flask were collected by filtration. The cellswere subsequently extracted for determination of TDC activityand protein content.

Incubation Time. Seven-d-old cultures (20 ml) were suppliedwith 100 MAg elicitor/g fresh weight, and TDC activity was meas-ured as a function of induction time.

Elicitor Concentration. Seven-d-old cell cultures (20 ml) wereinduced with various amounts of elicitor and incubated for 5 h.

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L-TYROSINE DECARBOXYLASE. I

Extraction and Purification of TDC from E. californica. Forthe purification of TDC, 7-d-old cell cultures of E. californicawere treated with 100 ,ug elicitor/g fresh weight for 5.5 h. Thecells were harvested by vacuum filtration and immediately usedfor extraction of TDC.

Preparation of Crude Extract. The cells (400-500 g freshweight) were homogenized in a Waring Blendor for 1 min at fullspeed in 200 ml of ice-cold buffer containing 100 mm bis-Tris-HCI (pH 6.8), 50 mm KC1, 1 mM EDTA, 12 mM 2-mercaptoeth-anol, 0.08 mM PLP, 1 mM L-tyrosine, and 0.5% (w/v) PVPP(extraction buffer). The homogenate was centrifuged at 28,000gfor 20 min.Ammonium Sulfate Fractionation. Solid (NH4)2SO4 was added

to the supernatant (510 ml) to give 60% saturation. After cen-trifugation (15 min, 15,000g), the resulting pellet was resus-pended in 20 ml of 25 mM bis-Tris-HCI (pH 7.1), 0.08 mm PLP,5 mM DTT, 55% saturation of(NH4)2SO4 and was recentrifuged.This pellet was redissolved in 15 ml ofthe same buffer containing10% saturation (NH4)2SO4 and, after 20 min, the suspension wascentrifuged at 40,000g for 15 min. All manipulations were carriedout at 0 to 4C. The supernatant was frozen in liquid nitrogenand stored at -70°C until used.

Desalting. The sample was thawed under shaking in a 30Cwater bath and then centrifuged at 40,000g for 15 min. DrySephadex G-25 (Pharmacia) was added to the supernatant toreduce its volume. The sample was desalted three times bycentrifugation (5 min, 650g) through 20 ml Sephadex G-25columns equilibrated with 25 mM bis-Tris-HCI (pH 7.1), 0.1 mmEDTA, 1 mm DTT. The eluate (5.5 ml) was filtered through a0.2,m cellulose acetate filter (FlowPore D26, Flow Laboratories)and used directly for anion-exchange fast protein liquid chro-matography.Ion-Exchange Chromatography. A Mono Q column (HR 10/

10, Pharmacia) was equilibrated with 25 mM bis-Tris-HCl (pH7.1), 0.1 mM EDTA, and 1 mM DTT at a flow rate of 3 ml/min.After injection of the sample (5.5 ml) with a Superloop-10 ml(Pharmacia), the column was developed with the following gra-dient from equilibration buffer to 0.35 M NaCl in the samebuffer: 0 to 4 min, O M NaCi; 4 to 6 min, O to 0.16 M NaCl; 6 to12 min, 0.16 M NaCl; 12 to 14 min, 0.16 to 0.21 M NaCi; 14 to19 min, 0.21 M NaCl; 19 to 22 min, 0.21 to 0.35 M NaCl; 22 to32 min, 0.35 M NaCl. TDC-active fractions were pooled anddiluted with 10 volumes of 25 mM bis-Tris-HCI (pH 7.1), 0.08mM PLP, 5 mM DTT. The solution was concentrated by ultrafil-tration (Diaflo YM 100, Amicon) to about 8 ml and rechroma-tographed on the Mono Q column under the same conditions.

Hydrophobic Interaction Chromatography. TDC-active frac-tions from the second Mono Q column were pooled and broughtto 0.8 M (NH4)2SO4 with solid salt and applied to a Phenyl-Superose column (HR 5/5, Pharmacia) that had been equili-brated at a flow rate of 0.5 ml/min with 25 mm bis-Tris-HCI(pH 6.0), 0.1 mM EDTA, 1 mM DTT, 0.8 M (NH4)2SO4 (startingbuffer). The proteins were eluted with the following gradientfrom starting buffer to 5 mM bis-Tris-HCl (pH 6.0), 0.1 mmEDTA, 1 mM DTT: 0 to 7 min, 0.8 M (NH4)2SO4; 7 to 10 min,0.8 to 0.4M; 10 to 26 min, 0.4 M; 26 to 30 min, 0.4 to 0.2M; 30to 38 min, 0.2 M; 38 to 40 min, 0.2 to 0 M; 40 to 50 min, 0 M(NH4)2SO4. The fraction containing most of TDC activity wassupplied with 1.6 mM PLP and solid (NH4)2SO4 to final concen-trations of 0.08 mm and 0.8 M, respectively. The sample wassubsequently rechromatographed on Phenyl-Superose. This timethe column was equilibrated with 25 mM Tris-HCl (pH 8.4), 0.1mM EDTA, 1 mM DTT, 0.8 M (NH4)2SO4 and was developedwith a gradient to 5 mm Tris-HCl (pH 8.4), 0.1 mM EDTA, 1mM DTT. The fraction with TDC activity was brought to 0.08mM PLP and stored at 2C. All chromatographic separationswere performed at room temperature.

Extraction and Purification of TDC from T. rugosum. Essen-tially the same purification procedure was used for purificationof TDC from cells of T. rugosum. Differences were as follows:300 g of cells were homogenized in 400 ml of extraction buffer.The proteins in the supernatant of the centrifuged homogenatewere precipitated with 50% saturation of (NH4)2SO4. After cen-trifugation, the pellet was resuspended with buffer containing35% saturation of(NH4)2SO4. Anion-exchange fast protein liquidchromatography was not performed twice since the enzymeactivity had already appeared after the first run on the Mono Qcolumn at 0.21 M NaCl. The Phenyl-Superose column wasequilibrated with 25 mm bis-Tris-HCl buffer (pH 7.1), 0.1 mMEDTA, 1 mm DTT, and 0.8 M (NH4)2SO4. The column wasdeveloped with a gradient to 5 mM bis-Tris-HCl (pH 7.1), 0.1mM EDTA, 1 mm DTT. The fractions containing TDC activitywere pooled and rechromatographed on the Phenyl-Superosecolumn under the same conditions (pH 7.1).Enzyme assay. The assay is based on '4C02 evolution from 1-

['4C]tyrosine and was carried out in glass vials (total volume: 14ml) designed by the authors (Fig. 1). Compartment A was filledwith 0.2 ml of the assay mixture containing 80 mm Tris-HCl(pH 8.4), 0.05 mM PLP, 5 mM DTT, 0.8 mM L-[l-'4C]tyrosine(0.78 MCi), and the decarboxylase (25 ,l), which was added afterhaving equilibrated the reaction vials at 30°C for 10 min. Com-partment B contained 0.2 ml of 4 M HC104. The vials wereclosed with rubber stoppers immediately after injection of theenzyme into the assay mixture and were incubated for 10 minat 30°C. To stop the reaction, the HC104 solution was stirredinto the assay mixture by vigorously shaking the vials on aVortex Genie 2. Then 0.25 ml of Protosol were injected with a1 ml syringe through the rubber stopper into compartment C.For CO2 absorption, the surface of the Protosol solution wasincreased by rolling the vials in a tilted position. After 30 min,the CO2 absorber was transferred into a scintillation vial contain-ing 5 ml of Aqualuma Plus (Lumac/3M), and the radioactivitywas determined with a Beckman LS 1800 scintillation counter.

Polyacrylamide Gel Electrophoresis. The purified TDC prep-arations were analyzed by slab gel electrophoresis under dena-turing conditions (0.1% SDS in running buffer and gel) accordingto the system described by Laemmli (12). The separating gel

-rubber stopper

A cC

70Jmt50mm

84*nmjm

-7mmmm

21mmFIG. 1. Schematic diagram of the glass reaction vial for the TDC

assay as described in "Materials and Methods."

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MARQUES AND BRODELIUS

contained10% polyacrylamide. Proteins were stained by thesilver method(14). The subunit mol wt ofTDC was determinedfrom a graph of migration distance versus log mol wt of standardproteins from Bio-Rad.Other Procedures. Protein content of samples was measured

by the method of Bradford (1) using the dye reagent concentrateand assay procedure from Bio-Rad. Gamma globulin was usedas a protein standard.

Cell dry weight was determined by drying vacuum-filtered cellson preweighed filter papers at 60°C to constant weight.

Sucrose concentration of the cultivation medium was meas-

ured as described previously (4).

RESULTS AND DISCUSSION

In this report we demonstrate how an unstable enzyme occur-

ring in very small amounts can be successfully extracted andpurified from plant cell suspension cultures. Of great importanceare the possible increase of enzyme activity (amount) by induc-tion with an elicitor and use of a rapid purification procedure.

Induction of TDC by Elicitor Treatment. Initially, the response

of the cell suspension culture of Eschscholtzia californica (usedas one of the sources of the enzyme) to elicitor treatment was

investigated. Under standard incubation conditions, the cell cul-ture reached stationary phase after 7 to 8 d (Fig. 2A). At thisstage the medium still contained glucose corresponding to morethan 5 g sucrose/L (Fig. 2A). Treatment of the cells with elicitorhad little effect on the total protein content throughout thegrowth cycle (Fig. 2B) but clearly increased TDC activity duringthe late exponential and early stationary growth phases (Fig. 2C).Relatively low TDC activities could be induced by elicitor treat-ment in the early exponential growth phase (d 2-5). The rela-tively high TDC activity observed on d 1 in nontreated cells maybe due to an induction of the enzyme upon inoculation of thecells into fresh medium. It may be pointed out that dilution withfresh medium has been used to induce TDC in cell suspensioncultures of Syringa vulgaris producing verbascoside (2). Further-more, on d 1 the cells appear to still be in a stationary growthphase from the previous growth cycle which may explain theinducibility of TDC. Cell suspension cultures of T. rugosum

respond similarly to elicitor treatment throughout a batch culti-vation (6). The highest TDC activities may be induced in cells

of T. rugosum or E. californica in late exponential or earlystationary growth phase.For the extraction of TDC, 7- and 10-d-old cultures of E.

californica and T. rugosum, respectively, were routinely em-

ployed. Optimal induction conditions, i.e. elicitor concentrationand incubation time after addition of elicitor, were establishedfor cultures of E. californica. As indicated in Figure 3, an

incubation time of 5 to 6 h (Fig. 3A) and a carbohydrateconcentration of at least 30 ,ug/g fresh weight (Fig. 3B) resultedin maximal TDC activity. The response of suspension culturesof E. californica to elicitor treatment was somewhat different

Table I. Comparison ofOptimal Conditions for Elicitor-Induced TDCFormation in Cell Suspension Cultures ofT. rugosum and

E. californica

T. E.rugosum californica

Age of culture (days) 10_1la 7-8Incubation after elicitation (h) 16-20a 5-6Amount of elicitor (,ug/g cells) 100-200a 30-40TDC activity in crude extract 0.66 2.2(nmol/min/g cells)

Specific TDC activity in crude ex- 0.18 1.7tract (nmol/min/mg protein)a These values are taken from Gugler et al. (6).

-c.- 0)

a)

.l,

a)

L-00 ^0

o N>

Ql ,a)

0'CU

0._

a-)

co

QE

0

.> cm0 \

EC 0 2 4

20

10a)0

0

I..

0

en

OD

incubation time (days)FIG. 2. Some characteristics of a cell suspension culture of E. califor-

nica during 8 d incubation at 26°C in the dark. A, Cell dry weight (0)and sucrose content (A) of the cultivation medium; B, protein content

of elicitor-induced (O) and noninduced (E) cells; C, TDC activity inextracts from elicitor-induced (E) and noninduced (l) cells.

100

.--

0

50

a)>

0 5 ~ 10 0 50 100

incubation time (h) elicitor concentration(pg/g fresh wt)

FIG. 3. TDC activity in a 7-d-old cell suspension culture of E. cali-fornica as a function of incubation time after addition of elicitor (A) andas a function of elicitor concentration (B).

from that ofT. rugosum cells (Table I). The latter culture required100 to 200 Mg carbohydrate per g fresh weight and an incubationtime of 16 to 20 h after addition of elicitor for maximal TDCactivity (6).

Purification of TDC. The purification of TDC to near homo-geneity could be performed successfully when utilizing elicitor-induced cells for extraction. Noninduced cells contained insuf-ficient levels ofTDC activity. Elicitor-induced Eschscholtzia cellscontained considerably more TDC activity than correspondingThalictrum cells (Table I). Therefore, E. californica cell cultures

A B

I

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L-TYROSINE DECARBOXYLASE. I

O-

a)

CZ.a)L,

a)

C)

a,

100

50-

0

100-

50

00 20 40 60 80 100

z

CZz

elution volume (ml)

FIG. 4. Elution profiles of anion exchange chromatography of TDCon a Mono Q column (HR 10/10). A, Chromatography of sample fromthe ammonium sulfate fractionation after desalting by gel filtration; B,rechromatography of the pooled TDC active fractions from A afterdesalting by ultrafiltration. ( ), A280; (@-), TDC activity; (---),

NaCl concentration.

were the most suitable source for TDC. However, for compara-tive purposes the enzyme was also purified from T. rugosum.

Table II summarizes a typical purification ofTDC from elici-tor-induced cells of Eschscholtzia. The enzyme was purified1540-fold to a final specific activity of2.6 Amol/min/mg protein.TDC represented approximately 0.06% of the protein extractedfrom induced E. californica cells. The highest specific activity,measured in another purification experiment, was 3.0 timol/min/mg protein (data not shown). The corresponding activity ofTDC from Thalictrum was 0.18 ,umol/min/mg protein. The lowspecific activity observed for the T. rugosum TDC may be dueto an intrinsically lower stability of the enzyme. The final yieldof TDC varied for different preparations between 2.5 and 9.5%.A specific activity of 1.4 nmol/min/mg protein after a 25-fold

purification ofTDC from barley (Hordeum vulgare L.) roots hasbeen reported (10). Purified mammalian aromatic amino acid

decarboxylase has a specific activity of 30 nmol/min/mg proteinfor tyrosine (3). Since TDC of plant origin appears to be quiteunstable (13), the low specific activity reported for barley rootTDC is not only due to a relatively low purification grade (10)but is, most likely, also caused by a significant loss of activityduring the relatively long time period required for purification.All chromatographic separations required for the purification ofTDC from Thalictrum or Eschscholtzia cells were carried outwithin 1 working day.Even though the two anion-exchange chromatography steps

in the purification of TDC from E. californica were performedunder identical conditions, a considerable purification could beobserved in the second step (Table II). This most likely resultsfrom the enzyme activity being eluted at different salt concentra-tions (0.16 and 0.21 M) during these two steps, as shown inFigure 4. We ascribe these fully reproducible, but different,elution patterns to the difference in the total amount of proteinin the applied samples. In this respect, it may be pointed outthat during the purification of TDC from T. rugosum, only oneanion-exchange chromatography step was included, since theenzyme was eluted directly at the higher salt concentration (0.21M) (data not shown).Hydrophobic interaction chromatography on a Phenyl-Super-

ose column was also performed twice. The degree of purificationwas improved considerably in the purification of TDC from E.californica by changing the pH of the elution buffer between thefirst and second run (pH 6.0 and pH 8.4) (Fig. 5). Obviously, thehydrophobicity of the proteins remaining after two anion-ex-change columns is influenced differently by changing the pH ofthe elution buffer. During the purification of TDC from T.rugosum, the two hydrophopic interaction chromatography stepswere carried out at the same pH (7.1).A major problem in the isolation of TDC was the rapid loss

of activity during purification. After extraction and during am-

monium sulfate fractionation, the enzyme was unstable at 2°C.Freezing and storage at -70°C prevented a decrease of activitybetween the first steps of purification (data not shown). PurifiedTDC was, on the other hand, inactivated by freezing but wasrelatively stable at 2°C. Further attempts to stabilize the purifiedenzyme are reported in the subsequent communication (13).SDS-PAGE. The purified preparation of Eschschohzia TDC

yielded one major protein staining band and a few minor bandsfollowing SDS-PAGE (Fig. 6, lanes A and B), while ThalictrumTDC appeared to be homogeneous (Fig. 6, lane C). Impuritiespresent in one preparation were absent in other preparations andvice versa (compare lanes A and B in Fig. 6). Furthermore, somefractions (preceding or following the peak fraction after hydro-phobic interaction chromatography) that did not show TDCactivity contained the same impurities but not the major bandat 56.3 kD seen in the TDC-active fractions. When comparingTDC-active fractions from the same purification experiment, the

Table II. Purification ofTDCfrom Elicitor-Induced Cell Suspension Cultures ofE. californica

Fraction Volume Protein Activity Specific Purification YieldActivity

ml mg nmol/ nmol/min/mg -fold %min proteinCrude supernatant 510 636 1082 1.7 1 100Ammonium sulfate 15 369 702 1.9 1.1 65Sephadex G-25 5.5 326 686 2.1 1.2 63Mono Q (I) 6 35 393 11.3 6.6 36Ultrafiltration 8 26 299 11.7 6.9 28Mono Q (II) 4.5 3.1 227 73 43 21Phenyl-Superose (pH 6.0) 2 107 9.9Phenyl-Superose (pH 8.4) 1.5 0.018 48 2620 1540 4.4

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MARQUES AND BRODELIUS

4L-'

CZ. _

0

01)4-'-CZ01)

L._

0Go

01)-I.

CZ._E

0EE

0Il0

>x

a)

4-'

0)

100

501-

0

-1-

CZa)

COE

0EE

elution volume (ml) elution volume (ml)FIG. 5. Elution profiles of hydrophobic interaction chromatography of TDC on a Phenyl-Superose column (HR 5/5). A, TDC-active fractions

from the eluate of the second Mono Q column (Fig. 4B) were pooled and chromatographed at pH 6.0; B, the peak fraction from A wasrechromatographed at pH 8.4. (-), A280; (-4*), TDC activity; (---) (NH4)2SO4 concentration.

Mr (kD)

1166.292.5--

FIG. 6. SDS-PAGE of purified TDC from elicitor-induced cell suspension cultures of E. californica(lane A and B) and T. rugosum (lane C). Each lane contained approximately 0.4 ,ug of protein.Lanes A and B represent preparations from two separate purification experiments. The positions ofmarker proteins are indicated by arrows. Gels were stained for protein by the silver method (14).

4 5

A B.

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L-TYROSINE DECARBOXYLASE. I

stain intensity of the major protein band, but not those of theimpurities, reflected the amount of TDC activity. These obser-vations suggest that the major band represents the enzyme. Thepresence of different impurities in separate preparations coulddepend on minor differences in the protein composition of theinduced cells and/or in the choice of fractions during purifica-tion. From Figure 6 it may also be concluded that the mol wt ofthe subunit is the same for TDC isolated from T. rugosum andE. californica, i.e. 56300 ± 300 (SD) (n = 5). The purifiedpreparations show one major band on a native PAGE gel (datanot shown).

LITERATURE CITED

1. BRADFORD MM 1976 A rapid and sensitive method for the quantitation ofmicrogram quantities ofprotein utilizing the principle ofprotein dye binding.Anal Biochem 72: 248-254

2. CHAPPLE CCS, MA WALKER, BE ELLIS 1986 Plant tyrosine decarboxylase canbe strongly inhibited by L-a-aminooxy-B-phenylpropionate. Planta 167: 101-105

3. CHRISTENSON JG, W DAIRMAN, S UDENFRIEND 1970 Preparation and proper-ties of a homogeneous aromatic L-amino acid decarboxylase from hogkidney. Arch Biochem Biophys 141: 356-367

4. FUNK C, K GUGLER, P BRODELIUS 1987 Increased secondary product forma-tion in plant cell suspension cultures after treatment with a yeast carbohy-drate preparation (elicitor). Phytochemistry 26: 401-405

5. GAMBORG OL, RA MILLER, K OJIMA 1968 Nutrient requirements of suspen-sion cultures of soybean root cells. Exp Cell Res 50: 151-158

6. GUGLER K, C FUNK, P BRODELIUS 1988 Elicitor-induced tyrosine decarbox-ylase in berberine synthesizing suspension cultures of Thalictrum rugosum.Eur J Biochem 170: 661-666

7. HAHLBROCK K, H GRISEBACH 1979 Enzymatic controls in the biosynthesis oflignin and flavonoids. Annu Rev Plant Physiol 30: 105-130

8. HAHN MG, P ALBERSHEIM 1978 Host-pathogen interactions XIV. Isolationand partial characterization of an elicitor from yeast extract. Plant Physiol62: 107-111

9. HEINSTEIN PF 1985 Future approaches to the formation of secondary naturalproducts in plant cell suspension cultures. J Nat Prod 48: 1-9

10. Hosoi K 1974 Purification and some properties of L-tyrosine carboxy-lyasefrom barley roots. Plant Cell Physiol 15: 429-440

11. KNOBLOCH K-H, B HANSEN, J BERLIN 1981 Medium-induced formation ofindole alkaloids and concomitant changes of interrelated enzyme activitiesin cell suspension cultures of Catharanthus roseus. Z Naturforsch 36c: 40-45

12. LAEMMLI UK 1970 Cleavage of structural proteins during the assembly of thehead of bacteriophage T4. Nature 227: 680-685

13. MARQUES IA, PE BRODELIUS 1988 Elicitor-induced L-tyrosine decarboxylasefrom plant cell suspension cultures. II. Partial characterization. Plant Physiol87: 53-56

14. MORRISSEY JH 1981 Silver stain for proteins in polyacrylamide gels: a modifiedprocedure with enhanced uniform sensitivity. Anal Biochem 117: 307-310

15. ROKEM JS, B TAL, I GOLDBERG 1985 Methods for increasing diosgeninproduction by Dioscorea cells in suspension cultures. J Nat Prod 48: 210-222

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