Planta (1984)162:334-341

P l a n t a 9 Springer-Verlag 1984

Purification and properties of a phenol oxidase derived from suspension cultures of Mucuna pruriens Harm J. Wichers, Geert J. Peetsma, Theo M. Malingr~ and Hindrik J. Huizing Laboratory of Pharmacognosy, State University of Groningen, Antonius Deusinglaan 2, NL-9713 AW Groningen, The Netherlands

Abstract. From cells of Mucuna pruriens, grown in suspension, a monophenol monooxygenase (EC 1.14.18.1) was purified to homogeneity, as deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme appeared to have a native molecular weight of 90 000 + 5 000 dalton, and consisted of two subunits, each of 42000~ 1000 dalton. High-performance liquid chromatography with electrochemical detection for specific measurement of catecholes, was used to determine separately the tyrosinehydroxylating and catecholase activities of the enzyme. For the enzymatic activities, pH optima of, respectively, 7.5 and 5.5-6.5 were found; the effects of some inhibitors on both activities appeared to be different. Michaelis-Menten characteristics for some mono- and o-dihydroxysubstrates were determined.

Key words: Cell culture (phenol oxidase) - ~-3,4Dihydroxyphenylalanine - Mucuna (suspension culture, enzyme) - Phenol oxidase - Tyrosinase.

Introduction

Suspension-grown cells of Mucuna pruriens synthesize and accumulate ~-3,4-dihydroxyphenylalanine (L-DOPA) endogenously (Huizing and Wichefs 1984). Therefore, M. pruriens might be expected to contain a phenoloxidase-like enzyme. However, a large number of plant species that contain phenoloxidases do not accumulate L-DOPA. In only a few plant species, besides M. pruriens, has the presence of L-DOPA been demonstrated, e. g. in the seeds of Viciafaba (Guggenheim 1913; von Schantz et al. 1977) and some species of Baptisia and Lupinus (Daxenbichler et al. 1971). These facts indicate regulatory differences between phenoloxidases in plants. In order to gain insight into the properties of the phenoloxidase-like enzyme in M. pruriens, this enzyme was purified from cells grown in suspension. Furthermore, a method employing a high-performance liquid chromatograph, equipped with an electrochemical detector for specific measurement of catecholes, was used for measurement of the formation of L-DOPA form L-tyrosine, independent of the oxidation of L-DOPA to the quinone by the same enzyme. As a consequence, a procedure for comparison of the kinetics of the two enzymatic activities of the same enzyme became possible.

Phenol oxidases in plants (EC 1.14.18.1) comprise a family of enzymes which are often difficult to purify. Therefore, relatively few data are available on their exact molecular weight and other properties, and most kinetic data have been obtained with partly purified enzyme preparations (for a review, see Mayer and Harel 1979). Even fewer data are available on the occurrence and properties of phenoloxidases in plant cell suspensions. (Volk et al. 1977, 1978).

Culture and maintenance of cell suspension cultures of Mucuna pruriens. Cell suspension cultures of M. pruriens L. DC f. utilis

Abbreviations:

Homogenisation ~?fcells and ammonium-sulfate precipitation. Af-

DEAE = diethylaminoethyl; HPLC = highperformance liquid chromatography; L-DOPA = L-3,4dihydroxyphenylalanine

Material and methods

(Wall. ex Wight) Back C V (Cell line MPL 1/I; Huizing and Wichers 1984) were cultured in MS medium (Murashige and Skoog 1962) supplemented with 1 mgl-1 indole-3-acetic acid, 1 mgl 1 Nt_benzyladenine and 4% (w/v) sucrose at ph 5.9 (prior to autoclaving). The cells were harvested, after a 7- to 8-d culture period, by filtration, and stored at - 18~C until further processing. ter thawing the cells, 370 g (fresh weight) were suspendend in 650 ml (final volume) ice-cold 10 mM sodium phosphate buffer

H.J. Wichers et al.: Phenol oxidase from Mucuna pH 6.0. The cells were homogenised with an Ultra Turrax (type T45/G45, Janke and Kunkel, Staufen, FRG) for 12 x 2.5 min, at 7.5-rain intervals, under cooling in ice. The homogenate was centrifuged for 15 rain at 1340g, 4~ The pellet (containing 90% of the catecholase activity, see Table 1) was resuspended in 10 mM sodium phosphate buffer pH 6.0 with 0.5 M NaC1 till a final volume of 500 ml and stirred overnight at 4~C. After this extraction, the cell debris was removed by centrifugation for 30 rain at 1600 g, 4~C. Preliminary work had shown that most of the enzymatic activity precipitated between 40 and 90% ammonium-sulfate saturation. Therefore, the supernatant, containing the solubilized catecholase, was brought to 40% saturation with ammonium sulfate. After stirring for 4-5 h at 4~C the precipitate was removed by centrifugation (60 rain, 1600g, 4~ Subsequently, the supernatant was brought to 90% saturation with ammonium sulfate, and after 4-5 h the precipitate was collected by centrifugation (60 min, 1600g, 4~ The precipitate was suspended into 10 ml 10 mM sodium phosphate pH 6.0 and dialysed against a volume of 5 x 2 1 of the same buffer at 4~

Column chromatography. The enzyme fraction obtained through ammonium-sulfate precipitation and dialysis was applied to a column (24 cm long, 2.5 cm inner diameter) of Celite 535 (Serva, Heidelberg, FRG). The column was washed with three column volumes of 10 mM sodium phosphate pH 6.0 and with one column volume of 200 mM sodium phosphate pH 6.0. Subsequently the catecholase was eluted with three column volumes 500 mM sodium phophate pH 6.0. The enzyme fraction was dialysed against a volume of 5 • 5 1 10 mM sodium phosphate pH 6.0 and concentrated by ultrafiltration on a PM 10 membrane (Amicon, Lexington, USA). This fraction was applied to a diethylaminoethyl (DEAE)-Sepharose CL-6B column (22 cm long, 1.7 cm inner diameter; Pharmacia, Uppsala, Sweden). After washing the column with two column volumes 10 mM sodium phosphate pH 6.0, the enzyme was eluted with a gradient of 0-0.25 M NaC1 in 10 mM sodium phosphate pH 6.0 (400 ml). The conductivity of the eluate was determined with a CG 857 conductometer (Schott Ger/ite, Hofheim, FRG). Determination of the native molecular weight. For estimation of the native molecular weight, a partly purified enzyme preparation (precipitated with ammonium sulfate and dialysed against 10 mM sodium phosphate pH 6.0) was applied to a Sephadex G-150 column (30 cm long 1.2 cm inner diameter; Pharmacia, Uppsala, Sweden) and eluted with 50 mM sodium phosphate pH 6.0. Dextran blue (MW 2 x 106 dalton, Pharmacia, Uppsala, Sweden), fl-glucosidase (MW 117 6000 and 66 000 dalton, Fluka, Buchs, Switzerland), glucose-oxidase (MW 160000 dalton, Boehringer, Mannheim, FRG) and horse-radish peroxidase (MW 40000 dalton, Sigma, St. Louis, USA) were used as molecular-weight markers.

335 with air, was incubated with 20 gl 1 M sodium ascorbate (final concentration 5 raM) and 0.1 ml of the sample, at 27 ~C. During the first 10 min of incubation samples of 0.4 ml of the incubation mixture were mixed with 0.4 m120% trichloroacetic acid (TCA), at timed intervals, to stop the reaction. After removal of the precipitate by centrifugation (15 min, 10000 g), the L-DOPA concentration in the incubate was determined by the HPLC method with electrochemical detection as described by Oosterhuis et al. (1980).

Preparation of N-formyl tyrosine. N-Formyltyrosine was prepared from L-tyrosine following the method of Sheehan and Yang (1958). Enzymatic activity in the presence of catalase. To distinguish between a phenoloxidase, and the possibility of peroxidase activity in the ammonium-sulfate-precipitated enzyme fraction being responsible for hydroxylation of L-tyrosine, the abovedescribed method for measurement of the tyrosine-hydroxylating activity was performed in the presence of 6500 U catalase (Boehringer, Mannheim FRG; cat. no. 106810).

Determination of extinction coefficients. The extinction coefficients of the enzymatic oxidation products of L-DOPA, D -DOPA, dopamine, D, L-a-methyl dopa, D, L-noradrenaline and L-adrenaline-bitartrate were determined at 475 nm according to the method of Waite (1976), using mushroom tyrosinase (Sigma, St. Louis, MO., USA). The measured values of the quinones produced from the following substrates were: L-DOPA 3313 M -1 cm -1, D-DOPA 3317 M - l c m -1, dopamine 2298 M - l c m -1, D, L-a-methyl dopa 2862 M -1 cm -1 D, L-noradrenaline 1861 M - lcm- J and L-adrenaline-bitartrate 2966 M - l c m -1.

Protein determination. Protein was determined according to the method of Lowry et al. (1951). Because L-DOPA, endogenously present in cells, interferes with the Lowry protein determination, proteins were precipitated from the samples with one sample volume 20% TCA; the precipitated proteins were washed twice with 1 ml 10% TCA and subsequently the precipitates were incubated for 1 h at 90~ in 0.5 ml 1N NaOH; insoluble material was removed by centrifugation and the supernatants were assayed for protein.

Sodium dodecyl sulfate poIyacrylamide gel electrophoresis. Sodium dodecyl sulfate-polyacrylamidegel electrophoresis was performed on 15% acrylamide slab gels with 2.6% crosslinking as described by Lugtenberg et al. (1975). The samples were heated for 5 rain at 100~ in the presence of 1% mercapto-ethanol prior to electrophoresis.

Results

Enzyme assays. During the purification procedure, catecholase

Purification o f the catecholase f r o m M . pruriens

activity was measured routinely spectrophotometrically: 3.9 ml 100ram sodium phosphate pH 6.0, saturated with air, was mixed with 1 ml L-DOPA (2 mg m l - 1) in the same buffer and 0.1 ml of the sample, at 27~C. The increase in absorbance at 475 nm was read. One unit of enzymatic activity was defined as the amount of enzyme that forms 1 lamol of quinone per rain under these conditions, assuming an extinction coefficient of 3313 M - l c m -J (see below) for the quinone formed from L-DOPA. For measurement of the tyrosine-hydroxylatingactivity, the formation of L-DOPA from L-tyrosine was determined with a method making use of high-performance liquid chromatography (HPLC) with electrochemical detection: 3.9 ml 2 mM L-tyrosine in 50 mM 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris)/HC1 pH 7.5 (final concentration 1.95 raM), saturated

Celite chromatography. T h e e n z y m e f r a c t i o n w h i c h precipitated with a m m o n i u m sulfate between 4 0 % a n d 9 0 % s a t u r a t i o n , after d i a l y s i s a g a i n s t 10 m M s o d i u m p h o s p h a t e p H 6.0, was a p p l i e d to a Celite 535 c o l u m n ; a f t e r w a s h i n g the c o l u m n t h o r o u g h l y (see M a t e r i a l a n d m e t h o d s ) a p p r o x . 16% o f the c a t e c h o l a s e activity, t o g e t h e r w i t h the b u l k o f the p r o t e i n , w a s w a s h e d off the c o l u m n . A p p l y i n g the e n z y m e f r a c t i o n to the c o l u m n i n 200 m M s o d i u m p h o s p h a t e p H 6.0 r e s u l t e d i n p o o r b i n d i n g o f the c a t e c h o l a s e a c t i v i t y to the c o l u m n . T h e e n z y m e f r a c t i o n t h a t was s u b s e q u e n t l y e l u t e d w i t h 500 m M

336

H.J. Wichers et al.: Phenol oxidase from Mucuna

0D280

201

enzyme activity 15 U.ml-1

200 mM

10 m M

12 1.5

10 I

05

'

!

. - --

''~

100

r. L .

,

-

200

300

"r'r

~_r-''r 0

~ ~"

-~

~',.t..

i

100

200

0

100

200

300

ELUATE,ml Fig. 1. Elution of catecholase activity from Celite 535. The catecholase activity was eluted with sodium phosphate buffer pH 6.0 of the molarities shown. , catecholase activity; - - - , ODzso

conductivity mS 15

enzyme activity U,m1-1 15 0D28~ xlO

I

10

40 7.5 30 5 20

10

I

I

I

I

I

I

I

I

0

10

20

30

40

50

60

70

~

I

I

80

90

~ll

I

I

100

110

0

fraction number Fig. 2. Elution of catecholase activity from DEAE-Sepharose CL-6B. The catecholase activity was eluted with a gradient of 0-0.25 M NaC1 in 10 mM sodium phosphate buffer pH 6.0. O - O , ODzso; A - A , catecholase activity; I1-11, conductivity of the eluents (measured in Siemens, S)

sodium phosphate pH 6.0 (Fig. 1) contained approx. 80% of the catecholase activity (Fig. 1).

Diethylaminoethyl-Sepharose chromatography. For a further purification, the enzyme fraction, which eluted from the Celite column, was applied to a DEAE-Sepharose CL-6B column previously equili-

brated with 10 mM sodium phosphate pH 6.0. After washing the column, a gradient of 0-0.25 M NaC1 in 10 mM sodium phosphate pH 6.0 was applied. The catecholase activity eluted as a single peak at a measured conductivity of the eluate of 12.5 mS (Siemens), corresponding to 0.135 M NaC1 (Fig. 2). The purification procedure for the cate-

H.J. Wichers et al.: Phenol oxidase from Mucuna

337

Table 1. Purification of the catecholase activity from suspension-grown cells of Mucuna pruriens Fraction

Volume

Total activity

Total protein

Specific activity Yield (%)

(ml)

(U)

(rag)

(U mg - 1)

Cell homogenate

650

9517

1365

Extracted pellet of cell homogenate Ammonium-sulfate precipitation, 40% pellet

285

8 642

Ammonium-sulfate precipitation, 90% pellet Enzyme fraction a after Celite chromatography Enzyme fraction a after DEAE chromatography

6.7

7

128

67.5

Protein

Enzyme

100

100

9.4

90.8

45.5

0.5

10.0

930

4.3

216

0.3

9.8

5

855

1.1

777

0.1

9.0

5.6

546

0.83

658

0.06

5.7

a After concentration through ultrafiltration

15 2•

6

160~00

116000

66000

40000

catecholase activrty (Urnl t) 10

O5

'

4

8

1

~

9

16

,

20

24

28

32

elubon volume(ml)

Fig. 4. Estimation of thb molecular weight, under non-denaturing conditions, of the catecholase from suspension-grown cells of Mucuna yruriens on a Sephadex G-150 column. Molecular weight and elution volume of standard proteins~are indicated. B B . catecholase activity

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the catecholase fi'action. The catecholase Fig. 3. Sodium dodecyl sulfatepoiyacrylamide gel-electrophoresis of the catecholase from suspension-grown cells of Mucuna pruriens. 1, catecholase; 2 standard proteins (molecular weight indicated, kdalton)

cholase activity is summarized in Table 1. The specific activity (U (rag protein)-~) is increased 94fold, when the cell homogenate and the DEAESepharose eluate are compared. However, during the purification procedure much of the enzymatic activity is lost. Therefore, it is not possible to determine which purification factor is reached for the catecholase activity.

fraction that eluted from the DEAE-Sepharose column was subjected to polyacrylamide-gel electrophoresis (Fig. 3). The enzyme appeared to be pure, as visual inspection of the gel revealed the presence of only one protein band. The molecular weight of this protein was estimated to be 42000 :k 1000 dalton.

Estimation of the molecular weight by gel filtration. A partly purified catecholase preparation (i. e. after ammonium-sulfate precipitation) was used to estimate the molecular weight of the catecholase under non-denaturing conditions on a Sephadex G-150 column. From two experiments the molecular weight was estimated to be 90000=k 5000 dalton under these conditions (Fig. 4).

338

H.J. Wichers et al.: Phenol oxidase from Mucuna 0D475

0.5 B

JJg L- DOPA. (ml.enz',me solution )-1

OmM

1000 Fig. 5A. Tyrosine hydroxylation by a

750

025

500 250

0

0

=

_

2

4

6

8

OO0 "-~ 10 0

J ;

5mM I

I

10 20

30 40 t(min)

t(min)

Measurement of the tyrosine-hydroxylating activity with the HPLC method. The method was based on the hydroxylation of L-tyrosine to L-DOPA in the presence of ascorbate. The amount of L-DOPA formed in the reaction mixture was quantitated using the HPLC method with electrochemical detection described by Oosterhuis et al. (1980). Ascorbate serves both as a cofactor for the tyrosinehydroxylating activity (Fig. 5 A) and as an efficient inhibitor of the catecholase activity (Fig. 5 B); both enzymatic activities were present in the ammoniumsulfate-precipitated catecholase fraction. Under standard conditions, 5 mM ascorbate was added to the reaction mixture, and this appeared to be sufficient to be non-limiting in the time course chosen for the tyrosine-hydroxylation reaction, and also to inhibit DOPA oxidation completely during this time course. Therefore a kinetic comparison between both enzymatic activities was possible. From the linear increase in DOPA concentration between 2 and 8 rain of incubation, the rate of synthesis was calculated.

Properties of the tyrosine-hydroxylating and catecholase activities; p H optima. In a partly purified enzyme preparation (precipitated with ammonium sulfate) the pH optimum for the tyrosinehydroxylating activity was 7.5, and for the catecholase activity a broad pH optimum of 5.5-6.5 was found (Fig. 6).

I

partly purified catecholase from Mucuna pruriens. DOPA Formation was measured with the HPLC-method. & - A , 0 mM ascorbate; I I - m , 0.1 mM ascorbate; A - A , 2 mM ascorbate; []-[~, 5 mM ascorbate; O - O , 8 mM ascorbate. B The effect of ascorbate on the formation of dopaquinone from L-tyrosine by a partly purified catecholase preparation from suspensiongrown cells of Mucuna pruriens. Upper curve, no ascorbate added; Lower curve, 5 mM ascorbate added

umol b DOPA min:1(ml.enzymesolution)1

jJmolquinone mintI (ml enzyme solutioni1

/ 0751

15

I

0.501

10

I

3

8

pH Fig. 6. Effect of pH on tyrosine-hydroxylating ( A - A ) and catecholase activities ( A - A ) of a partly purified catecholase preparation from suspension-grown cells of Mucuna pruriens

Specific activity. The specific activity of the catecholase fraction that eluted from the DEAESepharose column with L-DOPA as a substrate, was 658 ~tmol quinone rain -1 (mg protein) -1. From different experiments, a specific-activity range of 574-780 gmol rain -~ (mg protein) -1 was found. The

H.J. Wichers et al.: Phenol oxidase from Mucuna

339

Table 2. Michaelis-Menten characteristics for some mono- and o-dihydroxy substrates and a partly purified catecholase preparation from Mucuna pruriens Substrate

Km (raM)

Substrate

Vmax

(pmol.min - 1 (ml enzymesolution)-1)

Km (mM)

Vmax

(pmol.mi n - 1 (ml enzyme solution)-1)

L-Tyrosine

1.8

1.81

L-DOPA

6.66

30.0

N-Formyl-L-tyrosine

8.0

12.19

D-DOPA

5.55

33.3

Tyramine

0.18

6.36

Dopamine

1.09

18.2

a-Methyl DOPA

2.50

L-Adrenaline bi-tartrate

1.45

12.0

Noradrenaline

2.00

31.3

P-Hydroxyphenylacetic acid

31.5

22.2

2.63

Table 3. Effect of inhibitors on the tyrosine-hydroxylating and catecholase activities of the phenoloxidase from suspension-grown cells of Mucuna pruriens Compound

Concentration (mM)

Activitya 1

-

-

0.376

Diethyl-dithiocarbamic acid

0.01 0.05 0.2 1.0

Cystein-HCI Thiourea

Sodium azide

Sodium ascorbate

Inhibition (%)

Activity b 2

Inhibition (%)

0

3.25

0.279

52

2.92

10

0.018

97

0.56

83

0.537

7

2.32

29

0.467

19

1.30

60

0.2

0.487

16

2.04

37

0.2

0.474

18

2.69

17

1.0

0.384

33

1.86

43

0.1

-

0c

0

100

a Tyrosine-hydroxylating activity in ~tmol DOPA rain-1 (ml enzyme solution)-1 b Catecholase activity in gmol quinone min-1 (ml enzyme solution) 1 c induces a concentration-dependent lag-phase

same fraction, which was apparently pure (Fig. 3) showed a tyrosine-hydroxylating activity of 15 pmol L-DOPA min 1 (mg protein) -1. In several other experiments, a specific activity range of 11.2-16.5 gmol min -1 (mg protein) -I for the tyrosine-hydroxylating activity was found. These results indicate that both enzymatic activities are located on the same enzyme, and that the specific activity of the catecholase activity is 35-to 70-fold higher than the specific tyrosine-hydroxylating activity. In the course of the purification procedure, however, the tyrosine-hydroxylating activity was inactivated more rapidly than the catecholase activity. In an ammonium-sulfate-precipitated enzyme fraction, for example, the catecholase activity was only 5.5-to 8-fold higher than the tyrosinehydroxylating activity (see also Table 3).

Tyrosine-hydroxylating activity in the presence of catalase. The rate of formation of L-DOPA from

L-tyrosine by an ammonium-sulfate-precipitated enzyme fraction was not inhibited by the addition of 6500 units of catalase to the reaction mixture, as determined by the HPLC method.

Michaelis-Menten kinetics for some mono- and o-dihydroxysubstrates. Plant phenol oxidases are known for their broad substrate specificity. In order to test the substrate specificity of the enzyme from M. pruriens, Michaelis-Menten kinetics for some mono- and o-dihydroxy substrates were determined with, respectively, the HPLC-method (monohydroxysubstrates) and spectrophotometrically (o-dihydroxysubstrates, assuming the extinction coefficients mentioned under Material and methods). A partly purified enzyme preparation (ammonium-sulfate-precipitated) was used in the measurements. The results are summarized in Table 2. The values for Vma~ are expressed per ml of enzyme solution. Also, para-hydroxybenzoic acid

H.J. Wichers et al.: Phenol oxidase from Mucuna

340 and para-hydroxybenzaldehyde were tested as substrates, but the affinity of the enzyme for these compounds was very low. Only at very high substrate concentrations (20 m M or more) could a slow reaction be measured.

The effect of inhibitors on the tyrosine-hydroxylating and catecholase activities. Some well-known inhibitors of phenoloxidases were tested for their action upon both the tyrosine hydroxylation and the catechol oxidation. From Table 3 it can be concluded that the effect of a particular compound on the tyrosine-hydroxylating or catecholase activities can be quite different. Diethyl-dithiocarbamic acid has a much more pronounced effect on the tyrosine hydroxylation than on the catecholase activity, whereas with azide or cysteine-HC1 the opposite is the case. Thiourea affects both activities to roughly the same extent. Discussion With the relatively simple procedure described in this paper, it appeared to be possible to purify, from cell cultures of M. pruriens, an enzyme, which was able to hydroxylate L-tyrosine to L-DOPA, as well as to oxidize L-DOPA to dopa-quinone. The enzyme showed a broad substrate specificity towards mono- and o-dihydroxy phenolic substrates, with a relatively low affinity for these substrates. Furthermore, the enzyme was unable to oxidize ascorbate (not shown), and its tyrosine-hydroxylating activity was preserved in the presence of catalase. From these observations, we concluded that this enzyme can be classified as a monophenol monooxygenase, or by its classical name, tyrosinase. Comparison of the specific activities of both enzymatic conversions showed that the hydroxylation of L-tyrosine into L-DOPA was the ratelimiting step in the overall transformation of L-tyrosine into dopaquinone. It must be taken into account, however, that during the purification procedure the tyrosine-hydroxylating activity decreased more rapidly than the catecholase activity. Therefore, it is doubtful whether the observed ratio between the activities of the purified enzyme reflects the in-vivo situation correctly. The enzyme showed a native molecular weight of 90 000 dalton, whereas under denaturing conditions a molecular weight of 42000 dalton was found. Therefore, the native enzyme probably occurs as a dimer. The molecular weight of 42000 dalton for the subunit compared well with the molecular weight (43000 dalton) of the heavy subunit of Agaricus bispora tyrosinase (Strothkamp etal. 1976) and to the

molecular weight of 42000 dalton for Neurospora crassa tyrosinase (Lerch 1976). However, a broad range of molecular weights was reported for the catechol oxidases of higher plants (Mayer and Harel 1979, 1981). The use of the H P L C method for the measurement of the tyrosine-hydroxylation rate made a comparison of both enzymatic activities possible, without the disadvantage of measurement of an overall reaction, as is the case with oxygen or ascorbate-consumption measurements with a monohydroxy phenol as the substrate (Lerner et al. 1974; Patil and Zucker 1965), and without the need to use a radioassay (Pomerantz 1966; Motohashi et al. 1982). Using our method, we found a marked difference in p H optimum of 1-2 pH units for the tyrosine-hydroxylating and catecholase activities. In most reports, no such differences have been observed, probably as a result of the assay that was used; Pomerantz (1966) observed a difference in pH optimum for tyrosine hydroxylation and D O P A oxidation by mammalian tyrosinases. The differential effect of some inhibitors on the tyrosinehydroxylating and the catecholase activities might be an indication of different reaction centers for both activities, as is also indicated by the difference in pH optimum. Differential effects of inhibitors were also reported by Lerner et al. (1974) and by Vaughan and Butt (1970). The sulfur-containing compounds used in our study (thiourea, diethyldithiocarbamic acid and cysteine-ttC1) all exhibited a different effect on the radio of the enzymatic activities. Together with the difference in p H optimum, such a differential effect might be of practical importance when the eventual application of enzyme preparations for the production of cD O P A is considered. The authors would like to thank Mrs. B. Goedewaagen for technical assistance. These investigations were supported by the Committee of the "Van Leersumfonds", and by the Netherlands Foundation for Technical Research (STW), Technical Science Division of the Netherlands Organisation for the Advancement of Pure Research (ZWO).

References Daxenbichler, M.E., Van Etten, C.H., Hallinan, E.A., Fontaine, R.E. (1971) Seedsas sources of L-DOPA. J. Med. Chem. 14, 463~465 Guggenheim, M. (1913) Dioxyphenylalanin, eine neue Aminos/iure aus Viciafaba. Z. Physiol. Chem 88, 276 Huizing, H.J., Wichers, H.J. (1984) Production of L-DOPA by Mucunapruriens cell suspension cultures through accumulation or by biotransformation of tyrosine. In: Biotechnological research in The Netherlands, Houwink, E.H., van der Meer, R.R., eds. (Progressin industrial microbiologyseries.) Elsevier, Amsterdam (in press)

H.J. Wichers et al.: Phenol oxidase from Mucuna Lerch, K. (1976) Neurospora tyrosinase: molecular weight, copper content and spectral properties. FEBS Lett. 69, 157-160 Lerner, H.R., Mayer, A.M., Harel, E. (1974) Differential effect of phenylhydrazine on the catecholase and cresolase activities of Vitis catechol oxidase. Phytochemistry 13, 397-401 Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J. (195l) Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 265-271 Lugtenberg, B., Meijers, J., Peters, R., Van der Hoek, P., Van Alphen, L. (1975) Electrophoretic resolution of the major outer membrane protein of Escherichia coil K12 into four bands. FEBS Lett. 58, 254-258 Mayer, A.M., Hard, E. (1979) PolyphenoI oxidases in plants. Phytochemistry 18, 193-215 Mayer, A.M., Harel, E. (1981) Polyphenol oxidases in fruits Changes during ripening. Annu. Proc. Phytochem. Soc. 19, 161-180 Motohashi, N., Eguchi, H., Mori, I. (1982) Radioassay for oxidation of tyrosine by tyrosinase using L-[Ring-3H] tyrosine and L-[Carboxyl-14C]-tyrosine. Chem. Pharm. Bull. 30, 2094-2098 Murashige, T., Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15, 473-497 Oosterhuis, B., Brunt, K., Westerink, B.H.C., Doornbos, D.A. (1980) Electrochemical detector flow cell based on a rotating disk electrode for continous flow analysis and high performance liquid chromatography of catecholamines. Anal. Chem. 52, 203-205

341 Patil, S.S., Zucker, M. (1965) Potato phenolases. J. Biol. Chem. 240, 3938-3943 Pomerantz, S.H. (1966) The tyrosine hydroxylase activity of mammalian tyrosinase. J. Biol. Chem. 241, 161 168 Sheehan, J.C., Yang, D.D.H. (1958) The use of N-formylamino acids in peptide synthesis. J. Am. Chem. Soc. 80, 1154-1158 Strothkamp, K.G., Jolley, R.L., Mason, H.S. (1976) Quaternary structure of mushroom tyrosinase. Biochem. Biophys. Res. Commun. 70, 519-524 Vaughan, P.F.T., Butt, V.S. (1970) The action of o-dihydric phenols in the hydroxylation of p-coumaric acid by a phenolase from leaves of spinach beet (Beta vulgaris L.). Biochem. J. 119, 89-94 Volk, R., Harel, E., Mayer, A.M., Gan-Zvi, E. (1977) Catechol oxidase in tissue culture of apple fruit. J. Exp. Bot. 28, 82~830 Volk, R., Hard, E. Mayer, A.M., Gan-Zvi, E. (1978) Catechol oxidase in suspension cultures of apple fruit the effects of growth regulators. J. Exp. Bot. 29, 1099-1109 Von Schantz, M., Huhtikangas, A., Hiltunen, R., Hovinen, S. (1977) Zfichtung yon Pferdebohnensorten mit hohem L-DOPA-Gehalt. Sci. Pharm. 45, 201-212 Waite, J.H. (1976) Calculating extinction coefficients for enzymatically produced o-quinones. Anal. Biochem. 75, 211-218

Received 9 March; accepted 3 April 1984