Simple purification of aromatic l -amino acid decarboxylase from human pheochromocytoma using high-performance liquid chromatography

ANALYTICALBIOCHEMISTRY

19,408-414(1985)

Simple Purification of Aromatic L-Amino Acid Decarboxylase from Human Pheochromocytoma Using High-Performance Liquid Chromatography’ HIROSHI I~HINO~E,* KOHICHI KoJIMA,t’2 AKIFUMI ToGARI,t YOSHIO KATO,~: SIMONE PARVEZ,~ HASAN PARVEZ,~ AND TOSHIHARU NAGATSU**~ *Department of Biochemistry, Nagoya University School of Medicine, Nagoya 466, Japan; TDepartment of L&e Chemistry, Graduate School al Nagalsuta. Tokyo Institute of Technology, Yokohama 227, Japan; *Toy0 Soda Manufacturing Company, Ltd, Shin-nanyo-shi, Yamaguchi 746, Japan: and $XJnite’ de Neuropharmacologie, Vniversile’ de Paris XI, 91405 Orsay, France Received July 25, 1985 We purified aromatic L-amino acid decarboxylase (AADC) homogeneously and rapidly from human pheochromocytoma using high-performance liquid chromatography. HPLC with gel permeation and hydrophobic columns was highly effective, and the entire purification could be finished within 3 days. Purified AADC showed a single band with an M, of 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and decarboxylated L-3,4-dihydroxyphenylalanine, L-5-hydroxytryptophan, and L-three-3,4-dihydroxyphenykerine (a synthetic precursor of natural norepinephrine). Amino acid analysis of purified AADC was performed. o 1985 Academic Pres, Inc.

KEY WORDS: protein/enzyme purification; HPLC, proteins; aromatic L-amino acid decarboxylase; amino acid analysis; hydrophobic chromatography; human pheochromocytoma.

Aromatic L-amino acid decarboxylase (AADC?,EC 4.1.1.28) catalyzes decarboxylation of aromatic L-amino acids to produce aromatic monoamines (1). L-3,4-Dihydroxyphenylalanine (L-DOPA) and L-5-hydroxytryptophan (L-5-HTP) are supposed to be main natural substrates. The products, dopamine and serotonin, are neurotransmitters and precursors of norepinephrine, epinephrine, and melatonin. Norepinephrine is a neurotransmitter in sympathetic nerves and brain, while epinephrine and melatonin are hor’ Supported by grants from Ministry of Health and Welfare, and from Ministry of Education, Science, and Culture, Japan. 2 Present address; Hatano Research Institute, Food and Drug Safety Center, Hatano, Kanagawa 257, Japan. ’ To whom correspondence should be addressed. 4 Abbreviations used: AADC, aromatic L-amino acid decarboxylase; DOPA, 3,4-dihydroxyphenylalanine; 5HTP: 5-hydroxytryptophan; DOPS, 3,4-dihydroxyphenylserine; SDS, sodium dodecyl sulfate; DlT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis. 0003-2697185 $3.00 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

408

mones in adrenal medulla and pineal gland. Besides monoamine-producing cells such as adrenal medulla, brain, and pineal gland, many tissues contain AADC, and relatively high activity of AADC exists in kidney and liver (2). Pearse (3) termed a group of cells synthesizing both peptide hormones and amines as the APUD (amine precursor uptake and decarboxylation) system, and the tumors of APUD type as APUDoma. Cells belonging to the APUD system have a high activity of AADC. Pheochromocytoma is an APUDoma and has high AADC activity. Small-cell lung carcinoma is one of the tumors of APUD type, and is reported to have a relatively higher activity of AADC than other lung tumors and normal lung tissues (4-6). Recent reports suggest the presence of neurons in brain or in spinal cord which contain AADC but neither tyrosine hydroxylase nor serotonin (7,s). These neurons have the enzymatic capacity to catalyze directly the con-

CHROMATOGRAPHY

OF

L-AMINO

version of the amino acids tyrosine, tryptophan, or phenylalanine to their respective amines, tyramine, tryptamine, or phenylethylamine. Whether these amines are really produced, and how they act in these neurons, are the problems to be solved. For answering these problems, it is important to study characteristics of AADC from various tissues. The purification of AADC was performed from pig and guinea pig kidneys (9- 12). But purification from other tissues was difficult. In this paper, we report a rapid purification procedure of AADC by high-performance liquid chromatography from human pheochromocytoma and some characteristics of the purified enzyme. MATERIALS

AND

METHODS

Human pheochromocytoma was kindly supplied from Dr. Y. Shimosato and co-workers (National Cancer Center, Tokyo, Japan). I~-DOPA, L-5-HTP, D-DOPA, N-methyldopamine, and pargyline HCl were obtained from Sigma Chemical Company (St. Louis, MO.); pyridoxal phosphate was from Katayama Chemicals (Osaka, Japan); Amberlite CG-50 was from Rohm and Haas (Philadelphia, Pa.); EDTA was from Tokyo Kasei Company (Tokyo, Japan): and 3,4-dihydroxybenzylamine HBr was a kind gift from Eizai Pharmaceutical Company (Tokyo, Japan). Lrhreu-3,4-Dihydroxyphenylserine (DOPS) was a gift from Sumitomo Pharmaceutical Company (Osaka. Japan). Ail other chemicals were of analytical grade. Amberlite CG-50 (type 1, 100-200 mesh) was activated by cyclic washing with 2 M HCl and 2 M NaOH, and finally with water, equilibrated with 0.5 M potassium phosphate buffer (pH 6.5) and stored in the same buffer. Gel permeation HPLC was performed on a HLC-803D system with two columns of TSKGel G-3000 SW (i.d. 2.4 X 60 cm) (Toyo Soda, Yamaguchi, Japan). Hydrophobic chromatography with HPLC was done on a Gilson system 42 gradient HPLC with a model 111 uv detector (280 nm) using a column of

ACID

DECARBOXYLASE

409

Phenyl-5PW (i.d. 7.5 X 75 mm) (Toyo Soda, Yamaguchi, Japan). Amino acid analysis was performed by the PICO - TAG HPLC system (Waters Co., Milford, Mass.). The samples were hydrolyzed by HCl and combined with phenylisothiocyanate derivatives. Produced amino acid derivatives were analyzed by HPLC. DOPA and 5-HTP decarboxylase activities were assayed based on the measurement of enzymatically formed dopamine and serotonin by HPLC with electrochemical detection after isolation by an Amberlite CG-50 column (13,14). When the activity was high, the amount of dopamine in the Amberlite eluate was directly determined by its native fluorescence (excitation at 270 nm and emission at 320 nm) (1). The standard incubation mixture for DOPA as a substrate contained (total volume 400 ~1, in final concentrations) 30 mM sodium phosphate buffer (pH 7.2). 0.17 mM ascorbic acid, 0.1 mM pargyline HCI (a monoamine oxidase inhibitor), 0.01 mM pyridoxal phosphate, 1.O mM L-DOPA (or D-DOPA for the blank), and the enzyme (13). The standard incubation mixture for 5-HTP as a substrate contained (total volume 400 ~1, in final concentrations) 30 mM sodium phosphate buffer (pH 7.2). 0.1 mM pargyline HCl, 0.0 1 mM pyridoxal phosphate, 1.0 mM L-5-HTP (or D-5HTP for the blank). and the enzyme ( 14). The reaction was carried out at 37°C for 20 min with DOPA and for 90 min with 5-HTP, and was stopped by adding 1 ml of 0.4 M perchloric acid. The activity toward L-three-3,4-DOPS (a synthetic amino acid which produces I~norepinephrine) was measured as for DOPA decarboxylation, and norepinephrine formed was determined by HPLC with electrochemical detection. One unit of activity is defined as that amount of enzyme which produces I pm01 of amines per minute. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was carried out according to Laemmli ( 15) at room temperature. A gradient slab gel (4-l 5% polyacrylamide) was used. Gels were stained with Coomassie brilliant blue R-250.

410

ICHINOSE

Protein concentrations were estimated by the method of Bradford using bovine serum albumin as a standard (16). K,,, and V,,,,, values were calculated by the method of Wilkinson from the LineweaverBurk plot ( 17). RESULTS

PuriJication

of the Enzyme

All the procedures were carried out at 4°C. Step 1. Extraction. The tissue stored frozen at -80°C was homogenized by a blender in 4 vol of a solution consisting of 0.25 M sucrose, 1 mM dithiothreitol (DTT), and 0.1 mM EDTA. The homogenate was centrifuged at 100,OOOg for 1 h. The supernatant was passed through a glass-wool column to remove floating lipids. Step 2. Acid treatment. The pH of the supernatant was adjusted to 4.7 by adding 1 M acetic acid, and it was centrifuged at 30,OOOg for 15 min. The precipitate was removed and the pH of the supernatant that contained the enzyme was adjusted to 7.0 by the addition of 1 M Tris-HCl buffer (pH 7.2). Step 3. DEAE-Sephacel column chromatography. The enzyme solution was diluted with a half-volume of buffer A, consisting of 20 KIM Tris-HCl (pH 7.2), 8% sucrose, and 1 mM DTT, and applied to a column (i.d. 5.0

TABLE

PURIFICATIONOFAADCFROM

ET

AL.

X 23.5 cm) of DEAE-Sephacel previously equilibrated with buffer A. The column was washed with buffer A and the enzyme was eluted by a linear gradient of O-O.4 M NaCl in buffer A. The active fractions were pooled. Step 4. Gel permeation HPLC. Solid ammonium sulfate was added to the active fraction to 60% saturation, and the resulting precipitate was collected by centrifugation and dissolved in a small amount of buffer B, consisting of 50 mM potassium phosphate buffer (pH 6.8), 0.2 M NaCl, 0.1 mM pyridoxal phosphate, 1 mM DTT, and 0.1 mM EDTA. The enzyme preparation was applied to two columns connected sequentially of Toyo-Soda G3000 SW equilibrated with buffer B. Elution was carried out with the same buffer at a flow rate of 3 ml/min. The active fractions were pooled. Step 5. Hydrophobic HPLC with a Phenyl5PW column. To the enzyme solution solid ammonium sulfate was added to 15% saturation. The mixture was applied to a Phenyl5PW column for hydrophobic HPLC, previously equilibrated with buffer C consisting of 50 mM potassium phosphate buffer (pH 6.8), 15% ammonium sulfate, 0.1 mM pyridoxal phosphate, 1 mM DTT, and 0.1 mM EDTA. Then, the column was washed with buffer C containing 10% ammonium sulfate, and elution was carried out by a linear gradient of IO-O% ammonium sulfate at a flow rate of 0.5

1

HIJMANPHEOCHROMOCYTOMA

Steps

Total protein Cm)

Total activity NJ)

Specific activity (U/w)

Purification (-fold)

Yield (%I

Homogenate 100,000 g supernatant pH 4.7 supernatant DEAE-Sephacel G-3000 SW Phenyl-SPW (1) Phenyl-5PW (2)

llooo 8460 6930 221 20.8 1.45 0.87

44.8 21.7 12.5 21.0 17.9 12.4 9.02

0.004 0.003 0.002 0.12 0.86 8.57 10.3

1 0.7 40 287 2860 3430

100 45 98 65 45 33

Note. U: prnol dopamine formed/min.

CHROMATOGRAPHY

OF L-AMINO

ml/min. The same procedure was repeated in the second Phenyl-SPW HPLC. A typical purification procedure of AADC from 98 g of human pheochromocytoma tissue is summarized in Table 1. This purification procedure was reproducible and rapid. The entire purification was finished within 3 days. The specific activity of the final preparation with L-DOPA as a substrate was 10.3 pmol/ min/mg protein with 33% recovery. The typical chromatograms of each step were shown in Figs. 1 and 2. A single subunit with an M, of 50,000 was observed by SDS-PAGE (Figs. 3, 4). The M, of the enzyme was estimated to be approximately 100,000 by gel permeation chromatography. Properties of the Purified Enzyme

Substrate specificity of the purified enzyme from human pheochromocytoma was examined with L-DOPA. L-three-DOPS, and L-5HTP. The enzyme decarboxylated all three amino acids. Michaelis constants (Km) and maximum velocities ( Vmax)toward each substrate were shown in Table 2. L-DOPA was the best substrate with a high V,, (10 pmol/ min/mg protein) and a low K,,, (5 X lo-’ M). The orders of K,,, value and V,,,,, were DOPA

2.

0

411

ACID DECARBOXYLASE

Chromataqram

on o Column

of Phenyi-SPW

(21

FIG. 2. Purification of AADC from human pheochromocytoma by second phenyl-5PW HPLC. Elution was carried out with a linear gradient of ammonium sulfate starting at fraction No. 46, as described in the text. The flow rate was 0.5 ml/min and a total of 4.9 ml of fractions was collected.

a

b

c

d

e

f

.------

50 loo tractwn“umbert ,om,,t”be,

0

FIG. I. Purification of AADC from human pheochromocytoma by DEAE-Sephacel column chromatography (step 3). Elution was carried out with a linear gradient of sodium chloride starting at fraction No. I. as described in the text. The Bow rate was 200 ml/h and a total of 260 ml of active fractions was collected.

FOG.3. SDS-PAGE. Lanes: (a) crude extract of human pheochromocytoma: (b-e) active fractions from chromatography of DEAE-Sephacel (b), G-3000 SW (c). first phenyl-SPW (d), and second phenyl-SPW (e); (f and g) standard proteins. These fractions were subjected to electrophoresis in 4-15s gradient polyacrylamide gel in the presence of SDS. The slab gel was stained for protein with Coomassie brilliant blue R-250. Approximately I .8 c(gof purified enzyme (e) was applied to the gel.

412

ICHINOSE

ET AL.

IOOk

E .e ;

50k

b 7 : a I

I I

IOk 1 0

0.5 Relative

1.0 Mobility

FIG. 4. Determination of molecular weight ofthe subunit of AADC by SDS-PAGE. The molecular weights of the standard proteins used for calibration were: 1, &gaIactosidase, 116,W, 2, phosphorylase b. 92,500; 3, bovine serum albumin, 66,200; 4, ovalbumin, 45,ooO, 5, carbonic anhydrase, 31,000; 6, soybean trypsin inhibitor, 21,500.

< 5-HTP < DOPS, and DOPA > 5-HTP > DOPS, respectively. Results on the effect of pyridoxal phosphate (a cofactor of AADC) on enzyme activity are shown in Fig. 5. AADC had the activity in the absence of exogeneous pyridoxal phosphate, but the activity was stimulated about two- to threefold by the addition of 10-6-10-4 M pyridoxal phosphate. High concentration of pyridoxal phosphate inhibited the AADC activity due to formation of a Schiff base with LDOPA. These results are in good agreement with those of Christenson et al. (9). The pH dependence of the decarboxylation of DOPA by AADC is shown in Fig. 6. Optimum pH was observed at pH 7. The amino acid composition of AADC from human pheochromocytoma was summarized in Table 3. The amino acid composition of pig kidney enzyme (9) was also shown

FIG. 5. Effect of pyridoxal 5’-phosphate (PLP) on the activity of purified enzyme. The activity of AADC was measured as described under Materials and Methods, except that the concentration of PLP was varied as indicated.

for comparison. The compositions of the two enzymes are similar, but the amount of serine or glycine was more and that of leucine was less in human pheochromocytoma AADC as compared with pig kidney enzyme. DISCUSSION

In this study, we purified AADC from human pheochromocytoma by using HPLC. AADC had been purified from pig kidney (91 1) and guinea pig kidney ( 12), but it was difficult to purify the enzyme from other tissues. The reported specific activity of pig kidney enzyme (9,l I) and guinea pig kidney enzyme was 8.67, 3.07, and 9.35 pmol dopamine formed/min/mg protein, respectively. In 1983,

TABLE 2

v L Substrate

(M)

L-DDPA LJ-HTP r-three-DOPS

4.6 X 1O-5 6.7 x lo-’ 1.3 x 10-3

(pmol/Zn/mg protein) 10.3 1.55 0.56

7.0

0.0

9.0

FIG. 6. Effect of pH on the activity of AADC. The enzyme activity of purified enzyme was measured as described under Materials and Methods in 30 mM sodium phosphate (o), Tris-HCI (B), or glycine-NaOH (A).

CHROMATOGRAPHY

OF L-AMINO

TABLE 3 AMINO ACID COMFQSITION OF AROMATIC L-AMINO ACID DECARBOXYLASE

Amino acid Aspartic acidb Glutamic acid b Serine Glycine Histidine Arginine Threonine Alanine Proline Tyrosine Valine Methionine Isoleucine Leucine Phenylalanine Lysine Cysteine Tryptophan

Human pheochromocytoma (mol%) 7.3 12.9 17.6 15.3 2.5 3.0 4.4 9.4 3.1 1.4 4.3 2.8 3.2 6.0 2.9 3.2 -c -c

Pig kidney“ (mol%) 6.3 10.5 5.4 8.8 2.5 5.7 3.4 10.8 4.9 2.8 6.3 2.3 4.0 12.3 5.4 4.7 2.1 1.8

u These data were quoted from Christenson ef a/. (9). b The figures include both free and amidated residues of asparatic and glutamic acids. ’ This value was not determined.

Maneckjee and Baylin ( 18) reported successful purification of AADC from human pheochromocytoma with a specific activity of 0.15 pmol/min/mg protein (18). The enzyme preparation which we purified in this study had a high specific activity (10.3 pmol dopamine formed/min/mg protein). This purification was enabled by efficient and rapid resolution by the HPLC systems and by the stabilizing effect of pyridoxal phosphate in the buffers. Addition of pyridoxal phosphate to the buffers highly improved the recovery. In the DEAE-Sephacel column chromatography step, an increase of total activity was observed. This phenomenon was also seen in the purification from the heterotransplants of small-cell lung carcinoma and human ganglioneuroma tissues (unpublished data). Rahman et al. ( 19) has found that an endogeneous

413

ACID DECARBOXYLASE

inhibitor of AADC was present in monkey serum, and that the inhibitor could be removed by DEAE-Sephacel column chromatography. Hashimoto et al. (20) characterized endogeneous inhibitors of AADC in rat submandibular gland. These reports suggest that an endogeneous inhibitor of AADC is also present in pheochromocytoma cells and that its removal may have resulted in an increase of total activity. The previous results concerning the subunit structure of AADC are controversial. Voltattorni et al. (2 1) reported that AADC from pig kidney is a heterodimer consisting of 43,000and 50,000-Da subunits. On the other hand. Maneckjee and Baylin (18) reported that AADC from human pheochromocytoma is a homodimer with a 50,000-Da subunit from the study using a radiolabeled suicide substrate. Our results also support that human pheochromocytoma AADC is a homodimer of A4, 100,000 with a subunit of M, 50,000. It has been discussed whether DOPA decarboxylase and 5-HTP decarboxylase are the same enzyme or two isoenzymes. Christenson et al. (9) proved that AADC in pig kidney is a single enzyme and acts on both L-DOPA and L-5-HTP. In this study, a single AADC in human tumor tissues was also probed to decarboxylate both L-DOPA and L-5-HTP. But an inhibitor of AADC seems to be present in human tumor tissues and may modify the substrate specificity of the crude enzyme. Further study of the inhibitor of AADC is desired. ACKNOWLEDGMENTS The authors thank Dr. Yukio Shimosato (National Cancer Center, Japan) for his kind supply of human pheochromocytoma tissue, and Dr. Norio Kaneda (Department of Biochemistry, Nagoya University School of Medicine. Nagoya, Japan) and Nihon Waters LTD (Osaka, Japan) for their kind help in amino acid analysis by the PICO * TAG HPLC system. REFERENCES 1. Lovenberg, W.. Weissbach, H., and ( 1962) J. Bid.

Chem.

Udenfiiend.

S.

231, 89-93.

2. Rahman, M. K., Nagatsu, T., and Kato, T. ( 198 1) Biochem.

Pharmacol.

30,645~649.

414

ICHINOSE

3. Pearse, A. G. E. ( 1969) J. Histochem. Cytochem. 17, 303-3 13. 4. Baylin, S. B., Abeloff, M. D., Goodwin, G., Camey, D. N., and Gazdar, A. F. (1980) Cancer Rex 40, 1990-1994. 5. Baylin, S. B., Weisburger, W. R., Eggleston, J. C., Mendelsohn, G., Beaven, M. A., Abelolf, M. D., and Ettinger, D. S. (1978) New Engl. J. Med. 299, 105-I 10. 6. Nagatsu, T., Ichinose, H., Kojima, K., Kameya, T., Shimase, J., Kcdama, T., and Shimosato, Y. (1985) Biochem. Med., in press. 7. Jaeger, C. B., Ruggiero, D. A., Albert, V. R., Park, D. H., Joh, T. H., and Reis, D. J. (1984) Neuroscience 11, 691-713. 8. Jaeger, C. B., Teitelman, G., Joh, T. H., Albert, V. R., Park, D. H., and Reis, D. J. (1983) Science (WashingIon, D. C.) 219, 1233-1235. 9. Christenson, J. G., Dairman, W., and Udenfriend, S. (1970) Arch. Biochem. Biophys. 141,356-367. 10. Lancaster, G. A., and Sourkes, T. L. (1972) Canad. J. Biochem. 50,791-797.

ET AL. 11. Voltattomi. C. B., Minelli, A., Vecchini, P., Fiori, A., and Turano, C. (1979) Eur. J. Biochem. 93, 18 l188. 12. Srinivasan, K., and Awapara, J. (1978) B&him. Biophys. Acta 526, 597-604. 13. Nagatsu, T., Yamamoto, T., and Kato, T. (1979) Anal. B&hem. 100, 160-165. 14. Rahman, Md. K., Nagatsu, T., and Kato, T. (1980) J. Chromatogr. 221,265-270. 15. Laemmli, U. K. (1970) Nature (London) 227, 680685. 16. Bradford, M. M. (1976) Anal. Biochem. 72,248-254. 17. Wilkinson, G. N. (1961) B&hem. J. 80,324-332. 18. Maneckjee, R., and Baylin, S. B. (1983) Biochemisfry 22,6058-6063. 19. Rahman, M. K., Togari, A., Kojima, K., Takahashi, K., and Nagatsu, T. ( 1984) Mol. Cell. Biochem. 63, 53-58. 20. Hashimoto, S., Ikeno, T., Hasegawa, J., Nagatsu, T., and Kuzuya, H. (1980) Arch. Oral Biol. 25, 195199. 21. Voltattorni, C. B., Mine& A., Cirotto, C., Barra, D., and Turano, C. (1982) Arch. Biochem. Biophys. 217,58-64.