subunit structure of 3,4-dihydroxyphenylalanine decarboxylase from pig kidney

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 217, No. 1, August, pp. 58-64, 1982

Subunit Structure of 3,4-Dihydroxyphenylalanine Decarboxylase from Pig Kidney’

CARLA BORRI VOLTATTORNLz ALBA MINELLI, CARLO CIROTTO, DONATELLA BARRA, AND CARLO TURANO

Institutes of Biological Chemistry, Faculties of Pharmacy, Universities of Perugia, Camerino, and Rome; Institute of Histology and Embryo&g Faculty of Sciences, University of Perugiq

and CNR Center of IbMmdar Biology, Rame, Italy

Received December 8, 1981, and in revised form March 2, 1982

3,4-Dihydroxyphenylalanine decarboxylase from pig kidney has been shown to be a dimer of two nonidentical subunits. The existence of these subunits is proved by gel electrophoresis in 8 M urea or in 1% sodium dodecyl sulfate; their molecular weights are estimated to be 43,000 and 50,000. Separation of the two subunits has been achieved by chromatography on CM-cellulose in 8 M urea. Only the 50,000-dalton polypeptide chain contains bound pyridoxal-P. The differences shown by the amino acid compositions of the two subunits and in particularly by their tryptic peptide maps suggest that the 43,000-dalton subunit does not arise from protease attack on the 50,000-dalton subunit.

Dopa decarboxylase (L-3,4-dihydroxyphenylalanine carboxy-lyase, EC 4.1.1.28) from pig kidney, a pyridoxal-P-dependent enzyme which catalyzes the conversion of aromatic aminoacids into the corresponding amines, binds 1 mol of coenzyme per mole of protein (1, 2).

The purification and the analysis of the phosphopyridoxyl peptide obtained after reduction of the holoenzyme with NaBH4 and digestion with chymotrypsin show that pyridoxal-P is bound to a lysine side chain (3) as are other pyridoxal-P-dependent enzymes so far investigated.

Preliminary reports from other labo- ratories (1,4,5) of the behavior of the enzyme in polyacrylamide gel electrophoresis in the presence of sodium dodecyl

i This work was supported by CNR Grant 79.00903.04.

*To whom correspondence should be sent: Istituto di Chimica Biologica-Facolta di Farmacia via de1 Giochetto-C.P. 37, succ.3-06100 Perugia, Italy.

’ Abbreviations used; Dopa, 3,4-dihydroxyphenylalanine; SDS, sodium dodecyl sulfate.

sulfate (SDS), have indicated that Dopa decarboxylase is composed of subunits, although the number and the molecular weight of them is controversial. These ob- servations, together with the data on the stoichiometry of pyridoxal-P binding to the protein, prompted us to a more de- tailed investigation of the quaternary structure of the enzyme. We report here data showing that Dopa decarboxylase is comprised of two nonidentical subunits and give details of the separation and structural characteristics of these subunits.

MATERIALS AND METHODS

Pyridoxal-P and dithiothreitol were obtained from Sigma; @mercaptoethanol was from Koch-Light; acrylamide, bisacrylamide,N,N,iV’-N’-tetramethyleth- ylenediamine, SDS, and AG 501-X8D were purchased from Bio-Rad Labs; urea was from Merck; CM-cellulose (Microgranular CM 52) was from Whatman; and trypsin treated with L-1-tosylamido-2-phenyl- ethylchloromethyl-ketone was from Calbiochem. Urea was purified before use by deionization of 10 M solu- tions using a mixed-bed ion-exchange resin (Bio-Rad

0003-9861/82/090058-07$02.00/O Copyright Q 1982 hy Academic Press, Inc. All rights of reproduction in any form resewed.

58

SUBUNIT STRUCTURE OF PIG Dopa DECARBOXYLASE 59

FIG. 1. SDS-electrophoretic pattern of Dopa decarboxylase and its subunits on polyacrylamide gel. (A) enzyme in the presence of 100 mM @mercaptoethanol; (B and C) separated not fluorescent and fluorescent subunits, respectively; (D) B + C (1:l). Gel conditions: 5.6% acrylamide, 1% SDS.

AG 501-XSD) followed by filtration through a Mil- lipore filter.

All other reagents used were of analytical grade. For the standardization of gel electrophoresis a kit

(Boehringer-Mannheim) of the following proteins was used: trypsin inhibitor from soybean (Afr 21,500), albumin from bovine serum (iif? 68,000), RNA-poly- merase from Escherichio coli: a (Afr 39,000), fl (A& 155,000) and @’ (A& 165,000) subunits.

Fluorescence measurements were carried out with a Perkin-Elmer spectrophotofluorimeter MPF3, equipped with a Perkin-Elmer recorder.

Enzyme preparation The enzyme was prepared from pig kidney (2) and was essentially homogeneous on polyacrylamide gel electrophoresis and analytical centrifugation.

Protein determination Protein concentration was determined using the absorbance at 280 nm, assuming an absorbance of 1 for a concentration of 1 mg/ml.

Polyocrglamide gel electmphoresis. Disc gel electrophoresis in the presence of SDS or urea was performed as described by Fairbank et al. (6) and by Stegink et al. (7), respectively.

Gels were stained with Coomassie Brillant Blue R- 250. After destaining, the gels were scanned with a Varian Techtron spectrophotometer (Model 635) at 600 nm.

Fingerprint analysis. The protein was extensively dialyzed against 5% acetic acid and lyophilized. The resulting powder was suspended in 2.5 ml of 0.1 M

ammonium bicarbonate, pH 8.4, at a protein concentration of 1.5 mg/ml. Digestion with trypsin was carried out at 37°C according to Ingram (8). After 6 h the pH was brought to 6.4 with 1 M acetic acid and the clear solution was lyophilized. The lyophilized sample was dissolved in about 200 pl of water and 50 pl were spotted on Whatman 3MM paper. High- voltage paper electrophoresis was carried out at pH 6.4 (pyridine:acetic acid:water, 1:0.4:9) at 1700 V 2 mA/cm and descending paper chromatography was performed with 1-butanol:pyridine:acetic acid:water (15:10:3:12) as the solvent (9). After the bidimen- sional separation the paper was developed with a solution of ninhydrin in acetone (10).

Amino acid analysis. Amino acid analyses were carried out after hydrolysis in 6 M HCl at 110°C in evacuated tubes for 24 h. Nor leucine was added as an internal standard. Hydrolysates were analyzed using an LKB 4400 amino acid analyzer, equipped with a Spectra-Physics System I Computing Inte- grator. Cysteine plus cystine content was determined as cysteic acid after hydrolysis in the presence of 0.3 M dimethyl sulfoxide (11). Tryptophan was determined after hydrolysis with 4 N methanesulfonic acid containing 0.2% 3-(2-aminoethyl)indole (12).

RESULTS

Disc Gel Electrophoresis

When the native enzyme was pretreated with 1% SDS and 100 mM P-mercaptoethanol, two distinct bands, which stained with similar intensity, were observed on SDS-electrophoresis (Fig. 1A).

Results were essentially the same when preincubation and electrophoresis of the protein were done without P-mercaptoethanol.

The relative mobilities of these bands were compatible with proteins having a molecular weight of 43,000 and 50,000 in a linear plot of the relative mobilities of the proteins used as standards (see Ma- terial and Methods) vs the log of their molecular weights (data not shown).

When the enzyme, after treatment with 8 M urea in 5% acetic acid or in 5 mM citrate-sodium phosphate buffer, pH 3.7, containing 100 InM /3-mercaptoethanol, was subjected to disc gel electrophoresis in 8 M urea and 5% acetic acid it showed two clearly separated bands (Fig. 2A); the corresponding electropherogram shows two components of similar area.

60 VOLTATTORNI ET AL.

Separation of Subunits bg CM-Cellulose Chromatography

The subunits of Dopa decarboxylase were separated by CM-cellulose chromatography in the presence of 8 M urea at pH 3.7.

In a typical experiment, 300 ~1 (20 mg) of enzyme were reduced by NaBH4 according to Bossa et al (3) in order to bind irreversibly the coenzyme to the protein and were mixed with 10 ml of 5 mM citrate- sodium phosphate buffer at pH 3.7 containing 8 M urea and 100 IYIM @-mercaptoethanol.

This solution was dialyzed for 8 h against the same buffer. The sample was then ad- sorbed into a 1.5 X 15-cm CM-cellulose column which had been equilibrated with 8 M urea and 100 mM P-mercaptoethanol in 5 mM citrate-sodium phosphate buffer at pH 3.7. The column was eluted at a flow rate of 10 ml/h with a linear gradient: the mixing chamber contained 200 ml of the eluting buffer and the reservoir chamber the same volume of 0.08 M NaCl dissolved

A B C II

FIG. 2. Urea electrophoretic pattern of Dopa decarboxylase and its subunits on polyacrylamide gel. (A) The urea-treated enzyme in the presence of 109 mM ,9-mercaptoethanol; (B and C) separated not fluorescent and fluorescent subunits, respectively; (D) B + C (1:l). Gel conditions: 7.5% acrylamide, 8 M urea.

FRACTION NUflBER

FIG. 3. Separation of Dopa decarboxylase subunits by CM-cellulose chromatography. Volume of one fraction was 2.7 ml. (0) Absorbance at 280 nm; (0) fluorescence intensity at 395 nm (ext. 325 nm).

in the same buffer. The protein in each tube was measured by absorbance at 280 nm.

Figure 3 shows a typical pattern of separation of the enzyme into two protein peaks. The identity and homogeneity of these peaks were checked by SDS- or urea-polyacrylamide gel electrophoresis as shown in Figs. lB, C and 2B, C. The first eluted protein corresponded to the 43,000- dalton band, the second to the 50,000-dalton band. SDS or urea gel electrophoresis of a mixture of equal amounts of the two fractions gave two distinct bands which stained with approximately equal intensity (Figs. lD, 2D). About 74% of the protein applied to the column was recovered. Yields of the first and second peak were calculated to be 6.8 mg (0.158 pmol) and 8.1 mg (0.162 pmol), respectively, corresponding to l/l molar ratio.

Fluorimetric measurements of the fractions obtained by CM-cellulose chromatography have been carried out: an emis- sion at 395 nm (ext. 325 nm) has been found associated with only one of the two peaks (Fig. 3) corresponding to the 50,000- molecular-weight subunit. On this basis the fluorescent subunit has been named F (fluorescent) to distinguish from the 43,000-molecular-weight subunit, named NF (not fluorescent).

A separation identical to that shown in Fig. 3 was obtained when the enzyme, treated with urea and passed through a


TABLE I

AMINO ACID COMPOSITION OF Dopa DECARBOXYLASE AND ITS SUBUNITS

Subunit

Amino acid Native enzyme

Fluorescent @‘)

Not fluorescent (NV

Histidine Lysine Arginine Tryptopban” Cysteine + cystineb Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine

23.9 * 0.5 12.5 k 0.3 12.3 2 0.4 37.6 zk 0.5 19.8 + 0.7 17.8 + 0.5 41.0 + 0.8 23.9 If: 0.6 18.8 * 0.4 13.1 f c 5.1 If: c 5.0 + c 17.0 f c 10.2 f c 5.3 + c 58.8 k 0.3 28.1 + 0.9 30.1 AZ 1.2 35.3 f. 0.3 15.6 -t 0.3 21.2 f 0.6 43.5 f 0.1 21.0 + 0.7 21.1 + 0.7 91.3 k 0.2 48.0 It 1.2 48.0 f 3.4 64.2 f 0.2 31.6 2 1.2 31.7 + 1.7 61.2 -c 2.1 34.6 :i 0.8 28.2 f 1.1 92.6 f 0.3 54.2 k 1.0 38.0 f 1.2 51.4 + 0.5 27.0 :+ 1.3 28.7 k 1.2 11.1 f 2.4 9.9 * 0.4 2.0 + 0.1 28.2 It 0.3 16.9 ,k 0.7 12.5 f 0.5 74.1 k 0.4 41.6 + 0.9 29.7 + 1.3 18.5 + 0.5 9.8 * 0.3 7.1 f 0.7 37.2 + 0.5 20.4 + 0.4 17.4 f 1.2

Note. Values are expressed as residues per mole of protein, assuming 93,000, 50,000, and 43,000 as the molecular weight of the native protein, fluorescent, and not fluorescent subunits, respectively. Except when otherwise indicated, the values represent the means (*standard deviation) of three determinations.

’ Tryptophan has been determined after hydrolysis with 4 N methanesulfonic acid containing 0.2% 3-(2- aminoethyl)indole (12).

b Cysteine + cystine content has been determined as cysteic acid after hydrolysis in the presence of 0.3 M dimethyl sulfoxide (11).

’ Single determination.

CM-cellulose column, was not previously reduced by NaBH4; in this case, the fluorescence was not shown by the 50,000-molecular-weight subunit.

Amino Acid Composition

Amino acid composition of native protein compared with that of F and NF subunits is given in Table I. The results are expressed as number of residues, assuming 93,000, 50,000, and 43,000 as the molecular weight of the native protein, and F and NF subunits, respectively. The amino acid composition found for the native enzyme is in good agreement with that calculated by adding the compositions obtained from F and NF subunits, assuming

a molar ratio l:l, except for cysteine, tyrosine, and tryptophan. These amino ac- ids, however, are among those for which some losses during the hydrolysis can be expected, with the consequence of significant errors in their quantitative determination.

Peptide Maps

The tryptic digests of the native enzyme and of the two isolated subunits have no detectable “core.” In Figs. 4A-C the corresponding peptide maps are reported.

The patterns of these fingerprints show about 15 apparently homologous peptides in F and NF subunits in addition to many nonhomologous peptides.


DISCUSSION Christenson et al. (1) are consistent with the presence of three subunits with the

There are conflicting reports regarding molecular weights of 57,000, 40,000, and le number and the molecular weights of 21,000. Lancaster and Sourkes (4) suggest opa decarboxylase subunits. Data of that hog kidney Dopa decarboxylase con-

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FIG. 4. Fingerprint and schematic representation of tryptic digests of holoenzyme (A), fluorescent (B), and not fluorescent (C) subunits. Electrophoresis was in the horizontal direction with the cathode (-) to the right and the anode (+) on the left. Descending paper chromatography was done in the direction of the arrow. Spots marked with letters represent the most obvious homologous peptides in fluorescent and not fluorescent subunits. Dotted spots are only occasionally found.


sists of at least two unequal polypeptide chains weighing 48,000-50,000 and 40,000- 44,000. Finally Maycock et al. (5) indicate that the enzyme contains two subunits of approximately equal molecular weight (ca. 50,000). It should be noticed that the evidence for these results was based only on SDS-polyacrylamide gel electrophoresis analysis of the enzyme.

Our data obtained on either SDS-polyacrylamide or urea-polyacrylamide gels suggest the existence of two chains of different molecular weight and electric charge. These subunits are in a 1:l molar ratio and have apparent molecular weights of 50,000 and 43,000. As the presence of P-mercaptoethanol did not affect the dis- sociation in the presence of SDS, disulfide linkages are not apparently involved in the interaction between the subunits. Since the molecular weight of the native enzyme (recalculated from Ref. (2), using a partial specific volume of 0.732 ml/g, corresponding to the amino acid analysis reported here) is -100,000, we propose a dimeric structure for Dopa decarboxylase. These results confirm Lancaster and Sourke’s suggestion; it is interesting to note that Fragoulis and Sekeris (13) have also found two subunits of molecular weight of 50,000 and 46,000 for Dopa decarboxylase purified from Calliphwa vi&a larva integu- ments.

Moreover, chromatographic separation of the subunits not only provides further evidence that they occur in equal quan- tities (as evaluated by absorbance measurements) in the holoenzyme, but also has revealed by fluorescence measurements that the coenzyme is bound only to one chain, the one of higher molecular weight. These findings are consistent with the stoichiometry of pyridoxal-P binding of 1 per -100,000 molecular weight.

The amino acid compositions of the two subunits (see Table I) show significant differences as do their peptide maps. Al- though F and NF subunits share many identical peptides, other peptides are clearly different. This latter finding seems to exclude the possibility that the NF subunit could be produced by proteolytic digestion during the purification proce-

dure. Moreover, the enzyme purified in the presence of diisopropyl phosphofluoridate to inactivate proteases still shows two bands on SDS- or urea-gel electrophoresis. Attempts to determine the N-terminal residue of native enzyme and the separated subunits using the dansyl-chloride method (14) were unsuccessful.

In addition to the results reported and considering the fact that the calculated amino acid composition, based on the as- sumed dimeric structure, is consistent with that obtained by direct analysis of the native enzyme, it can be concluded that Dopa decarboxylase consists of two nonidentical subunits. Such an asymmetrical dimer is a rather unusual finding even though examples of this kind of quaternary structure have been described (15).

As far as pyridoxal-P-dependent enzymes are concerned, almost all the enzymes so far studied consist of a single polypeptide or of identical subunits with a pyridoxal-P binding site on each subunit (16, 1’7). However, there are examples of pyridoxal-P-dependent enzymes (16, 18) which consist of nonidentical subunits: it is remarkable that in these enzymes, pyridoxal-P is not bound to every subunit, which is also the case of Dopa decarboxylase. The absence of the coenzyme binding site on one of the two subunits of Dopa decarboxylase raises the question of the role played by the NF subunit in catalysis. Trials of recombination and renaturation of the chains to form active enzyme have been carried out, but removal of denatur- ating agent causes the protein to precip- itate.

Further investigations are needed to obtain a deeper insight into the quaternary structure-function relationship of Dopa decarboxylase.

ACKNOWLEDGMENTS

The authors are grateful to Professor F. Bossa for helpful discussion. The technical assistance of M. Codini is acknowledged.

REFERENCES

1. CHRISTENSON, J. G., DAIRMAN, W., AND UDEN- FRIEND, s. (1970) Arch. ~io&m. Biqphys. 141, 356-367.


2. BORRI VOLTATPORNI, C., MINELLI, A., VECCHINI, P., FIORI, A., AND TURANO, C. (1979) Eur. J. Biocha 93,181-187.

3. BOSSA, F., MARTINI, F., BARRA, D., BORRI VOL- TA’ITORNI, C., MINELLI, A., AND TIJRANO, C. (1977) Biochem. Biophys. Res. Commun 78, 177-183.

4. LANCASTER, G. A., AND SOURKES, T. L. (1972) Canad J. Biochem. 50,791-797.

5. MAYCOCK, A. L., ASTER, S. D., AND PATCHEIT, A. A. (1978) in Enzyme-Activated Irreversible Inhibitors (Seiler, N., Jung, M. J., and Kock- Weser, J., eds.), pp. 211-219, Elsevier/North Holland, Amsterdam.

6. FAIRBANK, G., STECK, T. L., AND WALLACH, D. H. F. (1971) Biochemistry 10,2606-2617.

7. STEGINK, L. D., MEYER, P. D., AND CHALXLEY, R. (1971) Anal Biochem 41, 351-359.

8. INGRAM, V. M. (1958) Biochim Biophys. Acta 28, 539-544.

9. CIRO~~O, C., SCOTTO DI TELLA, A., AND GERACI, G. (1975) Cell L&&r. 4.87-89.

10. CLEGG, J. B., NAUGHTON, M. A., AND WEATHER- ALL, D. J. (1966) J. Mel BioL 19,91-108.

11. SPENCER, R. L., AND WOLD, F. (1969) Ad Bio- them 32,1&j-190.

12. SIMPSON, R. J., NEUBERGER, M. R., AND LIU, T. Y. (1976) J. Bid C&-m 251. 1936-1940.

13. FRAGOLJLIS, E. G., AND SEKERIS, C. E. (1975) Arch. Biochem Biophys. 168,15-25.

14. GRAY, W. R. (1972) in Methods in Enzymology (McCormick, D. B., and Wright, L. D., eds.), Vol. 25, pp. 121-138, Academic Press, New York.

15. KLOTZ, 0. M., DARNALL, D. W., AND LANGERMAN, N. R. (1975) in The Proteins (Neurath H., ed.), Vol. 1, pp. 293-411, Academic Press, New York.

16. BRAUNSTEIN, A. E. (1973) in The Enzymes (Boyer, P. D. ed.), Vol. 9, pp. 379-481, Academic Press, New York.

17. SNELL, E. E., AND DI-MARI, S. J. (1970) in The Enzymes (Boyer, P. D., ed.), Vol. 2, pp. 335- 370, Academic Press, New York.

18. YAGI, T., MISONO, H., KURIHARA, N., YAMAMOTO, T., AND SODA, K. (1980) J. Biochem (Tokyo) 87, 1395-1402.

subunit structure of 3,4-dihydroxyphenylalanine decarboxylase from pig kidney

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