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Inhibitors binding to L-aromatic amino acid decarboxylase
Life Sciences, Vol. 31, pp. 1519-1524 Printed in the U.S.A.
Pergamon Press
INHIBITORS BINDING TO L-AROMATIC AMINO ACID DECARBOXYLASE
Erminia Barboni, Carla Borri Voltattorni*, Maria D'Erme, Anna Fiori, Alba Minelli*, Maria Anna Rosei I s t l t u t i di Chimica Biologica, Universita di Roma e Perugia* e Centro di Biologia Molecolare de] Consiglio Nazionale delle Ricerche. (Received in final form July 13, 1982)
S,mar~ The effect of a number of inhibitors of L-aromatic amino acid decarboxylase activity on the absorption spectrum of the enzyme-bound coenzyme has been studied. It has been observed that the compounds tested, even i f devoid of the amino function and therefore unable to form the Schiff base with the coenzyme, modify significantly the enzyme spectrum, indicating their binding to the coenzyme active site. Spectral modifications suggest that at least two kinds of binding of inhibitors to L-aromatic amino acid decarboxylase may occur, depending on their structural features. Moreover, from the spectra obtained at different concentrations of the inhibitors their a f f i n i t y constants have been determined: data indicate that the cathecol ring gives the largest contribution to the binding, while the presence of the carboxyl group, the aminic group and the aliphatic chain are responsible for a decrease in the binding, which could be relevant for the efficiency of the catalysis. Aromatic amino acid decarboxylase (AAD) (E.C.4.1.1.28) commonly known as Dopa decarboxylase, is an important enzyme in the biosynthetic pathway of dopamine, noradrenaline and adrenaline. The inhibition of the peripheral form of the enzyme has at present a great pharmacological importance in the treatment of Parkinson disease ( I ) , and more or less specific inhibitors are currently being used (2,3). The rational design of inhibitors of this enzyme is s t i l l an important task and the availability of less toxic inhibitors (4), possibly devoid of the substituted hydrazine function would be desirable. An extensive series of compounds have been examined for their inhibitory capacity (2,5,6), but often the usefulness of these studies is limited by the complicate kinetic properties of the enzyme (7), so that in most cases only a rough indication of the affinity for a certain compound for the enzyme (given as I~) is provided and a detailed analysis of the interaction between inhibitor and active site is precluded. We have started an investigation on this subject by using a purified pre paration of the enzyme, obtained in relatively high amounts, so that a direct spectrophotometric determination of the inhibitor binding to the enzyme is 0024-3205/82/141519-06503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
1520
L-aromatlc Amino Acid Decarboxylase
Vol. 31, No. 14, 1982
possible, beside the usual kinetic measurement. Furthermore, the effect of the inhibitor on the absorption spectrum of the enzyme-bound coenzyme has been measured so as to provide a further information on the nature of the i n h i b i t o r decarboxylase complex. Methods Materials L-Dopa, 5-OH-tryptophan, 2,4,6 Trinitrobenzen-l-sulphonic acid (TNBS) were obtained from Sigma Chemical Co., the inhibitors from Fluka. All other chemicals were reagent grade.
L-aromatic aminoacid decarboxylase was purified from pig kidney according to Borri Voltattorni et al. (8) and was homogeneous on polyacrylamide gel electrophoresis and analytical centrifugation, Each buffer contained lO'~M~-mer captoethanol and I0"~4 pyridoxal-P. The purified enzyme had a specific a c t i v i t y of about l umole/mg/min at 25 C° and pH 6.8. Enzyme concentration is expressed as bound coenzyme determined by releasing the bound pyridoxal-P into O.l M NaOH and by using ~ 388=6600 M -cm-- for pyridoxal-P concentration. Spectral data were obtained in a Cary 219 spectrophotometer equipped with a thermostated cell compartment. Enzymatic a c t i v i t y was followed according to Charteris and John (9) using TNBS as reagent for amine with a continuous extraction with benzene for 60' at 42 C° . Results and Qiscussion The addition to the enzyme of any of the compounds tested induces a spectral modification of the enzyme-bound coenzyme, as shown in table I and in Fig.l The compounds have been shown previously to be inhibitors of the decarboxylation reaction (lO); considering this fact, together with their a b i l i t y to modify the coenzyme absorption bands and their structural relationship with the substrates, i t is legitimate to assume that all of them bind to the enzyme active site. On the basis of the effect e l i c i t e d on the coenzyme spectrum, the compounds tested can be divided in two groups: those which induce an increase of intensity in the 420 nm band and a decrease in the 330 nm band, and those which show the opposite effects. Compounds belonging to the f i r s t group give a spectrum similar to that of the enzyme-substrate complex ( l l ) , characterized by an increased absorption at 420 rim. In fact a compound representative of this group is DOPAmethyl ester, which is capable, as the substrates are, to form a Schiff base with the aldeyde group of pyridoxal phosphate. The 420 nm peak, however, is increased also by 4-hydroxy-3-methoxy-phenyl-lactic acid, 4-hydroxy-3-methoxyphenyl-acetic acid, 3,4 dihydroxyphenylacetic acid, 5-Hydroxy indole-3-acetic acid, all of which lack the amino group and are therefore unable to form Schiff bases, but have as a common feature a polar group on the second carbon starting from the aromatic
Vol. 31, No. 14, 1982
L-aromatlc Amino Acid Decarboxylase
1521
TABLE I
COMPOUND 3,4-Dihydroxyphenylalanine methyl ester 3,4-Dihydroxyphenylacetic acid 4-hydroxy-3-methoxyphenyl acetic acid 4-hydroxy-3-methoxyphenyllactic acid 5-Hydroxyindole-3-acetic acid Caffeic acid (3,4-dihydroxycinnamic acid) Caffeic acid methyl ester 3,4-dihydroxyhydrocinnamic Hydrocinnamic Cathecol
acid
acid
420 nm
330 nm
increase " " " " decrease "
decrease
" " "
Resorcinol
"
5-Hydroxyindo]e
"
IB
ii
increase If ii II II II fl
Modifications induced on aromatic aminoacid decarboxylase absorption maxima by inhibitors. ring. I t should be noted that a polar group in this position is l i k e l y to be very close to the aldimine bond of the enzyme Schiff base i . e . that formed between pyridoxal phosphate and a lysine at the active site (12). Therefore i t can be assumed that these polar groups are able to stabilize the protonated form of the enzyme Schiff base, with i t s characteristic 420 nm absorption. Compounds which decrease the 420 nm band include those constituted only by the cathecol ring or the 5-hydroxy indole ring, and those with a substituent represented by a three carbon chain, with or without a carboxyl group at the end. The fact that compounds like cathecol and 5-hydroxyindole which should be bound at the active site at a certain distance from the coenzyme, are able to alter the absorption spectrum of the l a t t e r , can be explained assuming that these compound s h i f t the equilibrium between the 330 nm and the 420 nm absorbing forms (8) binding preferentially to the former. From the spectra obtained at d i f f e r e n t concentration of the inhibitory compounds their respective a f f i n i t y constants were determined (Table I I ) . Comparison of the K for the various inhibitors allows to evaluate the contribution to the binding of the different functional groups. The orto-di-hydroxy phenyl function appears to provide the largest contribution in the order of -6 Kcal/mole, as shown by the strong binding of cathecol i t s e l f . The stronger binding of 3,4-dihydroxycinnamic acid, of caffeic acid and i t s methyl ester shows that also the three-carbon aliphatic chain provides a significant contribution, which can be evaluated in the order of -I.5 Kcal/mole. The amino group is l i k e l y to provide a further contribution, which however cannot be quantitized by our data, since the only inhibitor which has been tested carrying this group, i . e . DOPAmethyl ester, has a dissociation constant
1522
L-aromatic Amino Acid Decarbo×ylase
Vol. 31, No. 14, 1982
O6 ° °°
*ee ~, :.e
~
dl~'';, " ..I 6,
AdAA66, •
11.6 & &
,D m 02
".B.O..~.~
e i
p •
4oo
O e O •
e~'. • e6"". ~ • • ".. %
)~ 1~1
5OO
FIG. 1 Spectral modification~ of Aromatic Amino acid decarboxylase by inhibitors • . AAD 5.8 x lO'-M in T.F. 6.80.Ol M --- AAD plus 3,4 dihydroxyphenylaceti~ acid 2.gx]O-4M oo AAD plus 5-Hydroxy indole 4.3xlOs'M • - AAD plus DOPAmethyl ester IxlO" M
so low to be unmeasurable by the spectrophotometric method. A kinetic determination of its dissociation constant gave a value of 1.88.10-7 M, which is to be considered with caution, taking into account the complexity of the kinetics of this enzyme. A carboxylate group instead does not favor the binding, as shown by a comparison of caffeic acid and i t s ester, and also by considering the binding of DOPAmethyl ester, which is certainly stronger than that of the real substrate L-DOPA. This fact can be interpreted assuming that the contemporary presence of the functional groups characteristic of the substrate, i.e. the hydroxylated aromatic ring, the amino group, the aliphatic chain and the carboxyl group, provides, rather than addictive binding contributions, some strain in the substrate, which could be of great importance for the subsequent catalytic process (13).
Vol. 31, No. 14, 1982
L-aromatic Amino Acid Decarboxylase
1523
TABLE I]
COMPOUND
K(M)
3,4-Dihydroxyphenylalanine methyl ester Caffeic acid methyl ester Caffeic acid 3,4-dihydro×yhydrocinnamic acid Cathecol 3,4 dihydroxyphenylacetic acid 4-hydroxy-3-methoxyphenyllactic acid 5-hydroxyindole-3-acetic acid Resorcinol 4-hydroxy-3-methoxyphenylacetic acid 5-Hydroxyindole Hydrocinnamic acid
(< <(
1 x 10-6 1 x lO"6_ 2 x I0"~ 7 x I0 "b 3 x I0"_b 4 . 5 x i 0 "b 9 x I0 "b. I .3xlO'_4 2 x 10-_ 3 6 x 10-~ j 9 x lO-3_ 2.2x10-z
Dissociation constants for inhibitors of aromatic aminoacid decarboxylase at 25 C°. This fact confirms previous observations on tyrosine decarboxylase, in which an unfavourable influence of the carboxyl group of the binding was also observed (14). This effect suggests that the catalysis operated by the amino acid decarboxylases is facilitated by the releasing of the strain which accompanies the removal of the carboxyl group from the active site-bound substrate. As a whole, these observations point out the usefulness of the determinations of the binding constants directly from spectral data whenever enzyme is available to make this possible. The advantage of this approach is particularly evident when kinetic measurements, as in the case of L-aromatic amino acid decarboxylase, are complicated by many side reactions such as the spontaneous decarboxylation of the substrate L-DOPA (15), the non-enzymic reaction of L-DOPA with free coenzyme (15) and the gradual inactivation of the enzyme following a product deamination reaction (7, 16). References I. 2. 3. 4. 5. 6. 7.
O. HORNYKIEWICZ. Br. Med. Bull. 29, ]72-178 (1973). G. BARTHOLINI and A. PLETSCHER. Pharmac. Therap. B l, 407-421 (1975). C.C. PORTER. Adv. Neurol. 2, 37-58 (1973). S.D. WELSON, J.R. MITCHELL, J.A. TIMBRELL, W.R, SNODGRASSand G.B. CORCORAN, Science 193, 901 (1976). Z. HUSZTI, E. ~SZTREINER, G. SZILAGYI, J. KOSATYand J. BORSYBiochem. Pharmacol. 22, 2267-2275 (1973). L'. ELLENBOGE~, R.G. KELLY, R.J. TAYLOR, Jr. C.S. STUBBSJr. Biochem. Pharmacol. 22 939-947 (1973). M.H. O'LEARY and R.L. BAUGHNJ. Biol. Chem, 252 7168-7173 (1977).
1524
8. 9. lO. II. 12. 13. 14. 15. 16.
L-aromatlc Amino Acid Decarboxylase
Vol. 31, No. 14, 1982
C. BORRI VOLTATTORNI, A. MINELLI, P, VECCHINI, A, FIORI, C, TURANO, Eur. J. Biochem. 93, 181-188 (1979). A. CHARTERISand R. JOHN, Anal. Biochem. 6.~6, 365-371 (1975). W.J. HARII~AN, R,I. AKAWIC and W.G. CLARK, J. Biol, Chem. 216, 507 (1955). A. FIORI, C. TURANO, C. BORRI VOLTATTORNI, A. MINELLI and'M. CODINI, FEBS Lett. 54, 122-125 (1975). F. BOSSA, F. MARTINI, D. BARRA, C, BORRI VOLTATTORNI, A. MINELLI and C. TURANO, Bioch. Bioph~s. Res. Commun. 78, 177-184 (1977). W.P. JENCKS, Adv. Enz~/m. A. Meister Ed. 4_~3,p. 241 (1975). A. ORLACCHIO, C. BORRI VOLTATTORNI and C. TURANO, Biochem. J. 185, 41-46 (I 980). G.J. CHRISTENSON, W. DAIRMAN, S. UDENFRIEND,Arch. Biochem. Biophys. 141, 356-367 (1970). C. BORRI VOLTATTORNI, A. MINELLI, C. TURANO,FEBS Lett. 17, 231-235 (1971)
Pergamon Press
INHIBITORS BINDING TO L-AROMATIC AMINO ACID DECARBOXYLASE
Erminia Barboni, Carla Borri Voltattorni*, Maria D'Erme, Anna Fiori, Alba Minelli*, Maria Anna Rosei I s t l t u t i di Chimica Biologica, Universita di Roma e Perugia* e Centro di Biologia Molecolare de] Consiglio Nazionale delle Ricerche. (Received in final form July 13, 1982)
S,mar~ The effect of a number of inhibitors of L-aromatic amino acid decarboxylase activity on the absorption spectrum of the enzyme-bound coenzyme has been studied. It has been observed that the compounds tested, even i f devoid of the amino function and therefore unable to form the Schiff base with the coenzyme, modify significantly the enzyme spectrum, indicating their binding to the coenzyme active site. Spectral modifications suggest that at least two kinds of binding of inhibitors to L-aromatic amino acid decarboxylase may occur, depending on their structural features. Moreover, from the spectra obtained at different concentrations of the inhibitors their a f f i n i t y constants have been determined: data indicate that the cathecol ring gives the largest contribution to the binding, while the presence of the carboxyl group, the aminic group and the aliphatic chain are responsible for a decrease in the binding, which could be relevant for the efficiency of the catalysis. Aromatic amino acid decarboxylase (AAD) (E.C.4.1.1.28) commonly known as Dopa decarboxylase, is an important enzyme in the biosynthetic pathway of dopamine, noradrenaline and adrenaline. The inhibition of the peripheral form of the enzyme has at present a great pharmacological importance in the treatment of Parkinson disease ( I ) , and more or less specific inhibitors are currently being used (2,3). The rational design of inhibitors of this enzyme is s t i l l an important task and the availability of less toxic inhibitors (4), possibly devoid of the substituted hydrazine function would be desirable. An extensive series of compounds have been examined for their inhibitory capacity (2,5,6), but often the usefulness of these studies is limited by the complicate kinetic properties of the enzyme (7), so that in most cases only a rough indication of the affinity for a certain compound for the enzyme (given as I~) is provided and a detailed analysis of the interaction between inhibitor and active site is precluded. We have started an investigation on this subject by using a purified pre paration of the enzyme, obtained in relatively high amounts, so that a direct spectrophotometric determination of the inhibitor binding to the enzyme is 0024-3205/82/141519-06503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
1520
L-aromatlc Amino Acid Decarboxylase
Vol. 31, No. 14, 1982
possible, beside the usual kinetic measurement. Furthermore, the effect of the inhibitor on the absorption spectrum of the enzyme-bound coenzyme has been measured so as to provide a further information on the nature of the i n h i b i t o r decarboxylase complex. Methods Materials L-Dopa, 5-OH-tryptophan, 2,4,6 Trinitrobenzen-l-sulphonic acid (TNBS) were obtained from Sigma Chemical Co., the inhibitors from Fluka. All other chemicals were reagent grade.
L-aromatic aminoacid decarboxylase was purified from pig kidney according to Borri Voltattorni et al. (8) and was homogeneous on polyacrylamide gel electrophoresis and analytical centrifugation, Each buffer contained lO'~M~-mer captoethanol and I0"~4 pyridoxal-P. The purified enzyme had a specific a c t i v i t y of about l umole/mg/min at 25 C° and pH 6.8. Enzyme concentration is expressed as bound coenzyme determined by releasing the bound pyridoxal-P into O.l M NaOH and by using ~ 388=6600 M -cm-- for pyridoxal-P concentration. Spectral data were obtained in a Cary 219 spectrophotometer equipped with a thermostated cell compartment. Enzymatic a c t i v i t y was followed according to Charteris and John (9) using TNBS as reagent for amine with a continuous extraction with benzene for 60' at 42 C° . Results and Qiscussion The addition to the enzyme of any of the compounds tested induces a spectral modification of the enzyme-bound coenzyme, as shown in table I and in Fig.l The compounds have been shown previously to be inhibitors of the decarboxylation reaction (lO); considering this fact, together with their a b i l i t y to modify the coenzyme absorption bands and their structural relationship with the substrates, i t is legitimate to assume that all of them bind to the enzyme active site. On the basis of the effect e l i c i t e d on the coenzyme spectrum, the compounds tested can be divided in two groups: those which induce an increase of intensity in the 420 nm band and a decrease in the 330 nm band, and those which show the opposite effects. Compounds belonging to the f i r s t group give a spectrum similar to that of the enzyme-substrate complex ( l l ) , characterized by an increased absorption at 420 rim. In fact a compound representative of this group is DOPAmethyl ester, which is capable, as the substrates are, to form a Schiff base with the aldeyde group of pyridoxal phosphate. The 420 nm peak, however, is increased also by 4-hydroxy-3-methoxy-phenyl-lactic acid, 4-hydroxy-3-methoxyphenyl-acetic acid, 3,4 dihydroxyphenylacetic acid, 5-Hydroxy indole-3-acetic acid, all of which lack the amino group and are therefore unable to form Schiff bases, but have as a common feature a polar group on the second carbon starting from the aromatic
Vol. 31, No. 14, 1982
L-aromatlc Amino Acid Decarboxylase
1521
TABLE I
COMPOUND 3,4-Dihydroxyphenylalanine methyl ester 3,4-Dihydroxyphenylacetic acid 4-hydroxy-3-methoxyphenyl acetic acid 4-hydroxy-3-methoxyphenyllactic acid 5-Hydroxyindole-3-acetic acid Caffeic acid (3,4-dihydroxycinnamic acid) Caffeic acid methyl ester 3,4-dihydroxyhydrocinnamic Hydrocinnamic Cathecol
acid
acid
420 nm
330 nm
increase " " " " decrease "
decrease
" " "
Resorcinol
"
5-Hydroxyindo]e
"
IB
ii
increase If ii II II II fl
Modifications induced on aromatic aminoacid decarboxylase absorption maxima by inhibitors. ring. I t should be noted that a polar group in this position is l i k e l y to be very close to the aldimine bond of the enzyme Schiff base i . e . that formed between pyridoxal phosphate and a lysine at the active site (12). Therefore i t can be assumed that these polar groups are able to stabilize the protonated form of the enzyme Schiff base, with i t s characteristic 420 nm absorption. Compounds which decrease the 420 nm band include those constituted only by the cathecol ring or the 5-hydroxy indole ring, and those with a substituent represented by a three carbon chain, with or without a carboxyl group at the end. The fact that compounds like cathecol and 5-hydroxyindole which should be bound at the active site at a certain distance from the coenzyme, are able to alter the absorption spectrum of the l a t t e r , can be explained assuming that these compound s h i f t the equilibrium between the 330 nm and the 420 nm absorbing forms (8) binding preferentially to the former. From the spectra obtained at d i f f e r e n t concentration of the inhibitory compounds their respective a f f i n i t y constants were determined (Table I I ) . Comparison of the K for the various inhibitors allows to evaluate the contribution to the binding of the different functional groups. The orto-di-hydroxy phenyl function appears to provide the largest contribution in the order of -6 Kcal/mole, as shown by the strong binding of cathecol i t s e l f . The stronger binding of 3,4-dihydroxycinnamic acid, of caffeic acid and i t s methyl ester shows that also the three-carbon aliphatic chain provides a significant contribution, which can be evaluated in the order of -I.5 Kcal/mole. The amino group is l i k e l y to provide a further contribution, which however cannot be quantitized by our data, since the only inhibitor which has been tested carrying this group, i . e . DOPAmethyl ester, has a dissociation constant
1522
L-aromatic Amino Acid Decarbo×ylase
Vol. 31, No. 14, 1982
O6 ° °°
*ee ~, :.e
~
dl~'';, " ..I 6,
AdAA66, •
11.6 & &
,D m 02
".B.O..~.~
e i
p •
4oo
O e O •
e~'. • e6"". ~ • • ".. %
)~ 1~1
5OO
FIG. 1 Spectral modification~ of Aromatic Amino acid decarboxylase by inhibitors • . AAD 5.8 x lO'-M in T.F. 6.80.Ol M --- AAD plus 3,4 dihydroxyphenylaceti~ acid 2.gx]O-4M oo AAD plus 5-Hydroxy indole 4.3xlOs'M • - AAD plus DOPAmethyl ester IxlO" M
so low to be unmeasurable by the spectrophotometric method. A kinetic determination of its dissociation constant gave a value of 1.88.10-7 M, which is to be considered with caution, taking into account the complexity of the kinetics of this enzyme. A carboxylate group instead does not favor the binding, as shown by a comparison of caffeic acid and i t s ester, and also by considering the binding of DOPAmethyl ester, which is certainly stronger than that of the real substrate L-DOPA. This fact can be interpreted assuming that the contemporary presence of the functional groups characteristic of the substrate, i.e. the hydroxylated aromatic ring, the amino group, the aliphatic chain and the carboxyl group, provides, rather than addictive binding contributions, some strain in the substrate, which could be of great importance for the subsequent catalytic process (13).
Vol. 31, No. 14, 1982
L-aromatic Amino Acid Decarboxylase
1523
TABLE I]
COMPOUND
K(M)
3,4-Dihydroxyphenylalanine methyl ester Caffeic acid methyl ester Caffeic acid 3,4-dihydro×yhydrocinnamic acid Cathecol 3,4 dihydroxyphenylacetic acid 4-hydroxy-3-methoxyphenyllactic acid 5-hydroxyindole-3-acetic acid Resorcinol 4-hydroxy-3-methoxyphenylacetic acid 5-Hydroxyindole Hydrocinnamic acid
(< <(
1 x 10-6 1 x lO"6_ 2 x I0"~ 7 x I0 "b 3 x I0"_b 4 . 5 x i 0 "b 9 x I0 "b. I .3xlO'_4 2 x 10-_ 3 6 x 10-~ j 9 x lO-3_ 2.2x10-z
Dissociation constants for inhibitors of aromatic aminoacid decarboxylase at 25 C°. This fact confirms previous observations on tyrosine decarboxylase, in which an unfavourable influence of the carboxyl group of the binding was also observed (14). This effect suggests that the catalysis operated by the amino acid decarboxylases is facilitated by the releasing of the strain which accompanies the removal of the carboxyl group from the active site-bound substrate. As a whole, these observations point out the usefulness of the determinations of the binding constants directly from spectral data whenever enzyme is available to make this possible. The advantage of this approach is particularly evident when kinetic measurements, as in the case of L-aromatic amino acid decarboxylase, are complicated by many side reactions such as the spontaneous decarboxylation of the substrate L-DOPA (15), the non-enzymic reaction of L-DOPA with free coenzyme (15) and the gradual inactivation of the enzyme following a product deamination reaction (7, 16). References I. 2. 3. 4. 5. 6. 7.
O. HORNYKIEWICZ. Br. Med. Bull. 29, ]72-178 (1973). G. BARTHOLINI and A. PLETSCHER. Pharmac. Therap. B l, 407-421 (1975). C.C. PORTER. Adv. Neurol. 2, 37-58 (1973). S.D. WELSON, J.R. MITCHELL, J.A. TIMBRELL, W.R, SNODGRASSand G.B. CORCORAN, Science 193, 901 (1976). Z. HUSZTI, E. ~SZTREINER, G. SZILAGYI, J. KOSATYand J. BORSYBiochem. Pharmacol. 22, 2267-2275 (1973). L'. ELLENBOGE~, R.G. KELLY, R.J. TAYLOR, Jr. C.S. STUBBSJr. Biochem. Pharmacol. 22 939-947 (1973). M.H. O'LEARY and R.L. BAUGHNJ. Biol. Chem, 252 7168-7173 (1977).
1524
8. 9. lO. II. 12. 13. 14. 15. 16.
L-aromatlc Amino Acid Decarboxylase
Vol. 31, No. 14, 1982
C. BORRI VOLTATTORNI, A. MINELLI, P, VECCHINI, A, FIORI, C, TURANO, Eur. J. Biochem. 93, 181-188 (1979). A. CHARTERISand R. JOHN, Anal. Biochem. 6.~6, 365-371 (1975). W.J. HARII~AN, R,I. AKAWIC and W.G. CLARK, J. Biol, Chem. 216, 507 (1955). A. FIORI, C. TURANO, C. BORRI VOLTATTORNI, A. MINELLI and'M. CODINI, FEBS Lett. 54, 122-125 (1975). F. BOSSA, F. MARTINI, D. BARRA, C, BORRI VOLTATTORNI, A. MINELLI and C. TURANO, Bioch. Bioph~s. Res. Commun. 78, 177-184 (1977). W.P. JENCKS, Adv. Enz~/m. A. Meister Ed. 4_~3,p. 241 (1975). A. ORLACCHIO, C. BORRI VOLTATTORNI and C. TURANO, Biochem. J. 185, 41-46 (I 980). G.J. CHRISTENSON, W. DAIRMAN, S. UDENFRIEND,Arch. Biochem. Biophys. 141, 356-367 (1970). C. BORRI VOLTATTORNI, A. MINELLI, C. TURANO,FEBS Lett. 17, 231-235 (1971)