differential inhibition of activated tyrosine hydroxylase

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 22’7, No. 2, December, pp. 562-569, 1983

Differential Inhibition of Activated Tyrosine Hydroxylase’

D. B. BENNETT AND C. J. COSCIA’

Edward A. Daisy Department of Biochemistry, Saint Lacis University School of Medicine, 1.402 South Grand Boulevard, St. Louis, Missouri 63104

Received March 31, 1983, and in revised form August 1, 1983

Under conditions of cyclic AMP-dependent protein phosphorylation, tyrosine hydroxylase (EC 1.14.16.2; TH) is activated. Kinetic analysis reveals that, upon activation the affinity of the enzyme cofactor tetrahydrobiopterin, V,,,, as well as the Ki of its putative feedback inhibitor dopamine, are increased. Catecholic inhibitors of rat striatal TH have been assessed for the structural requirements that impart differential sensitivity to activated and control enzyme. By varying cofactor and inhibitor concentrations, K;s were generated from Dixon plots. Structural analogs of dopamine in which the amino group was fixed in a oi.s conformation, i.e., 6,‘7-dihydroxytetrahydroisoquinolines, exhibit the same Ki for activated and nonactivated TH. However, 2-amino-6,7-dihy- droxytetralin (ADTN), in which the nitrogen is extended in a fixed trams conformation of the /3-rotamer, exhibited a fourfold increase in Ka upon assaying tyrosine hydroxylase under phosphorylation conditions. By systematically increasing the hydrophobicity of the substituent at C-l of 1-carboxy-6,7-dihydroxytetrahydroisoquinolines the inhibitory potency was enhanced, suggesting the presence of a hydrophobic region near the catecholic binding site. If the hydrophobic group was rigid as in the catechol estrogens, 2-hydroxy-estradiol and 2-hydroxyestrone, the Ki was relatively low (2 X 10p5M) despite the absence of an amino group. Upon activation the Ki increased fourfold. These studies provide insight into the topography of the catecholic binding site on TH and to attendant changes occurring upon activation. The results suggest that the catechol binding site includes both amino group-interacting and hydrophobic regions which are influenced by enzyme activation.

Regulation of enzyme activity by protein phosphorylation and dephosphorylation represents a common phenomenon of con- siderable physiological significance. Acti- vation of mammalian striatal tyrosine hydroxylase [L-tyrosine, tetrahydropteridine, oxygen:oxidoreductase (3-hydroxylating); TH3, EC 1.14.16.21 occurs by cyclic AMP-

i Supported by NIH Grant NS12342. * To whom correspondence should be addressed. a Abbreviations used: ADTN, 2-amino-6,7-dihy-

droxy-tetralin; CAMP, adenosine-X5’-cyclic phosphate; DNLCA, 3,4-deoxynorlaudanosolinecarboxylic acid; 2- hydroxyestradiol, 2,3,5-estratrien-2,3,17+triol; 2- hydroxyestrone, 1,3,5-estratrien-2,3-diol-17-one; 6MPHI, 6-methyl-tetrahydropterin; NLCA, norlau- danosolinecarboxylic acid; TIQ, tetrahydroisoquinolines; TH, tyrosine hydroxylase; TCA, trichloroacetic acid; Pipes, 1,4-piperazinediethanesulfonic acid.

dependent protein phosphorylation and this process may be involved in the normal sequella of impulse flow in catecholami- nergic neurons (l-6). Under phosphorylation conditions in vitro, a pH-dependent increase in V,,, of TH and a decrease in the Km of its cofactor appear to be responsible for activation. At pH 7 the change in V,,, is greater than the effect on Km whereas at pH 6 activation has been attributed primarily to a change in cofactor affinity (7). Furthermore, the Ki of dopamine, a putative feedback inhibitor of TH, increases threefold under conditions of cyclic AMP-dependent protein phosphorylation at pH 6.7 (8). As a result of these observations it has been postulated that feedback inhibition by catecholamines may be overcome by activation of TH in vivo.

000%9861/83 $3.00 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.

562

INHIBITION OF TYROSINE HYDROXYLASE 563

While there have been numerous investigations on the structural requirements for inhibition of TH (5,9-14), until recently little attention has been given to the sus- ceptibility of the activated form of TH to various catechol inhibitors. Having found that catecholic tetrahydroisoqui- noline (TIQ) carboxylic acids (15, 16), in contrast to dopamine, were equipotent inhibitors of control and activated TH, we initiated a more extensive structure activity study. Our objective is to gain some insight into the molecular features required for inhibition of the activated form of the enzyme. In undertaking an analysis of the topology of the catechol-binding site in this fashion we have made the reason- able assumption that TH contains a single region to which catechols, that are cofactor-competitive by kinetic analyses, bind. It has not been proven that the catechol- and cofactor-binding sites on TH are iden- tical.

In this report we investigated the inhibition of rat striatal TH by a series of catechols. We found that 2-amino-6,7-di- hydroxytetralin (ADTN), which is fixed in a bans p-rotamer conformation, exhibits a fourfold increase in Ki under phosphorylation conditions. In addition we provide evidence for the presence of a hydrophobic region near the catechol-binding site which may explain the high affinity of catechol estrogens for this domain and the similar change in their Ki under protein phosphorylation conditions.

MATERIALS AND METHODS

Materials. The (+)-TIQs were synthesized in our laboratory (17) and their purity was determined by high-pressure liquid chromatography. On the basis of peak height analysis the TIQs used gave a single band which was judged to be contaminated with no more than 0.1% impurities. Salsolinol and ‘I-O-methyl salsolinol were gifts from Dr. Michael Collins. ADTN was provided by Dr. T. Westfall. Most biochemicals were purchased from Sigma Chemical Co., St. Louis, Missouri. Calbiochem-Behring, La Jolla, California, provided the GMPH(.

Enzyme preparation and assay. These were con- ducted as previously described (8, 15, 16). Striatum were dissected from Sprague-Dawley rats and ho- mogenized in buffer containing 0.2% Triton X-100.

After centrifugation at 40,OOOg the supernatant was passed over a Dowex 50 cation-exchange column to remove endogenous catecholamines. In assays of control TH, incubation mixtures contained 100 PM L-tyrosine (425,090 cpm L-[3,+!G3H$yrosine), 850 pM 6MPHI (in a few experiments 700-984 PM cofactor was used), 20 mM ascorbate, 1 mM ferrous ammonium sulfate, 3200 units of catalase, 50 mM potassium phosphate buffer (pH 6.6), inhibitor, and enzyme in a total volume of 150 al. After a 30-min incubation at 37°C the reaction was terminated with 5% TCA and the amount of tritiated water released was measured (18). Under the conditions of our assay enzyme activity was linear for 30 min. In these assays cofactor was chemically reduced, so that inhibition of an endogenous dihy- dropteridine reductase by TIQs (19) would have no effect on the TH activity.

For assays of activated TH, incubations were performed as described above except that a phosphorylating system (7,8,15) was included in the mixture; 33 mM Pipes buffer, pH 6.6, replaced the phosphate buffer; and the reaction was terminated after 10 min. In addition to the components listed above, phosphorylating conditions were produced by the inclusion of MgClz (13 mM), NaF (13 mM), theophylline (533 PM), ethylene glycol bis @-aminoethyl ether)-N,N’- tetraacetic acid (80 PM), disodium ATP (666 pM, in Pipes buffer), and cyclic AMP (133 PM) in the assay (150 aI total). TH was activated by a 3-min prein- cubation with the phosphorylating system (+cAMP). In controls (-CAMP) the CAMP and ATP were omitted from this system. Catalysis was then initiated by addition of tyrosine, 6MPHI, and inhibitor. In most experiments a 1.4- to 3.4-fold activation was observed depending upon the cofactor concentration. Kinetic data were derived by fitting data to linear functions by least square analyses.

In previous investigations of activated TH no at- tempts were made to obviate the possibility that the reduction of inhibition by dopamine was caused by phosphorylation-dependent metabolism of the catecholamine to a less inhibitory intermediate. In a control experiment the inhibition of TH by 10 pM dopamine was determined in the presence and absence of phosphorylation conditions and the dopamine con- tent was measured by HPLC (17) after the incubation. The final dopamine concentration had decreased but to the same extent under both conditions.

RESULTS

Before kinetic analyses were performed, we surveyed various TIQs for their inhibition of TH (Table I). These results were obtained at saturating levels of tyrosine (100 PM). Concentrations of the biopterin cofactor analog GMPH, (700-984 PM) were

564 BENNETT AND COSCIA

maintained near its Km for the unactivated enzyme since many of these TIQs are cofactor competitive.

By changing substituents in the heterocyclic ring of TIQ particularly at positions 1, 3 and 4 we came to the following con- clusions concerning the catechol-binding site. While both 1-alkyl-and 1-benzyl TIQs were of comparable inhibitory potency, in- sertion of a carboxyl at C-l diminished the inhibition. Introduction of a 3-carboxyl gave inhibitors of intermediate efficiency as evidenced by the results with 3-carboxysalsolinol and DNL-3-CA (Table I). A series of 1-alkyl-1-carboxy TIQs were synthesized from dopamine and the corre- sponding cr-keto acids of the branch-chain amino acids and glutamate. As the hydrophobicity of the 1-alkyl group increased in this group of compounds, inhibition of TH was enhanced. This was also observed for the 1-benzyl-1-carboxy-TIQ series, i.e., the Ki of DNLCA, which contains an unsub- stituted benzyl, was less than derivatives oxygenated in the 3 and 4 positions of the moiety, e.g., NLCA (see Ref. (5) and Table I). These data suggest the existence of at least one hydrophobic region near the catechol-binding site. This hydrophobic domain may also be responsible for the high affinity of catechol estrogens for TH (see below).

In the benzyl TIQ series, introduction of a hydroxyl group at C-4 reduced inhibitor potency. Although aromatization of the heterocyclic ring elicited a similar dimi- nution of inhibition, the 3,4-dihydro derivative appeared to be as inhibitory as the 1,2,3,4-tetrahydro derivative. Finally, in- sertion of one or more methyl groups on the catecholic hydroxyls (‘7-O-methyl salsolinol or 3,4-dihydropapaverine) abolished inhibition of TH entirely as previously observed (9, 10).

The source of TH for the above studies was a 40,OOOg supernatant, prepared from rat striatal homogenates treated with Tri- ton X-100 and passed through an ion-exchange resin to remove endogenous catecholamines (7,8). This preparation has the advantage of containing the necessary enzymes for protein phosphorylation result- ing in activation of TH. By addition of ATP

and cyclic AMP, 3- to lo-fold activation of TH is observed. Although not as enriched in TH as a previously prepared loo-fold purified enzyme from bovine adrenal me- dulla (15), we found this rat striatal preparation gave similar kinetic patterns suggesting that side reactions of substrates and products were not significantly different. Therefore this enzyme preparation was used for inhibition studies on activated TH. Since we demonstrated that the ma- jority of the inhibitors used in this study were competitive with the reduced pterin cofactor 6MPH4, we obtained the Ki for activated and control enzyme from Dixon plots in which inhibitor and cofactor concentrations were varied (Figs. 1 and 2). Ty- rosine was maintained at 100 PM which was found to be saturating for the activated enzyme in previous experiments (15).

In our initial study (15), we observed that 1-benzyl-1-carboxy TIQs maintained the same inhibitory capacity for activated and control enzyme. But certain 1-benzyl TIQs gave kinetic patterns in Lineweaver-Burk plots suggesting that they were not pure cofactor-competitive inhibitors. These results raised two questions. First, we won- dered whether 1-benzyl-1-carboxy TIQs could bind at different sites because of the similarity of the benzyl moiety to a substituted phenylalanine. Secondly, it is pos- sible that both the TIQ and benzyl moieties of certain substituted-benzyl TIQs could interact with the catecholic binding domain. Thus it was of interest to examine the inhibition of 1-alkyl TIQs which have only one aromatic ring. From the results in Table II it is clear that 1-alkyl TIQs having carboxyl groups in the l- or ~-PO- sitions also exhibit no change in K, upon activation of TH by protein phosphorylation.

Since the amino group in all of these compounds is fixed in a rigid cis conformation, we previously speculated that dopamine (which is free to adopt different conformational states) may be bound to the enzyme in a trams conformation (15, 16). To test this possibility we used two dopamine analogs in which the amino group is fixed in a tram conformation. Both of these, ADTN and apomorphine, proved


TABLE I

INHIBITION OF TH BY TIQs

Compound

1-alkyl TIQs Salsolinol (R’=CHa,R”,R=H)

3-Carboxysalsolinol (R”=COOH,R’=CHa,R=H)

l-Ethyl-l-carboxy-6,7-dihydroxy TIQ (R”=H,R’=COOH,R=CzH5) l-Isopropyl-1-carboxy-6,7-dihydroxy TIQ

(R”=H,R’=COOH,R=(CH,)zCH-) 1-Isobutyl-l-carboxy-6,7-dihydroxy TIQ

(R”=H,R’=COOH,R=(CHa)zCHCHz-) 1-set-Butyl-1-carboxy-6,7-dihydroxy TIQ

(R”=H,R’=COOH,R=CH,CH(CH,)CHz-) 1-(2-Carboxy-ethyl)-1-carboxy-6,7-dihydroxy TIQ

(R”=H,R’=COOH,R=HOOC CH,CH,-) ‘T-O-Methyl salsolinol (R’=CH3,R”=R=H)

1-Benzyl TIQs Tetrahydropapaveroline (R’=R”=H,R=(H0)zC6H3CHz-)

3,4-Dihydropavaveroline(A1,R”=H,R=(HO)&H&Ha-) Papaveroline(A’,A3R”=H,R=(HO)&,H&Hz-) 3,4-Dihydropapaverine

(6,7-OCH3,A’,R”=H,R=(CH30)2C6H3CH,-) NLCA (R”=H,R’=COOH,R=(OH)&H&HZ-) DNLCA (R”=H,R’=COOH,R=CsH&Hz-) DNL-3-CA (R”=COOH,R’=H,R=CsH&H,)

I-Hydroxy-DNLCA (4-OH,R”=H,R’=COOH,R=CaH&Hz) Apomorphine

Concentration

(PM)

Percentage inhibition

(&SE)

100 98.5 10 72.6 f 7.7

100 57.5 10 12.8 f 7.7

100 32.8

100

100

100 58.9

100 100

100 99.3 10 72.3 f 10.2 10 74.35 10 26.5

100 2.9 100 30.2 100 62.7 100 82.1

10 56.9 f 4.3 100 39.3 100 100

10 86.5 f 9.5

35.9

53.4

18.0 1.2

Note. Control velocities were in the range of 7 pmol/min/mg striatum. Percentage inhibition values are the averages of at least two to three experiments in which triplicate determinations were made.

to be potent inhibitors of TH. However, though it had the greatest inhibitory po- ADTN, which is fixed in a tram /3-rotameric tency (Table II). These results suggest that conformer, exhibited a four-fold increase dopamine might interact with activated in Ki under conditions of protein phos- TH in the trans @onformation. phorylation (Fig. 1). Apomorphine, which As seen in Fig. 1 in the absence of in- is a rigid tram a-rotamer, showed no sta- hibition, the activation of the enzyme is tistically significant change in Ki even cofactor dependent with greater differ-


5-

4-

-CAMP

176

0 2 4 6 6 10

0.5 1 +cAMP

r 1 r I I 1 0 10 20 30 40 50

ADTN, uM

FIG. 1. Dixon plot of the inhibition of control and activated TH by ADTN. See Methods for incubation and assay conditions.

Materials and

ences in velocity between activated and control occurring at lower concentrations of GMPH,. For a given cofactor concentration some variation in the activation was observed. For ADTN we found that, in the experiment in which greater activation was observed for a given cofactor concentration, the difference in Ki was greater. On the other hand for apomorphine there was no correlation of the change in Ki with that in activation.

It has been recognized that for this group of cofactor-competitive inhibitors only the catechol moiety is essential for inhibition of TH (9,lO). Catechol estrogens represent fairly potent members of this class that lack an amino group, and their inhibition may have physiological significance (20- 21). Furthermore, inspection of Dreiding models of the catecholic estrogens and l-

see-butyl-1-carboxy-6,7-dihydroxy TIQ reveals that a part of the steroidal hydrophobic moiety may occupy the same space into which the 1-alkyl group is free to ro- tate. Since this suggests that these estrogens could bind to the same catechol and putative hydrophobic domains on TH, they were logical candidates to test in our system. In contrast to the 1-alkyl TIQs, both 2-hydroxyestrone and 2-hydroxyestradiol showed a four-fold increase in Ki under phosphorylation conditions (Table II). A representative Dixon plot used to obtain the Ki is illustrated in Fig. 2.

DISCUSSION

The objective of this study was to de- termine the structural features of the cat- echo1 inhibitor that were required to im-


6MPH4, pM

-CAMP

1.2 -

0 40 60 120 160

2-Hydroxyestrad~d, pM

FIG. 2. Dixon plot of the inhibition of control and activated TH by 2-hydroxyestradiol. See Materials and Methods for incubation and assay conditions.

part a sensitivity to phosphorylation of the enzyme. To undertake such an analysis

sible but less plausable that a phosphate

several assumptions were made. First, group is inserted directly on an amino acid

since the inhibitors tested in this inves- residue that is at the catechol-binding site. Within this theoretical framework we can

tigation give cofactor-competitive kinetic plots with both control and activated TH,

interpret our data as indicating the pres-

we assumed that they all interact exclu- ence of at least three critical binding re-

sively at the same binding site of the en- gions as shown in Fig. 3: (i) As a result of

zyme. Secondly, upon introduction of a extensive studies on TH inhibition (g-14), it is well documented that a catechol-bind-

phosphate group through a covalent bond (4, 23) it is conjectured that the confor-

ing site exists. Methylation of the phenolic

mation of TH changes with a resultant al- hydroxyls totally abolishes binding to this site. (ii) A polar or an ionic second locus

teration in this binding domain. It is pos- that interacts with the presumably posi-


TABLE II EFFECT OF PHOSPHORYLATION ON TYROSINE HYDROXYLASE INHIBITION BY CATECHOLS

Compound -CAMP +cAMP

3-Carboxysalsolinol 19.9 f 2.3 21.1 f 1.7 1-set Butyl-l-carboxy-6,7-dihydroxy TIQ 39.2 -+ 3.6 32.6 f 5.9 ADTN 3.36 f 0.47 14.6 3~ 3.2* Apomorphine 0.823 + 0.119 1.79 f 0.59 2-Hydroxyestrone 21.1 f 3.7 30.7 f 4.7** 2-Hydroxyestradiol 17.6 f 0.03 76.0 5 8.7*

Note. K, values represent the average derived from three to four kinetic plots in which cofactor concentrations were varied as shown in Fig. 1 and 2. In the absence of inhibitor Km values were 717 and 164 PM and V,,,,, values were 5.1 and 7.4 pmol/mg striatum for control and activated enzymes, respectively.

* P i 0.05 one-tailed Student’s t test. **p < 0.005.

tively charged amino group is inferred by the enhanced binding of catecholamines over simple catechols such as dihydroxy- phenyl acetic acid, catechol, and other compounds (9, 10, 16). The differential ef- fects of catechol analogs fixed in the cti and tram conformation, e.g., 3-carboxysalsolinol versus ADTN, upon protein phosphorylation (Table II) are consistent with this notion. Equally supportive are the differences observed between apomorphine and ADTN (Table II). These results suggest that dopamine binds to TH in a tram /3-rotameric conformation. (iii) Fi- nally, several lines of evidence indicate hydrophobic interactions. There was an increase in inhibitory potency upon system- atic increases in the hydrophobicity of the 1-alkyl group of the TIQs studied (Table I). In addition a sensitivity of the inhibition of TH by catechol estrogens to phosphorylation was observed. These results suggest

FIG. 3. Hypothetical catechol-binding sites on TH. A juxtaposition of amine and hydrophobic groups on TH as shown could explain why both estrogens and TIQs with hydrophobic residues may be highly inhibitory. However, there could also be more than a single interacting hydrophobic region on TH.

the existence of a hydrophobic region in TH located near the catechol-binding site as illustrated (Fig. 3).

In contrast to the catechol estrogens, the Ki of 1-see-butyl-1-carboxy TIQ did not change upon phosphorylation (Table II). These results could be interpreted to indicate that the affinity of the &fixed amino group for a site on TH offsets the effect of the hydrophobic see-butyl group. Alternatively, a comparison of Dreiding models of this TIQ and the catechol estrogens reveals that, although their hydrophobic groups can occupy similar positions, the see-butyl group possesses considerably more freedom of rotation than the C and D rings of the steroid nucleus. Thus a phosphorylation-induced conformational change of the hydrophobic pocket on TH may affect the binding of the less flexible catechol estrogen more than 1-alkyl or l- benzyl TIQs. Another possibility would be that the differences are due to molecular size. The synthesis of appropriate rigid analogs of the 1-alkyl and 1-benzyl TIQs and suitable tetralins with flexible alkyl groups will permit assessment of the relative im- portance of the cis amino group and the hydrophobic moiety.

It has been documented that estrogens are hydroxylated to catechol derivatives in the hypothalamus (22). Since estrogens also deplete hypothalamic catecholamine levels in viva, the possibility exists that this may be mediated via an inhibition of TH by the newly synthesized catechol es-


trogens (20-21). One complication with this hypothesis has been the inability to de- termine levels of catechol estrogens (21, 22). If the Ki of the catechol estrogens for activated TH observed in this study had been less than the 2 X 1O-5 M value obtained for the control enzyme, this hypothesis would be more tenable. Instead, the fact that the catechol estrogens exhibit a lower affinity for activated TH weakens the the- ory. Since rat striatal TH was used in this investigation, however, there is the possibility that hypothalamic TH may respond differently to catechol estrogens.

In summary, we have demonstrated that 1-alkyl TIQs which contain an amino group fixed in a cis conformation are equipotent inhibitors of control and activated TH. ADTN, which has an amino group in a rigid trans-p-conformation, and catechol estrogens, which lack an amino group, exhibit fourfold lower affinity for the activated form of the enzyme. These results suggest that a region which interacts with an amino group and possibly a hydrophobic domain near the catechol-binding site of TH are influenced upon activation by protein phosphorylation. Confirmation of this hypothesis awaits the availability of large amounts of highly purified forms of the activated and control enzyme.

ACKNOWLEDGMENTS

We would like to thank B. Ence, Dr. J. M. Lasala, and Dr. T. Hudlicky for synthesis of TIQs used in this study; J. Fitch, T. Ryan, and T. Consler for per- forming certain enzyme inhibition experiments; and Dr. H. F. Gilbert for reading this manuscript.

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differential inhibition of activated tyrosine hydroxylase

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