Vol. 216, No.3Printed in U.S.A.

ABSTRACT

0022-3565/81/2163-0647$02.OO/OTHE Jouaa*i. OF P,u.ucacol.ooY AND Exi’uuau�smL TsaaapxuTicaCopyright C 1981 by The American Society for Pharmacology and Experimental Therapeutics

Tyrosine Hydroxylase: Studies on the Phosphorylation of aPurified Preparation of the Brain Enzyme by the Cyclic AMP-Dependent Protein Kinase1

ARTHUR M. � JOACHIM D. RAESE, MITCHELL A. LAZAR and JACK 0. BARCHAS4

Nancy Pritzker Laboratory of Behavioral Neurochemistry, Department of Psychiatry and Behavioral Sciences, Stanford University School ofMedicine, Stanford, California

Accepted for publication December 1 0, 1980

Edelman, Arthur M., Joachim D. Raese, Mitchell A. Lazarand Jack D. Barchas: Tyrosine hydroxylase: Studies on thephosphorylation of a purified preparation of the brain enzymeby the cyclic AMP-dependent protein kinase. J. Pharmacol.Exp. Ther. 21 6: 647-653, 1 981 .

Tyrosine hydroxylase [L-tyrosine, tetrahydropteridine: oxygenoxidoreductase (3-hydroxylating); EC 1 .1 4.1 6.2] (TH) was pu-rified from bovine corpus striatum. The purification involvedsequential DEAE cellulose, hydroxylapatite and CM SephadexC-50 chromatography, followed by glycerol density gradientcentrifugation. Final preparations appeared to be 90 to 100%pure as judged by polyacrylamide gel electrophoresis under

denaturing conditions in acetic acid-urea. The enzyme wasestimated to have a minimum molecular weight of approxi-mately 60,000 daltons. Purified TH could be activated in vitroby incubation with magnesium adenosine triphosphate and thecatalytic subunit of cyclic AMP-dependent protein kinase(ATP/protein phosphotransferase; EC 2.7.1 .37). When thefinal purified preparation of TH was incubated under theseconditions utilizing [y-32P]ATP, it was found to incorporate 0.7to 0.9 mol of phosphorus/mol of protein. These results suggestthat the activation of TH in the presence of phosphorylatingconditions is due to its phosphorylation by cyclic AMP-depen-dent protein kinase.

Tyrosine hydroxylase (TH) catalyzes the initial and most

probably rate-limiting step of catecholamine biosynthesis (Na-

gatsu et al., 1964; Levitt et al., 1965). Knowledge of the regu-

lation of this enzyme is crucial, therefore, for an understanding

of catecholamine synthesis in the adrenal gland and in the

peripheral and central nervous systems.

TH is subject to multiple modes of regulation. The enzyme

is activated in vitro by phospholipids (lloyd and Kaufman,1974; Raese et al., 1976) and polyanions such as heparin

(Kuczenski and Mandell, 1972; Katz et al., 1976) and is inhibited

by the catecholamine end products (Nagatsu et al., 1964). TH

is also activated by phosphorylation reactions catalyzed by the

cyclic AMP-dependent protein kinase (Harris et al., 1975; Lloyd

and Kaufman, 1975; Lovenberg et al., 1975; Morgenroth et al.,

1975; Raese et al., 1977) and by a cyclic nucleotide independent

protein kinase (Raese et al., 1979b). We have previously pre-

sented evidence using a partially purified preparation of TH

Received for publication December 12, 1978.

1 � work was supported by National Institutes of Mental Health Program-

Project Grant MH 23861 as well as by National Institutes of Drug Abuse GrantDA 01207 and the ONR. A preliminary report of portions of this work waspresented at the Fourth International Catecholamine symposium in PacificGrove, CA, Sept. 17-22, 1978.

2 Present address: Department of Pharmacology, SJ-30, University of Wash-ington &hool of Medicine, Seattle, WA 98195.

3 Held a National Institutes of Mental Health Biosciences Fellowship from

Grant MH 15147.

4 Recipient of Research Scientist Development Award MH 24161.

that the enzyme can be phosphorylated in vitro (Raese et aL,

1977). We here present further data indicating the capability

for phosphorylation of bovine corpus striatal TH by cyclic

AMP-dependent protein kinase. Some of these results have

been previously reported in preliminary form (Edelman et al.,

1978; Raese et al., 1979a,b).

Enzyme Purifications

Methods

TH. Bovine corpora striata were freshly dissected at a local slaugh-terhouse and frozen on Dry Ice. The tissue was either homogenized onthe same day or stored at -70#{176}Cfor subsequent purifications. All ofthe following steps were performed at 4#{176}C.Up to 1 kg of tissue washomogenized in 3 volumes of a buffer containing 25 mM Tris HCI(pH 7.5), 15 mM /3-mercaptoethanol, 1 mM disodium(ethylene dini-

trilo)tetraacetic acid (EDTA), 0.05 M NaC1 and 10% glyceroL Thehomogenate was centrifuged 2.5 hr at 30,000 x g. The supernatant wasdirectly applied to a DEAE Cellulose (Sephacel; Pharmacia Fine Chem-icals, Piscataway, NJ) column equilibrated with the homogenizationbuffer. The packed bed volume of the column was approximately equalto the volume of supernatant applied. The column was washed withthe homogenization buffer until the absorbance at 280 am of theeffluent was �O.O& absorbance units (-4 column volumes). This wasfollowed by approximately 2 column volumes of this buffer containing0.1 mM cyclic AMP to elute the catalytic subunit of protein kinase(Kinzel and Kubler, 1976). TH was then eluted with the homogenization

buffer containing a linear gradient of NaCl, 0.05 to 1.0 M. The totalvolume of the gradient was 2.5 times that of the packed bed. Enzymat-

647

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ically active fractions, eluting between 0.13 and 0.35 M NaC1, were

pooled and dialyzed exhaustively against a buffer containing 25 mMpotassium phosphate (pH 6.8), 15 mM fl-mercaptoethanol, 1 mMEDTA and 20% glyceroL Insoluble material was removed by centrifu-gation and the resultant supernatant was applied to a hydroxylapatite(Bio-Rad Laboratories, Richmond, CA) column (2.6 cm X 28 cm)equilibrated with the same buffer. The column was washed with this

buffer until the absorbance at 280 am ofthe effluent was approximately�O.O6 absorbance units. The enzyme was eluted with a linear potassium

phosphate gradient (1.4 1), 25 to 400 mM, pH 6.8, containing 15 mM

fl-mercaptoethanol, 1 mM EDTA and 20% glycerol. Active fractions,eluting between 0.05 and 0.16 M potassium phosphate, were pooled and

dialyzed exhaustively against a buffer containing 50 mM potassium

phosphate (pH 6.8), 1 mM dithiothreitol (DTT), 1 mM EDTA and 20%

glyceroL The enzyme was applied to a CM Sephadex C-50 (Pharmacia)column (2.6 cm x 60 cm) equilibrated with the same buffer. The columnwas washed with this buffer until the absorbance at 280 am was <0.04

absorbance units. The enzyme was then eluted with a linear potassium

phosphate gradient (1.5 1), 50 to 250 mM, pH 6.8, containing 1 mM

DTT, 1 mM EDTA and 20% glycerol. TH activity was eluted between

approximately 0.10 and 0.14 M potassium phosphate.Enzyme was either stored at 4#{176}Cor concentrated (approximately 20-

fold) by vacuum dialysis and purified further after exchange of buffercomponents and reduction of the glycerol concentration by density

gradient centrifugation in 9 to 34% (w/v) linear glycerol gradientscontaining 50 mM potassium phosphate (pH 6.8), 1 mM DTT and 1

mM EDTA. Centrifugation was performed in a Beckman L2-65B

ultracentrifuge at 40,000 rpm for 20.5 hr at 4#{176}Cusing a Spinco SW4O

rotor. Fractions containing TH activity (sedimenting at -9S) from theglycerol gradient were pooled and stored at 4#{176}C.

Several aspects of the purification could be modified without appar-

ent effect. DEAE Sephadex could be substituted for DEAE cellulose;DTT (0.5-2 mM) could be substituted for /3-mercaptoethanol; andEDTA could be used at either 0.1 or 1.0 mM. However, during thehydroxyl.apatite chromatography, 10% glycerolgave better performancethan 20% in terms of binding of the enzyme to the column.

Protein kinase. The catalytic subunit of cyclic AMP-dependentprotein kinase is here referred to simply as protein kinase. All stepswere performed at 4#{176}C.Active fractions from the cyclic AMP elutionfrom the DEAE column (see TH Purification) were pooled, dialyzedagainst a buffer containing 50 mM potassium phosphate (pH 6.8), 1mM DTT, 0.1 mM EDTA and 30% glycerol and applied to a CMSephadex C-50 column (2.6 cm x 15 cm) equilibrated with the same

buffer. The column was washed with this buffer and the enzyme elutedwith a buffer containing 250 mM potassium phosphate (pH 6.8), 1 mM

DTT, 0.1 mM EDTA and 30% glycerol. The specific activity of this

preparation was 180,000 pmol/min/mg of protein using histone assubstrate (see Enzyme Assays below). This source of protein kinasewas used routinely for activation experiments. Alternatively, the en-zyme after CM Sephadex chromatography was incubated at 30#{176}Cfor30 mm with 9 mM MgCl2 and 94 �tM [y-�P]ATP and chromatographedon Sephadex G100 (2.6 x 95 cm) in a buffer containing 50 mM potassium

phosphate (pH 6.8), 1 mM DTT, 1 mM EDTA and 30% glyceroL Thisprocedure allowed the identification and removal of phosphate accept-lag contaminants present in the protein kinase preparation at the CM-Sephadex stage. The removal of minor contaminants by the G-100column produced essentially no change in the specific activity of the

enzyme. Protein kinase prepared by this method was homogeneous as

judged by polyacrylamide gel electrophoresis in either acetic acid-urea(Zahler, 1974) or in the sodium dodecylsulphate (SDS)-phosphatesystem of Weber and Osbom (1975). This preparation was used in theexperiments illustrated in figures 3 to 5. The enzyme was stored at 4#{176}C.At this temperature, the rate of decay of enzyme activity was less than10% per month.

Enzyme assays. TH was assayed essentially according to themethod of Waymire et al. (1971). A typical assay mixture contained:sodium 2-[N-morpholino]ethane sulfonate (pH 6.0, 0.24 M); beef livercatalase, 860 units (Worthington Biochemical Corp., Freehold, NJ);

excess sheep liver dihydropteridine reductase in 10 mM Tris-HC1, pH,7.4, purified through the second ammonium sulfate step, 2000 units

(defined according to Method I ofCraine et al., 1972); reduced NADPH,

0.1 �imol; L-[1)4C]tyrosine (51.9 mCi/mmol), 39 �zM; tetrahydrobiop-term, 1.2 mM and 25 �tl of enzyme from the various stages of purifica-

tion. Bovine serum albumin (BSA; Sigma Chemical Company, St.Louis, MO) was added to bring the protein concentration in each tubeto at least 2 mg/mi before the start of the reaction. The volume during

this phase of the reaction was 85 4 After 25 mm at 30#{176}C,0.5 �imol of3-iodotyrosine in 5 mM HC1 was added to stop TH activity. Excesspartially purified hog kidney decarboxylase (Waymire et al., 1971) wasadded with pyridoxal phosphate (final concentration, 0.1 mM) in 0.5 Mphosphate buffer (pH 8) containing 10% glycerol to bring the volume to

160 4 ‘4C02 was trapped and counted essentially according to theprocedure of Ichiyama et al. (1970). Biopterin (Regis Chemical Co.,

Morton Grove, IL) was reduced to tetrahydrobiopterin according to theprocedure of Lloyd and Weiner (1971).

For experiments illustrated in Figures 1 and 3, the assay conditions

employed were as follows: 0.1 to 0.2 M sodium acetate (pH 6) was thebuffer, the concentrations ofsubstrates were: tyrosine (15 �tM, fig. 1; 21

1iM, fig. 3) and tetrahydrobiopterin (160 �tM, fig. 1; 800 tiM, fig. 3). BSAwas not included. The reaction proceeded for 30 to 45 mm in a total

volume of 75 to 125 i1, after a 30-mm preincubation in the case of theexperiment shown in figure 1. The decarboxylase-pyridoxal phosphatesolution was added in 5 mM phosphate buffer (pH 7) containing 10%glycerol to bring the final volume to 150 to 200 �d. Other aspects of theassay were as outlined above.

Protein kinase was assayed according to the procedure of Witt andRoskoski (1975) with 0.12 mM ATP, 4.3 mM MgCl2 and 4.8 mg/mI ofHistone LI-A (Sigma). One unit of protein kinase activity is defined asthe amount of enzyme which catalyzes the transfer of 1 pmol of �Pfrom [y�nP]ATP to histone per rain.

Protein assays. Protein concentrations shown in table 1 were

determined by the fluorescamine procedure (Bohien et al., 1973). Insome experiments (illustrated in figs. 1-5), TH was radioactively meth-ylated using [‘4C]formaldehyde according to the procedure of Rice and

Means (1971) in order to quantitate small amounts of protein duringanalytical procedures. In general, this procedure, which involved con-

centration and dialysis against 0.2 M borate buffer (pH 9), caused

relatively minor losses of enzyme activity. ‘4C-labeling did not preventthe activation response of TH to phosphorylating conditions. For thestudies to determine the molar ratio of aP�labehag, both methylatedand unmethylated TH was utilized. For studies using unmethylatedenzyme, protein was determined by the fluorescamine procedure.

Electrophoresis in acid-urea gels. The procedure used was amodification of that of Zahler (1974). Gels containing urea and aceticacid were polymerized to be 6 or 6.5% acrylamide, using N,N’-diallyl-

tartardiamide as the cross-linking reagent (at 15% of the total acryl-amide). Samples (2-5 ,ig of protein) were prepared by dilution with anequal volume ofa dissociating solution containing 25% (v/v) acetic acid,50% (w/v) phenol, 2% (v/v) $-mercaptoethanol and 4 M urea, followedby incubation at 30#{176}Cfor 2 hr. Boiling was avoided as this tended to

produce additional bands, presumably due to acid hydrolysis. Electro-phoresis was performed as described by Zahier (1974). Staining was in0.25% (w/v) Coomassie blue in 10% (v/v) acetic acid and 10% (v/v)isopropanol for 4 to 6 hr. Gels were destained electrophoretically.

Molecular weights of enzyme subunits were estimated by a semilogplot of molecular weight (ordinate: log scale) vs. relative mobility

(abscissa: standard scale). Mobility was calculated relative to the mo-bility of deoxyribonuclease I. Molecular weights of the standards used

were taken as: rabbit muscle phosphorylase a: 92,500 pig heart fumar-am: 48,500; ovalbumin: 44,000 and deoxyribonuclease I from beefpancreas: 31,000 (Lindberg, 1967; Castellino and Barker, 1968; Klotz etal., 1975). For estimation of relative purity of TH, gels were scannedspectrophotometrically at 550 nra with a Gilford 2000 spectrophotom-eter equipped with a linear transport attachment. For liquid scintilla-

tion counting, gels were sliced into approximately 1.5 mm slices. Eachslice was digested at room temperature overnight in 1 ml of 2% (w/v)

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1981 Brain TH Phosphorylation 649

a Picomoles of dopa per minute.b Dialyzed against homogenization buffer.

periodic acid and counted in 10 ml of Hydromix (Yorktown Research;Piscataway, NJ).

Determination ofphosphorus incorporation. The purified prep-

aration of TH (7 �g) was incubated for up to 240 rain at 30#{176}Cwith: 600units (3.6 big) of protein kinase purified through the Sephadex G-100step, 10 mM MgCl2 and 90 �M [y�P]ATP (35.3 �Ci/umol). The incu-bation volume was 0.32 ml. A 20-sal aliquot of the incubation mixturewas added to 100 �il of a solution of 2 mg/ml of BSA followed immedi-ately by 1 ml of 11% (w/v) ice-cold trichioroacetic acid (TCA). ‘4C-labeled purified TH (1 x i0� cpm, 3.0 ig) was then added for estimation

of recovery. The mixture was incubated at 0#{176}Cfor 30 rain and centri-fuged. The pellet was redissolved in 300 �il of ice-cold 0.25 N NaOH.The TCA precipitation and NaOH resolubilization was repeated twicemore. The final pellet was redissolved in 300 �l of 1 N NaOH at room

temperature. One hundred microliters were counted in 10 ml of Hydro-mix. The blank was a parallel procedure identical in all respects to thatdescribed above except that the appropriate buffer was substituted forTH. The TH used for these determinations was approximately 95%pure as judged by electrophoresis under denaturing conditions. Theminor contaminants (approximately 5% of the protein) did not detect-ably incorporate phosphorus.

Results

Table 1 illustrates a purification of bovine striatal TH. The

yield of this particular preparation was atypically low. A final

yield of 1 to 4% would be expected for this scheme. The

preparation showed good stability (approximately 30% loss per

month) when stored at 4#{176}C.

The final step in the purification scheme involved density

gradient centrifugation as is ifiustrated in figure 1. TH from the

CM-Sephadex step centrifuged in a linear 5 to 20% sucrose

density gradient yielded a sedimentation coefficient (S�,w) of

8.9S corresponding to a molecular weight by reference to known

standards (Schachman, 1959; Martin and Ames, 1961; Sober,

1968) of approximately 180,000. This will be referred to as the

9S form. A smaller peak of enzyme activity at 48 was also

observed. We have previously presented evidence that this

represents the dissociation of TH into subunits (Raese et al.,

1977). The size of the 4S form (45-50,000) suggested a tetra-

meric structure for the enzyme. The sedimentation behavior of

TH in linear 9 to 34% glycerol gradients was not detectably

different from that in linear 5 to 20% sucrose gradients. Glycerol

gradients were routinely used in the purification.

The purity of the 9S form of the purified preparation of TH

was evaluated by polyacrylamide gel electrophoresis in acetic

acid-urea. Figure 2 illustrates an enzyme preparation which was

apparently homogeneous. For this particular preparation, a

second density gradient was employed to remove minor con-

taminants. We have subsequently found that a second gradient

is effective to only a slight degree in this regard and therefore

this procedure was not included in subsequent preparations (for

example, table 1). The minimum molecular weight of the pun-

fled preparation determined by electrophoresis (as described in

“Methods”) was found to be approximately 60,000. The purity

of each preparation was quantified by spectrophotometric scan-

ning of the stained gels at 550 run. By this criterion, most

preparations were about 90% pure. Polyacrylamide gel electro-

phoresis was also performed according to the SDS-phosphate

system of Weber and Osborn (1975). This technique produced

similar results as the acid-urea gels.

Fig. 1 . Sucrose density gradient centrifugation of TH and activation byphosphorylating conditions. TH from the CM-Sephadex step was radio-actively methylated using [14C]formaldehyde (Rice and Means, 1971).The enzyme (70 ,.tg) was centrifuged in a 5 to 20% (w/v) linear sucrosedensity gradient. The gradient contained 50 mM potassium phosphate(pH 6.8), 0.1 mM EDTA, 1 mM DTT and 0.1 5 M KCI. Aliquots ofgradient fractions were preincubated with 1 25 U of protein kinase,purified through the CM-Sephadex step (see � ‘Methods’ ‘), 50 �M ATPand 2 mM MgCI2 for 30 mm at 30#{176}Cand then assayed for TH activity(#{149}.left ordinate) as described in “Methods.” The same fractionsincubated without protein kinase, ATP and MgCI2 served as control(A, left ordinate). Protein (0, right ordinate) was determined by the ‘4C-content of the fractions.

TABLE 1

Purification of bovine corpus striatal TH

Fraction Volume Protein Activity� Specific Activity Fold Purification Yield

ml mg units units/mg %

I. Supernatant of crude 2420 1 3794.0 765,688 56 1 .0 100.0homogenateb

II. DEAE celulose 400 2720.0 242,880 89 1 .6 31.7III. Hydroxylapatite 206 255.0 1 1 7,008 459 8.3 15,3IV. CM-Sephadex C-50 136 7.3 22,413 3070 55.3 2.9

V. Glycerol density gra- 14.8 0.74 5,482 7408 1 33,0 0.7dient

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650 Edelman et al. Vol. 216

A representative result of a labeling experiment with the

purified preparation is shown in figures 4 and 5. Figure 4

illustrates the incorporation of 32P into the purified TH prepa-

ration after incubation with protein kinase, Mg� and [-y-32PJ-

ATP. In addition, a fainter second protein band was phos-

phorylated. As is shown in a control gel (fig. 5), this represented

an autophosphorylation of the protein kinase present in the

reaction mix. This autophosphorylation of the catalytic subunit

of protein kinase occurred in a nonstoichiometric fashion (max-

imally 0.2 mol ofphosphorus per mol ofprotein). The low molar

ratio of protein kinase autophosphorylation could theoretically

be explained by the formation ofan acid-labile phosphohistidine

bond which has been reported to occur with the brain enzyme

by Kochetkov et al. (1976). In some gels (for example, fig. 4),

there was nonmigrating, apparently precipitated, material on

top of the gel which is characteristic of this electrophoretic

system (Zahler, 1974). The amount of this material varied

between electrophoretic runs of the same protein fraction and

did not represent proteins aside from the two major bands.

In order to determine the molar ratio of 32P incorporation,

the purified preparation of TB was incubated with [‘y-32P]ATP

and protein kinase and incubation conditions were adjusted so

that the reaction went to completion. The amount of phospho-

fl’s incorporated was determined by TCA precipitation as de-

scribed in “Methods” (Determination of Phosphorus Incorpo-

ration). Assuming a MW of 60,000, the purified enzyme prepa-

ration was found to incorporate 0.7 to 0.9 mol of phosphorus

per mol of protein. Under similar conditions but with a different

preparation of TB, a value of 0.7 was obtained.

Discussion

Fig. 2. Acid-urea polyacrylamide gel electrophorsis of the purifiedpreparation of TH. TH from the CM-Sephadex step was radioactivelymethylated and centrifuged in a linear glycerol gradient as describedin � Methods. � � The gradient contained 50 mM sodium phosphate (pH

6.8), 1 mM EDTA and 1 mM DTT. The 9S protein peak was againsubjected to glycerol gradient centrifugation in the same manner (after

removal of glycerol and exchange of buffer components by dialysis).

The second gradient contained 20 mM Tris-HCI (pH 8.5), 1 mM EDTA

and 1 mM DTT. Peaks of enzyme activity at 9S and 4S were obtained.Fractions were stored at - 20#{176}Cbefore electrophoresis. The 9S peakfrom the second gradient with a specific activity of approximately 5000pmol of dopa formed per mm/mg of protein was electrophoresed in

acid-urea gels as described in � � Methods. �

As illustrated in Figure 1, the final preparation ofTH retained

its ability to be activated by incubation with MgATP and the

catalytic subunit of cyclic AMP-dependent protein kinase. We

have noticed no systematic changes in the extent of this acti-

vation as a function of the stage of purification of TH. The

mechanism of the activation seems to involve an increase in the

affmity of the enzyme for the reduced pterin cofactor without

a change in Vmax (Lloyd and Kaufman, 1975; Lovenberg et al.,

1975; Edelman et al., 1978). By contrast, Joh et al. (1978) found

a Vmax increase without any Km changes for the rat striatal

enzyme.

TB from the CM-Sephadex step was incubated with protein

kinase and [‘y-32P]ATP, exhaustively dialyzed to remove labeled

ATP and centrifuged in a 9 to 34% glycerol gradient, a procedure

which separates TB from the bulk of the protein present at the

CM Sephadex step, from protein kinase, and from any residual

traces of [-y-32P]ATP. The result, shown in figure 3, was that

32P was associated with TH activity at 9S, indicating phos-

phorylation of the enzyme.

On density gradient centrifugation, bovine corpus striatal TB

may be present in catalytically active multiple forms depending

on the buffer composition in the gradient (Raese et al., 1977).

All forms retained the ability to be activated by phosphorylat-

ing conditions. The smallest form (4S) obtained had a MW of

45 to 50,000 by reference to the sedimentation of known stan-

dards included in the gradient and has been hypothesized to

represent monomeric TB. The data presented in this commu-

nication indicate that the 9S form had a minimum MW of

about 60,000 as estimated by gel electrophoresis under dena-

turing conditions. The 4S form, however, had the same electro-

phoretic mobility as the 9S form on SDS gels (data not shown).

This indicates that, although the methods do not precisely

agree with respect to subunit molecular weight, the 4S form

apparently represents a monomeric form of TB and not a

proteolytic product.

Several lines ofevidence indicate the possibility that a certain

portion of the TB molecules are recovered in the final prepa-

ration in an inactive state. 1) On density gradients, the protein

and TB activity peaks were not precisely superimposable. The

small protein peak at 9S reproducibly had a slightly greater

sedimentation coefficient than the enzyme activity peak (fig. 1

and 3). For example, the ratio of enzyme activity to protein

concentration calculated for the fractions through the 9S peak

of figure 1 is (taking fraction 14 as equal to 1.0 in arbitrary

units): 14, 1.0; 15, 1.0; 16, 2.4; and 17, 5.8. SDS gels run on the

density gradient fractions through the 9S peak area yielded

essentially identical staining patterns. That is, no extra protein

bands were observed whose presence could have accounted for

the lack of completely constant specific activity on the density

gradient (A. Edelman, J. Raese and J. Barchas, unpublished

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Brain TH Phosphorylation 651

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FRACTION NUMBER

Fig. 3. Association of 32P with TH activity on glycerol density gradient centrifugation. Two milliliters of TH (860 �sg) from the CM Sephadex step,radioactively methylated with 14C (Rice and Means, 1 971 ), was incubated with 1 2.3 mM MgCI2, 21 .3 1�M [y.-32P]ATP (64 1�Ci) and 2000 U ofprotein kinase (1 2 f.Lg) in a total incubation volume of 3.24 ml for 45 mm at 30#{176}C.After the incubation, [-y-32P]ATP was removed by exhaustivedialysis against 50 mM potassium phosphate (pH 6.8) containing 1 mM OTT and 1 mM EDTA. A 600-gil aliquot (1 40 �tg) of the dialyzed solutionwas applied to a linear 9 to 34% glycerol gradient containing the above buffer and centrifuged at 40,000 rpm for 20 hr. The gradient wasfractionated into 32 fractions (0.4 mI/fraction). Fraction 32 is the top fraction of the gradient. 32P and protein (14C content of the fractions) were

analyzed by double-label liquid scintillation spectrometry and plotted (ordinate) against fraction number. Also plotted is TH activity of thosefractions found to be enzymatically active (assayed as described in � � Methods’ ‘). The absolute values for protein concentrations in this experiment

are approximate.

12.(

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Fig. 4. Phosphorylation of the purified preparation of TH. TH (the same source of enzyme as is shown in figure 2; 5000 pmol of dopa formed permm/mg of protein; 1 .9 �zg of protein) was incubated for 1 hr at 30#{176}Cwith: 1 00 U (0.6 �zg) of protein kinase purified through the Sephadex G-1 00

step (as described in ‘ ‘Methods’ ‘), 1 4 mM MgCI2 and 6.3 �M [‘y-32P]ATP (1 0 DCi; 1 6.9 �iCi/nmol) in a total volume of 94 il, The reaction mixturewas then diluted with an equal volume of dissociating solution, divided in half and electrophoresed in duplicate gels as described in � Methods,”After staining, one gel was photographed and one was sliced and counted as described in �‘Methods. ‘‘ Quantitation of protein staining was bydensitometry of the photograph shown using a Helena Quick Scan Densitometer. -, densitometric tracing of the photograph of the stained

gel; , 32P radioactivity.

results). 2) Upon isoelectnic focusing in polyacrylamide gels, the

purified preparation of TB showed heterogeneity with two

main peaks of enzyme activity. The more basic form had a

much higher specific activity than the more acidic form (J.

Raese, G. Makk, A. Edelman and J. Barchas, unpublished

results). 3) Data obtained for the specific activity ofthe enzyme

appeared to underestimate the purification achieved. For ex-

ample, TB activity sedimenting on the density gradient at 9S

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Fig. 5. Autophosphorylation of protein kinase, Incubation was as described in the legend to figure 4, except that the appropriate buffer wassubstituted for TH. Processing of duplicate gels was as described in figure 4. -, densitometric tracing of the photograph of the stained gel;

, 32P radioactivity.

40

652 Edelman et al. Vol. 216

was usually cleanly separated from the bulk of the protein

sedimenting at 4S (fig. 1), suggesting that a much greater

purification was achieved by this procedure than that indicated

by the calculated increase in specific activity (Table 1). In

addition, the fmai specific activity is lower than that reported

for preparations from other tissues (Joh et al., 1978; Vulliet et

al., 1980). For example, Joh and co-workers give a specific

activity of approximately 252 nmol/min/mg for TH they purify

from rat striatum.

There are a number of possible explanations to account for

these observations. One would be that contaminating protein(s)

have not been adequately removed during the purification.

Another is the possibility of a lower turnover number for the

bovine stnatal enzyme as compared with TB isolated from

other tissues. Another report of a lower specific activity for the

bovine striatal enzyme was that of Uoyd and Kaufman (1974)

whose 40% pure preparation of TB from bovine caudate had a

specific activity of 0.95 to 2.2 nmol of dopa per mg/mm. Finally,

it may be that the enzyme as isolated contains a fraction of

denatured molecules (perhaps due to covalent modification for

example, by carbohydrate attachment or partial phosphoryla-

tion or perhaps due to lability under the conditions of the

purification) and a fraction of “native” molecules. We have in

fact observed an increased lability of TB after exposure to

phosphorylating conditions. That is a fraction of isolated TB

molecules may be inactive due to their endogenous phos-

phorylation. The molecular weights of these two forms are

expected to be the same; however, the sedimentation coeffi-

cients (perhaps due to a change in partial specific volume) and

isoelectric points could differ (points 1 and 2 above).

The association of 32P with TB activity and the phosphoryl-

ation of the purified preparation of TB by protein kinase are

illustrated in figures 3 and 4. Since the molar ratio was found

to be 0.7 to 0.9 mol of phosphorus per mol of protein, it is

therefore unlikely that a minor contaminating protein could

account for the observed phosphorylation. The molar ratio of

32P-labeling was similar with methylated or unmethylated TB

indicating that methylation with [‘4C] formaldehyde did not

artifactually introduce an ability of TB to be phosphorylated.

Bylund and Krebs (1975) observed that, with some proteins,

denaturating treatment resulted in a greater susceptibility to

phosphorylation while, with other proteins, the susceptibility

to phosphorylation was reduced. That TB might belong to the

latter class is suggested by the report of Markey et al. (1980)

who found that pheochromocytoma TB lost its ability to be

phosphorylated concomitant with loss of activity upon storage.

This suggests that the phosphorylation reported here with the

striatal preparation cannot be simply explained as a nonspecific

effect due to denaturation.

Although initial reports were negative (Lovenberg et al.,

1975; lloyd and Kaufman, 1975), evidence has now accumulated

from a number of laboratories that TH from a variety of species

and tissues is capable of being directly phosphorylated (Le-

tendre et al., 1977a,b; Raese et al., 1977, 1979a; Edelman et al.,

1978; Joh et al., 1978, 1979; Vulliet et al., 1979, 1980; Waymire

et al., 1979; Yamauchi and Fujisawa, 1978, 1979a,b; Markey et

al., 1980; Edelman et al., this communication). Most of these

reports presented evidence for phosphorylation specifically by

the cyclic AMP-dependent protein kinase. Letendre and co-

workers reported (1977a,b) that TB immunoprecipitated from

adrenals and superior cervical ganglia cultured in the presence

of 32P1 incorporated radioactive phosphate. The possibility ex-

ists that in vivo phosphorylation may not exclusively be cata-

lyzed by the cyclic AMP-dependent protein kinase. Data of

Yamauchi and Fujisawa (1979) indicated that in bovine adrenal

meduilary cytosol, the cyclic AMP-dependent protein kinase

was involved. A recent report (Raese et al., 1979b), however,

demonstrating phosphorylation in vitro by a cyclic nucleotide

independent kinase raises the possibility of regulatory controls

other than cyclic AMP being involved in TB phosphorylation.

An as yet unresolved issue is that ofthe subunit stoichiometry

of TB. The report of Joh et al. (1978) suggested that TH was

composed of three nonidentical subunits only one of which

could be phosphorylated. Similarly, Markey et al. (1980) re-

ported that one (offour) subunit is phosphorylated. Preliminary

data from our laboratory using a better resolving electropho-

retic system than in this communication suggest that the sub-

units of TB are closely related in size but nonidentical (J. D.

Raese, 0. Makk and J. D. Barchas, manuscript submitted). The

prediction from the work of Joh and co-workers and that of

Markey et al., however, would be that 0.2 to 0.3 mol of phos-

phorus per mol of protein (assuming subunits are present in

equimolar ratios) should have been obtained here rather than

0.7 to 0.9. Our results are in agreement with those of Vulliet et

al. (1980). The cause of this discrepancy is, at present, unclear.

To summarize, our results show that a purified preparation

of striatal TB can be phosphorylated in vitro by cyclic AMP-

dependent protein kinase which also activates the enzyme.

Furthermore, this is the first report on the stoichiometry of

phosphorylation of TH from brain. Although the purification

we describe results in low yield, it should allow for the routine

preparation of several milligrams ofthe striatal enzyme purified

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1981 Brain TH Phosphorylatlon 653

to 90 to 100% of electrophoretic homogeneity. This method

should prove a useful tool for the study of the biochemical

properties of brain TH.

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