Journai OJ hrrurochenisrry. 1976 Vol. 26. pp. 871-878. Pergamon Press. Printed in Great Britain.

SEROTONIN BINDING PROTEIN : ENHANCEMENT OF BINDING BY Fe2+ AND INHIBITION OF BINDING BY DRUGS' , 2 HADASSAH TAMIR,ATHALIAKLEINand MAURICE M. RAPPORT Division of Neuroscience, New York State Psychiatric Institute and Department of Biochemistry, Columbia University, College oC Physicians and Surgeons, New York, NY 10032, U.S.A. (Received 14 July 1975. Accepted 24 September 1975)

Abstract-The binding of serotonin to a soluble, high affinity binding protein, present in synaptosomes and associated with serotonergic tracts, has now been studied for the effects of metallic ions and various drugs. At optimal concentration ( 1 0 - 4 ~ of ) Fez'. the enhancement of binding was close to 20-fold. A much smaller effect was noted with Cu2+. With other ions (Fe3+, M n 2 + , Coz+, Ni2+, Cr3+, Mg2+,Caz') little or no effect was seen. For the effect with Fez*. preincubation was required M were inhibitory. Studies based on equilibrium (10 min, 25°C) and concentrations higher than dialysis show that the effect of Fez+ was on the affinity of the binding of serotonin to the protein, rather than on the binding capacity. In polydcrylamide gels at pH 8.6 the migratory properties of thc serotonin-protein complex formed in the presence of Fez+ differ from those of the complex formed without Fe2+.Nucleotides (ATP, GTP, ADP, AMP) inhibited thc binding. The effects of several classes of drugs (inhibitors of biogenic amine storage and uptake, psychotomimetics, M A 0 inhibitors and drugs binding to contractile proteins) were also studied. The only effective inhibitors of serotonin binding were reserpine, vinblastine and CZ-74, which caused 50% inhibition at 2 x 1 0 - 6 ~ , M respectively. 7.5 x lo-" M and 0.2 x

IN RECENT communications we reported the detection in synaptosomes of rat brain of a soluble protein with high affinity for serotonin (TAME& HUANG,1974). The association of this protein with serotonergic tracts was confirmed by regional distribution and lesion experiments VAMIR & KUHAR,1975). Placement of lesions in the midbrain raphe nuclei resulted in a substantial decrease in the serotonin binding capacity in the cerebral cortex, associated with the loss of serotonergic fibers. The present study was undertaken to further characterize the interaction of the protein with serotonin. Partial purification of the protein resulted in loss of binding capacity; this loss could be restored by addition of Fe2+ ions and to a lesser extent by Cuz+. The possible function of the protein was studied by testing the effects of drugs with established sites of action. Two drugs, reserpine and vinblastine, were found to be very elfective in inhibiting the binding. Their mode of action suggests that the binding protein may be a contractile protein with storage and/or transport functions. I A preliminary report of part of this work was presented at the Fourth Annual Meeting of the Society for Neuroscicncc. (TAMIR& RAPPORT, 1974.) 2Supported in part by a grant from the Benevolent Foundation of Scottish Rite Freemasonry, Northern Jurisdiction, U.S.A. Abhreiiation used: LSD, lysergic acid diethylamide.

A heterogeneity of the storage or transport forms of biogenic amines has been suggested by several lines of evidence. Thus autoradiographic studies failed to show an association of norepinephrine or serotonin with most of the vesicles in axon terminals in rat CNS and this led to suggestions that the amines were either bound to a macromolecule or preferentially stored in vesicles nearest to the plasma membrane (DESCARRIES & DROZ,1970; GERSHON et al., 1974). Amine storage forms also responded differently to drugs such as reserpine and amphetamine. Soluble storage forms of serotonin and norepinephrine were more susceptible to inhibition by reserpine than particulate forms, whereas the reverse was observed with amphetamine (GLOWINSKI& BALDESSARINI, 1966; GARATTINI & VALZELLI. 1965). Heterogeneity of storage forms is also implied by observations that newly injected or newly synthesized biogenic amine is more rapidly utilized than that already present in tissue et al., 1972). The evidence implicating a (GLOWINSKI non-particulate storage form suggests that such a form may be found in a high affinity binding protein. METHODS AND MATERIALS Partial purification of the binding protebi from whole bruin

All procedures were carried out at W I T . The brains of young male rats (125-200g), without the cerebellum, were homogenized with 2vol of 0.32~-sucrose in 871

872

HADASSAH TAMIR,ATHALIAKLEINand MAURICE M. RAPPORT

M-KPO,, pH 7.5. The particulate fraction was removed by centrifugation at 14,500g for 30min. The pellet was reextracted with 1/2 the original volume of buffered sucrose and the centrifugation was repeated. The supernatant and wash were pooled and centrifuged (100,ooOg; 60min) to remove vesicles, microsomes and fragments of membranes. The buffer was brought to 0.02 M and the protein was fractionated with ammonium sulfate. The fraction precipitating between 20 and 60% saturation contained the serotonin binding protein. The precipitate was collected by centrifugation (l5,ooO g; 20 min) and dissolved in 0 . 0 2 ~ - K P 0 , buffer, pH 7.5. The solution was dialyzed against 0.02 M-KPO,, (pH 7.5; 3 h) and the protein was reprecipitated with ammonium sulfate. In this second fractionation. the binding protein precipitated between 0 and 30% saturation. The precipitate was collected and dissolved as before and then dialyzed against 31 of 0.02 M-KPO,, pH 7.5, containing 10% glycerol, for 3 h. At this stage the binding protein was stable for several months if kept frozen. Binding assay. Portions of protein (0.1mg or less) were combined with FeSO, ( 1 0 - , M o r as indicated) and allowed to remain at room temperature for 8 min in buffer (O.O~M-KPO,,pH 7.5), final vol 0.5ml. Then r3H]serotonin was added ( 1 x lo-' M or as indicated), and incubation was continued for an additional 15 min. The incubation mixture was applied to a column of Sephadex G-50 (1 x IOcm) equilibrated with buffer. After collecting the void volume, the protein-serotonin complex was collected and counted in Bray solution (6 ml). Nonspecific binding was determined by carrying out the measurement in the presence of a lMH)-fold excess of cold serotonin. Inhibition studies by drugs in vitro. The effects of drugs and of various indole derivatives were studied by adding the drug together with the labelled serotonin and incubating for 15 min. Equilibrium dialysis was performed as described previously (TAMIR & HUANG,1974). When the effect of Fez+ was studied by equilibrium dialysis, the metal was added with the Iigand. Gel rlectrophoresis. The complex of protein-ligand (100fig protein) with or without Fez+ was subjected to electrophoresis in polyacrylamide gels (6.5%; 10 cm; 4"C, pH 8.6, 3 ma/gel). The gels were sliced (2mm/slice) and solubilized with tissue solubilizer (TS-I; 0.2 ml) by heating at 50°C overnight. Additional tissue solubilizer was added (0.2 ml); the heating was continued for 2 h. Solutions were counted in 6 ml toluene base scintillation liquid (6 g PPO, 0.075 g POPOP/1 of toluene). Gels were stained with either Coomassie B l u e ( F ~ i R s a N rt ~ sal., 1971) or for lOmin with cc,x-dipyridyl (0.7% in 8"/, acetic acid + 804 mercaptoethanol). Radioactivity measurements. These were done in a Packard counter (Model 3320). As judged by channel ratio

PURIFICATION TABLE1. PARTIAL

RESULTS

Binding activity in the 100,000 g Ammonium suEfate fractionation

100,000g supernatant. dialyzed 2&60u/, sat. (NH,),SO, fraction Fraction reprecipitated at 0-30'j/, sat. (NH,),SO,

OF SEROTONIN BINDING PROTEIN

S.E.M.

400 k 25 145 f 14

92

supernatant.

A partial purification of the binding protein from the homogenate was achieved in three steps: extensive dialysis of the 100,OOOg supernatant a n d two successive fractionations with ammonium sulfate (Table 1). Before dialysis, the specific binding capacity in the 100,OOOg supernatant was very low. Dialysis increased the specific binding 5- t o 7-fold a n d non-specific binding only slightly (1.5- to 2-fold). The increase was probably due to removal of the free a n d some of the bound endogenous serotonin. T h e complete loss of binding capacity which resulted from prolonged (overnight) dialysis may have been caused by proteolytic activity. When the 100,OOO g supernatant was fractionated with ammonium sulfate. thc specific binding capacity of the protein precipitated at 0-30% saturation increased Cfold with n o loss of total capacity. Occasionally total capacity increased, presumably d u e t o removal of an inhibitor of binding (bound endo-

Total protein mg

Fraction

Each value is a mean

count, quenching in all samples was the same and therefore the values were not corrected. Chemicals. Chemicals and their sources were as follows: [G-3H]Serotonin and L-[G-3Hjtryptophan (9 Ci/mmol): Amersham Searle Corp. ; $6- and 5,7-dihydroxytryptamines as creatinine sulfate salts, Regis Chemical Co. (Morton Grove, Ill.); 6-hydroxytryptamine-creatinine sulfate, colchicine, harmaline and nialamide, Sigma Chemical Co., ( S t . Louis, MO.); FeSO, (analytical grade), Mallinckrodt Chem. Works (St. Louis, MO.); l,lO-phenanthroline, Eastman Kodak CO. (Rochester, NY); cc,a-dipyridyl, Fisher Scient. Co. (Fairlawn, NJ); ammonium sulfate (spccial enzyme grade), Schwarz-Mann (Orangeburg, NY); pargyline hydrochloride, Abbot Labs. (North Chicago, IL,); tissue solubilizer (TS-l), Research Products International Corp. (Elk Grove Village, IL.); cytochalasin B, Aldrich Chem. Co. (Milwaukee, WI.). Reserpine phosphate, imipramine, desimipramine and benzoctamine (as hydrochlorides) were a gift of Ciba-Geigy (Summit. NJ). Chlordiazepoxide was a gift from Hoffmann-La Roche (Nutley, NJ). Chlorgyline hydrochloride was a gift of May and Baker Ltd. (Bismuth, Dagenham, England). Vinblastine sulfate was a gift from Lilly Res. Lab. (Indianapolis, IN). Deprenyl (dl) was a generous gift of Dr. J. Knoll (Semmclweis University of Medicine. Hungary). CZ-74 and fenfluramine were a generous gift of Dr. S. H. Snyder (Johns Hopkins University).

+ 9.5

Binding capacity Specific Nonspecific c.p.m./mg protein 8.0 f 1.6 x lo4 21.0 k 2.4 x lo4 31.0

3.8 x

of 6 experiments. 16 animals each

lo4

3.6 k 0.4 x lo4 3.1 0.3 x lo4

+

2.8 f 0.25 x

lo4

873

Serotonin binding protein TABLE 2. EFFECTOF

Metal

M)

None Fe2

+

cu2+

MnZ+ Mg'+ Fe' +

co2+

Ni2* Cr'

+

Ca' Zn2

METALS ON BINDING OF SEROTONIN TO SOLUBLE PROTEIN*

Serotonin binding c.p.m./mg protein Relative binding 1.0 20.4 4.6 2.4 1.2 1.1 1.o 1.o 1.0 1.o 0.52

6300 k 470 129,000 ? 6450 29,000 2c 3 100 15,000 f 1600 7500 k 700 6900 k 650 6280 k 470 6310 2c 470 6280 ? 470 6300 2c 470 3300 i 350

* Reprecipitated at G30% sat. (NH4)2S0,; 0.2mg protein/ml. Each value is the mean f S.E.M. of three experiments minimally. genous serotonin?). The nonspecific binding in this fraction decreased relative to that of the 100,OOOg supernatant. Occasionally a white precipitate of translucent material was seen after freezing and thawing. This insoluble material, soluble at high ionic strength (0.3 M-salt) showed a high degree of binding capacity and was probably an aggregated form of the binding protein. In the initial fractionation most (go"/,) of binding capacity was found in the 20-60% saturated ammonium sulfate fraction. In the second fractionation the binding protein was recovered in the G30% fraction. Removal of inert protein apparently changed the solubility of the ' binding protein. The binding capacity of the reprecipitated material was stable for several months at -20°C in 10% glycerol; 95% of the total binding of this preparation was specific and was enhanced markedly by Fez'. At this stage, other methods of purification, such as ion exchange chromatography, molecular sieve chromatography

iI

O?

10

5

15

20

MINUTES

FIG.2. Time dependence of Fe2+ enhancement: Protein was incubated at room temperature with M-FeSO, for the indicated time. Then [3H]serotonin was added and the assay was carried out as dcscribed in Fig. 1. Each point represents the mean of three experiments.

and differential heat treatment caused excessive loss of activity. Effect of metals

When chelators such as thioglycerol, ascorbic acid, o r EDTA (10- M)were added to the incubation mixture, the binding was inhibited completely. Therefore the effect of various metal ions was studied. The binding (at 1 0 - 4 ~ was ) greatly enhanced by Fe2+, to a much smaller extent by CU" and still less by M n Z + (Table 2). Other metals (Mg2+, Fe3', Co2+, Ni2+ Cr3+, Ca2+ and Zn2+) had no effect. The effect of Fez on enhancement of specific binding was 20-fold whereas the enhancement of nonspecific binding was only 2- to 3-fold. +

P

50

/

4.0

, 0.I

0.2

Fe+* CONCENTRATION (mM) FIG. 1. Enhancement of serotonin binding by Fc2+.Protein, reprecipitated at @30% sat. ammonium sulfate; 0.1 mg, was combined with FeSO, and incubated for 8 min at room temperature in buffer in a total vol of 0.5ml. [3H]Serotonin was added (1 x ~ O - ' M ) and incubation was continued for 15 min. The mixture was applied to a column of Sephadex G-50 to separate thc serotonin-protein complex from free serotonin. In a typical experiment 100% binding corresponds to 200,000 c.p.m./mg protein or 20 pmol serotonin/mg protein. Each point represents the mean of three experiments.

m

I

I

5.0

10.0

I

15.0

' x107

I

EEROTON IB FIG.3. Double reciprocal plot of bound vs free serotonin. Protein (0.2 mg) was dialyzed for 18 h at 4°C against increasing concentrations of [3H]serotonin in a total vol of 1 ml. The difference between c.p.m./ml inside and outside the dialysis bag is the measure of bound ligdnd. Each point reprcsents the mean of two experiments.

874

HADASAHTAMIR,ATHALIA KLEINand MAURICE M. RAPPORT

SLICE NUMBER

FIG. 4. Polyacrylamide gel electrophoresis of protein-serotonin complexes: with Fez', without Fez' and with serotonin + iron only. Samples of protein (0.2 mg) were incubated in duplicate with and without Fez+ ( 1 0 - 4 M ) for 8 min. Then C3H]serotonin (1 x lo-' M) was added. After incubation (15 min, 25°C) the complex was subjected to electrophoresis (6.5%; 4°C; pH 8.6; 3 ma/gel). One gel was stained with a,u-dipyridyl and the other was sliced and counted. The same pattern of migration was observed in 5 separate experiments.

The effect of Fe2+as a function of its concentration (Fig. 1) showed optimal enhancement of serotonin binding at 1 0 - 4 ~at; higher concentrations, the enhancement was less, indicating some degree of inhibition by excess ferrous ion. The effect of Fe2+ as a function of time of incubation with the binding protein (Fig. 2) showed a rather sharp optimum at 8-10min, after which the binding activity decreased rapidly, falling to less than half of its maximal value in 15min. FeSO, and FeCI, were equally effective but ferrous ethylenediammonium sulfate was only 1/6 as active, since the Fez+ is chelated. In the presence of Fez+ the binding of serotonin was proportional to protein concentration (up to 0.3 mg protein/ml). The mechanism of enhancement of binding by Fe2+ was by increasing the binding affinity of the protein for serotonin, rather than by increasing its binding capacity. The evidence was obtained from a study based on equilibrium dialysis of binding as a function of serotonin concentration in the presence and absence of FeSO, (~O-,M). The affinity of the ligand for the protein was calculated from a double reciprocal plot (Fig. 3): in the presence of Fe2+ the value of the dissociation constant was 1.6 x lo-' I, in its absence 5.0 x lo-' M. The capacity (intercept on the ordinate) was not affected. However, the effect

;

:

of the metal might be more complex since the small difference in affinity does not fully-explainthe 20-fold enhancement of binding. The mobility of the protein-serotonin complex on gel electrophoresis was studied in the presence and absence of Fez+ (Fig. 4). In the presence of Fe2+ a much greater degree of binding was associated with high molecular weight proteins that do not penetrate a 6.5% gel even when the pore size was increased by raising the concn. of N,N'-methylene-bisacrylamide to 15%. Since the fraction contained endogenous iron. some of these high molecular weight proteins were present before addition of Fez+. The low molecular weight peak (slices 4-5) decreased slightly after adding Fez+. It thus appears that Fez+ enhances the binding to high molecular weight proteins probably by increasing their concentration. However it will not be possible to resolve this question until conditions are found that allow penetration of the complex into the gel. The complex did not dissociate, even in the presence of 6M-urea. By staining the gel with ccp-dipyridyl, 5 bands of ironcontaining proteins could be discerned: a major one at the origin and four minor ones migrating close to the dye marker. The rapidly migrating iron-containing proteins did not bind serotonin. These observations indicate that the protein that does bind serotonin in the presence of Fe2+ is specific. Staining with Coomassie Blue showed that most of the protein did penetrate the gel. Efect of iron chelators. Both a,a-dipyridyl and 1,lO-phenanthroline ( lo-, M) failed to inhibit the binding. In some preparations binding was enhanced in the presence of these compounds, probably by removal of excess iron. Prolonged dialysis against a,adipyridyl(4 h, 5 x lo-, M) caused 80% loss of binding and this could be restored by the addition of Fe2+, indicating dissociation of the complex does occur very slowly. Effect of nucleotides on serotonin binding It was proposed by ~OLBURN & MAAS(1965) that biogenic amines are stored in nerve endings in the form of ternary metal-ATP-biogenic amine complexes

:

'O0i

8

25

1

I0-4

I0-3 0-3

10-2

4TION (MI NUCLEOTIDE CONCENTRATION

FIG. 5. Nucleotide inhibition of serotonin binding. Protein was incubated with FeSO, M) for 8 min at room temperature and then nucleotide was added followed by [3H]serotonin (lo-' M). The binding was assayed as in Fig. 1. Each point represents the mean of three experiments.

Serotonin binding protein

TABLE 3. INHIBITION BY

DRUGS

875

in vitro OF mE BINDING OF SEROTONIN TO BINDING

PROTEIN

Inhibition* Drug

Molar concentration x lo5

(%I

0.2

50 None None None None

Drugs affecting storage Reserpine Benzoctamine Chlordiazepoxide Amphetamine Fenfluramine

1

1 5 10

Inhibitors of contractile proteins Vinblastine C ytochalasin-b Colchicine

0.75

5 100

Psychotomimetic drugs CZ-74 (mescaline analogue) D-LSD L-LSD

0.02

5 5

so 50 None 50 None None

M A 0 Inhibitors Nialamide Chlorgyline Harmaline Deprenyl Pargyline

25 None None None None

Drugs affecting uptake Amitriptyline Imipramine Desimipramine

None None None

* Drug was added immediately before serotonin, and incubation with the protein and metal was continued for 15 min. The drugs were tested at three different concentrations; experiments were run in duplicate. with either Fez+,Cu2+,or Mg2+.Therefore the effect No high molecular weight complex between seroof nucleotides on serotonin binding was studied. tonin, Fez', and ATP was seen in the absence of The nucleotides ATP, ADP and AMP were found protein, at these concentrations, by either molecular to inhibit the binding of serotonin to the binding pro- sieve chromatography or electrophoresis. Since the tein: 50% inhibition was observed at 7 x 1 0 - 4 ~ , association of serotonin with a high molecular weight 20 x 1 0 - 4 ~and 50 x 1 0 - 4 ~respectively (Fig. 5). protein is dependent on the protein, it is not related GTP had the same inhibitory effect as ATP. This in- to the observations of COLBLJN & MAAS (1965); hibition could not be overcome by increasing the w n - DAPRADA et al., (1971) in which aggregation of biocentration of Fez+ and it is therefore unlikely that genic amines was dependent on divalent ions and a the nucleotides inhibit by chelating this metal. nucleotide. EfSect of drugs on binding

loor

A?

-8

Log Drug Concentration M

FIG.6. Dependence of serotonin binding on drug concentration. Protein (0.1 mg) was incubated with ReSO, ( 1 0 - 4 ~ )for 8min at room temperature. The drug was added followed by [3HJserotonin (1 x l o - ' ~ ) . The binding was assayed as in Fig. 1. Each point represents the mean of two experiments.

Various drugs were studied to determine their effectiveness in inhibiting the binding of serotonin to the binding protein. The drugs were tested at three different concentrations; experiments were run in duplicate. The results (Table 3) indicate that although few were effective, reserpine, CZ-74 and vinblastine were very good inhibitors. Reserpine, known to impair serotonin storage, (BRODIE et al., 1957) caused 50% inhibition of serotonin binding at 2 x M (Fig. 6). This result suggests that the binding protein may have a storage function. CZ-74, a psychotomimetic drug, was a very potent inhibitor (Fig. 6), but other psychotomimetic drugs, such as d-LSD which are believed to compete with serotonin for receptor sites, were not effective inhibitors.

876

HADASSAH TAMIR, ATHALIA KLEINand MAURICE M. RAPPORT

A very interesting observation was the inhibition lase (EC 1.14.17.1) and indoleamine dioxygenase, of binding by agents that bind to contractile proteins proved to be a very potent inhibitor of serotonin M. and change their degree of aggregation: vinblastine binding, producing 50% inhibition at 5 x caused 50% inhibition at 0.75 x 10- M (7 fig/ml) Ascorbic acid can act in several ways, since it is not (Fig. 6) and cytochalasin b caused 50% inhibition at only a reducing agent, but is also a chelator and a 5 x 1 0 - 5 ~The . degree of inhibition was the same source of free radicals. Also it m a y act in its lactone when the drug was added either together with Fez+ as well as free acid forms. Dehydroascorbic acid was . chelators, such or after incubation with the metal. Colchicine, on the without effect at 2 x 1 0 - 6 ~ Other as EDTA and Cleland's reagent, were found to be other hand, did not affect the binding at M. M A 0 inhibitors did not affect binding (only niala- potent inhibitors of binding. These results indicate mide showed a small inhibitory effect) indicating that that the most probable explanation for inhibition by the binding protein is not MAO. Drugs that are in- ascorbic acid is chelation of the metal. It is unlikely volved in blocking reuptake, such as tricyclic antide- that oxidation is involved in serotonin binding. pressants and amitriptyline, had no effect on binding, nor did drugs that are known to affect the turnover Is the binding protein an enzyme in the biosynthetic of serotonin, such as amphetamine and fenfluramine. pathway of serotonin? Neither tryptophan 5-hydroxylase (EC 1.14.16.4) Amphetamine and fenfluramine have also been reported to affect storage, but presumably at a site nor 5-hydroxytryptophan decarboxylase (EC 4.1.1.28) & different from that affected by reserpine (MORGAN et are regulated by serotonin concentration (MCGEER PETERS, 1969; LIN et al., 1969; JEQUIER et al., 1969; al., 1972; COSTA& REVUELTA, 1972). personal communication) and thereThe results of these experiments exclude several A. S. PERUMAL, possible physiological roles for the binding protein. fore these enzymes probably do not have a binding These include enzymes involved in synthesis or degra- site for serotonin. Nevertheless, the question arises dation of serotonin, carriers in the reuptake with sufficient frequency to make it important to rule mechanism affected by imipramine and desimipra- out their involvement. The &30"/, saturated mine, or some soluble form of receptor. The effects ammonium sulfate fraction was incubated with of reserpine and vinblastine suggest that the binding C3H]tryptophan and subjected to electrophoresis on protein may serve as a storage or transport form and polyacrylamide gel. No binding was detected either may be a neurotubulin-like macromolecule which is close to the origin or in slices 4 5 where the serotonin-protein complex migrates. The tryptophan-proalso sensitive to reserpine. EfSect of indole derivatives. It was observed pre- tein complex was located in slices 2&24. Furtherviously (TAMIR & HUANG,1974)that a hydroxy group more, the affinity of tryptophan for the binding proon the indole was essential for binding since trypto- tein'is a 1000-fold smaller than that of serotonin. phan and tryptamine were not inhibitory. Also a free DISCUSSION amino group enhanced binding, since bufotenin was bound less effectively than serotonin. 5,7-DihydroxyThese studies show that the ability of partially puritryptamine was a very potent inhibitor of binding fied binding protein to bind serotonin is greatly (50% inhibition at 2 x lo-' M). In the present study enhanced by Fe2+.The effect of Fe2+ is very specific. other indole derivatives were tested. 5,6-Dihydroxy- Other divalent ions, with the exception of Cu2+,have tryptamine, 6-hydroxytryptamine and 5-hydroxytryp- little effect, and Cu2+ is very much less effective than tophan were all found to inhibit binding, the concen- Fez+. The optimum concentration of Fe2+ needed trations producing 50% inhibition being 7 x lo-' M, for enhancement of serotonin binding is M. This 30 x ~ O - ' M and 200 x 1 0 - ' ~respectively. is the concentration of iron (Fe2+ + Fe3+) that is Examination of oxidation-reduction mechanisms. found in synaptosomes; whole brain has about 10 Since dihydroxytryptamine had a very high affinity times more (COLBURN & MAAS,1965). The question for serotonin binding protein, it was important to of whether Fez+ is oxidized to Fe3+ in the process study the effects of oxidizing enzymes and their cofac- of enhancing the binding of serotonin remains open. tors on the binding. H,O, (or a generating system Possibly electron spin resonance measurements may for it) is known to enhance several monohydroxylases. permit this question to be answered. whereas catalase (EC 1.11.1.6) and peroxidase (EC The need for incubation of protein with the metal 1.11.1.7), which catalyse the reduction of H,O,, in- to achieve maximum enhancement suggests that some hibit them. At concentrations up to 5 X 1 0 - 7 ~ , change in protein conformation and/or state of aggreH,Oz did not alter the binding of serotonin. b t a l a s e gation may be involved. This is supported by the find(up to 950 units) also did not affect the binding. How- ing that binding affinity rather than binding capacity ever, horseradish peroxidase was found to enhance is affected by Fe2+.The failure of the serotonin-prothe binding, both in the absence of Fez+ and in its tein-metal complex to penetrate even a porous gel presence. This enhancement was observed even with suggests either that the complex has a low net charge an enzyme preparation that had been heated at 100" or that it is an aggregate of very high molecular weight. The first possibility is unlikely since the serofor 20min and was no longer active. Ascorbic acid, a cofactor for dopamine-/?-hydroxy- tonin binding protein binds very tightly to D F A E - e l -

Serotonin binding protein

877

lulose, indicating its negative charge. With respect to tion of 5,6-dihydroxytryptamine. Cytochalasin was et al., size, its molecular weight should be much higher than shown to inhibit cellular movement (WESSELS that of thyroglobulin (6.6 x lo5) which migrates into 1971) as well as release of neurotransmitters (THOA this gel. This assumes that asymmetry of the mol- et al., 1972; NICKLAS& BERL, 1974). Colchicine is also known to bind to neurotubulin, but its ineffectiecules is comparable. Two observations make it unlikely that the binding veness in inhibiting the binding of serotonin shows protein promotes the formation of a metal-nucleotide- either that the serotonin binding protein differs from biogenic amine complex of the type proposed by C ~ L - neurotubulin or that a free colchicine binding site is not essential for serotonin binding. BURN & MAAS(1965), or as demonstrated in rabbit Two ultrastructural entities that may be related in platelets by DAPRADAet al. (1971). In their type of complex, protein was not required and in the type some way to the serotonin binding protein were de(1972) has of binding reported here nucleotides were inhibitory. scribed recently in the literature. TRANZER A n effort to determine the functional role of the described an “electron dense fine ultrastructure” in the binding protein was based on examining the effect axons of CNS and peripheral nerve tissue: it is disof different drugs with accepted sites of action, tinct from microtubules and disappears after treatalthough it is well known that drugs may have more ment with reserpine. GRAY(1973) described a webthan one such site. An interesting finding was that llke cytoplasmic component (cytonet) surrounding serotonin binding was inhibited by low concent- synaptic vesicles. It is different from microtubules or rations of reserpine microfilaments and inserts into the dense material of M). It has been shown (GLOWINSKI & AXELROD,1966) that reserpine rapidly the membrane. GRAYsuggested that actin-like materdepletes some of the norepinephrine found in the ial is part of the cytonet, a structure he assumes to supernatant fraction, a fact which they interpret as be involved in storage and transport. Although much more work will be needed to eluciindicating a ‘soluble’ storage form distinct from storage vesicles. Our experiment is consistent with the date the nature of the serotonin binding protein and view that the serotonin binding protein may be such its physiological role, the present study suggests that a soluble storage rorm which is sensitive to reserpine. it is a contractile-like protein that may act as a storMARCHBANKS (1967) has observed that reserpine, at age form for serotonin. Further purification of the 5 x 1 0 - 6 ~inhibits , (34%) the binding of serotonin protein and studies of the nature of its interactions to rat brain synaptosomes. However, the use of are in progress. synaptosomes to study reserpine action does not perREFERENCES mit unambiguous interpretation, since the observed E. G., KUNTZMAN R. & SHOREP. effects may be due to action of the drug at a number BRODIEB. B., TOMJCH A. (1957) J . Pharmac. exp. Ther. 119, 461-467. of sites including the post-synaptic receptor. R. W. & MAASJ. W. (1965) Nature, Land. 208, Neither L-LSD nor D-LSD affected binding. This COLBURN 37-41. finding further serves to distinguish the membraneCOSTAE. & REVUELTAA. (1972) Biochem. Plarmuc. 21, bound, LSD-sensitive post-synaptic receptor of sero23852393. tonin separated by affinity chromatography (SHE et DAPRADA M., PLETSCHER A. & TRANZER J. (1971) J . Phya[., 1974; MEHL& WEBER,1974) from the soluble prosiol., Lorid. 217, 677-688. tein we are studying. It is also unlikely that the sero- DESCARRIES L. & DROZB. (1970) J . Cell Biol. 44, 385-399. tonin binding protein is the presynaptic serotonin FAIRBANKS G., STECKT. L. & WALLACHD. F. 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HADASSAH TAMIR,ATHALIAKLEINand MA~JRICE M. RAPFQRT

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TAMIRH. & RAPPORTM. M. (1974) Abstracts, 4th Annual Meeting Soc. Neuroscience, p. 447. TAMIRH. & KUHARM. J. (1975) Brain Res. 83, 169-172. THOAN. B., WOOTENG. F., AXELRODJ. & KOPINI. J. (1972) Proc. nutn. Acad. Sci. U.S.A. 68, 2227-2230. TRANZER J. P. (1972) Nature, Lond. 237, 57-58. M. WESSELSN. K., SPOONERB. S., ASH J. F., BRADLEY O., LUDUENAM. A., TAYLORE. L., WRENNJ. T. & YAMADA K. M. (1971) Science 171. 135-143. S. (1969) Biophys. Soc. Abstr. WEISENBERG R. & TIMASHEFF 9, 174. WILSON L., BRYANJ., RUBYA. & MAZIAD. (1970) Proc. mtn. Acad. Sci. U.S.A. 66, 807-814.

Serotonin binding protein:enhancement of binding by Fe2+ and inhibition of binding by drugs.

Journai OJ hrrurochenisrry. 1976 Vol. 26. pp. 871-878. Pergamon Press. Printed in Great Britain. SEROTONIN BINDING PROTEIN : ENHANCEMENT OF BINDING B...
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