Membrane by Binding of [2-3H]Dihydrotetrabenazine (Adrenal Medulla/Catecholamine Uptake/Scatchard Analysis/Neuroleptics) D

Proc. Natt Acad. Sci. USA Vol. 80, pp. 584-588, January 1983 Neurobiology

Characterization of the monoamine carrier of chromaffin granule membrane by binding of [2-3H]dihydrotetrabenazine (adrenal medulla/catecholamine uptake/Scatchard analysis/neuroleptics) D. SCHERMAN, P. JAUDON*, AND J. P. HENRY Service de Biochimie-Physique, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France Communicated by PierreJoliot, June 28, 1982

ABSTRACT [2-3H]Dihydrotetrabenazine (2-hydroxy-3-isobutyl-9, 10-dimethoxy-1,2,3,4,6,7-hexahydro-llb-H-benzo[a]- quinolizine), a derivative of the neuroleptic tetrabenazine, binds to the membrane ofpurified bovine chromaffin granules. Specific CH30 binding was characterized by Kd and B.., values of 3.1 nM and H 62 pmol/mg ofmembrane protein, respectively. It was reversible, with association and dissociation rate constants of 0.22 x 106 M'1 (CH3)2 s-1 and 1.8 x 10-3 s-1, respectively. Binding sites were present in extracts of medulla but not in corticoadrenal extracts; in the medulla they were restricted to chromaffin granule membranes. [2-3H]Dihydrotetrabenazine binding occurred on the catechol- Tetrabenazine amine carrier ofthe chromaffin granule membrane because it was clearly correlated with inhibition of norepinephrine uptake. In addition, inhibitors and substrates ofthe uptake reaction displaced [2-3H]dihydrotetrabenazine from its binding sites, and their potency as displacers was qualitatively correlated with their IC50 or Km. These results suggest that use of [2-3H]dihydrotetrabenazine CH30' binding might be an interesting technique in the study of the ve- sicular monoamine carrier. The chromaffin granules of the adrenal medulla and the ghosts derived from their membranes accumulate catecholamines by 3H a two-step ATP-dependent process (1-4): (i) inward transloca- [3H]Dihydrotetrabenazine tion of protons by an electrogenic ATP-dependent H' pump (5-10), and (ii) a monoamine carrier driven by the H' electro- MATERIALS AND METHODS chemical gradient (9, 11-15) and specifically blocked by reser- TBZOH was obtained by reduction oftetrabenazine (Fluka) by pine or tetrabenazine (16). Whereas the H' pump has been NaBH4 in methanol (25). [3H]TBZOH (12 Ci/mmol; 1 Ci = 3.7 functionally and structurally related to the mitochondrial ATP- x 1010 Bq) was prepared as described (24). Its purity was pe- ase complex (17, 18), the catecholamine carrier has not yet been riodically checked by TLC and, when necessary, the product characterized. It is assumed to be a protein because the trans- was repurified (24). Stock solutions (80 ,AM) were made in 100 port is temperature sensitive, shows some stereoselectivity (19), mM HCl and diluted in water. and is affected by the histidine-specific reagent diethylpyro- Bovine chromaffin granule membranes were prepared by carbonate (20). The carrier has been solubilized and reconsti- osmotic lysis of granules isolated by centrifugation on a 1.6 M tuted in lipid vesicles (21, 22), but this technique has not al- sucrose layer (26, 27). Membranes were frozen in liquid nitro- lowed purification. The carrier is difficult to gen and were stored at -80°C. They were rapidly thawed at activity assay 370C, centrifuged at 100,000 x g for 15 min, and resuspended because vesicles have to be energized to accumulate catechol- -in 20 mM Hepes KOH buffer containing 0.3 M sucrose. amines, thus introducing possible artifacts. For [3H]TBZOH binding studies, membranes (5-15 ,ug of An alternate approach to the study of the carrier is the use protein per ml) were incubated at 25°C with various concen- ofbinding techniques. We have recently shown that, ofseveral trations of [3H]TBZOH in 0.3 M sucrose/20 mM KOH Hepes, inhibitors (tetrabenazine, reserpine, haloperidol, and chlor- pH 7.5. Bound ligand was measured by filtration on Millipore promazine), tetrabenazine has the most specific interaction with HAWP filters or by centrifugation at full speed in a Beckman the carrier (23). In the present communication, we describe the Airfuge with cellulose propionate tubes. For filtration, 0.2- to binding of 2-[2-3H]hydroxy-3-isobutyl-9, 10-dimethoxy-1,2,3, 2.0-ml aliquots of the incubation mixture were diluted in 4 ml 4,6,7-hexahydro-llb-H-benzo[a]quinolizine (dihydrotetraben- of ice-cold 0.3 M sucrose/10 mM KOH Hepes, pH 7.5, con- azine, [3H]TBZOH), a derivative of tetrabenazine to bovine taining 125 ,AM tetrabenazine and were filtered on filters pre- chromaffin granule membrane. A preliminary account of some viously washed with 4 ml of the same buffer. The filters were of these experiments has been published (24). then washed twice with 4 ml of buffer and their radioactivity

The publication costs ofthis article were defrayed in part by page charge Abbreviation: TBZOH, dihydrotetrabenazine. payment. This article must therefore be hereby marked "advertise- * Present address: Ecole Polytechnique, 91128 Palaiseau Cedex, ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. France. 584 Downloaded by guest on September 25, 2021 Neurobiology: Scherman et aL Proc. Nati. Acad. Sci. USA 80 (1983) 585 was measured by liquid scintillation counting in Aqualuma (Lumac, Schaesberg, The Netherlands). Controls indicated that 1.0 the filters bound 8% of the total radioactivity. The addition of 10 or 125 AM tetrabenazine to the washing buffer decreased 0.8e this value to 5% or 1%, respectively. Alternatively, incubation 2 mixture aliquots (200 were centrifuged for 5 min, and the c _~~~~~ A4) z pellets were washed with 200 /l of ice-cold sucrose. Free 0 0.6~ [3H]TBZOH was determined from the radioactivity of the su- mN pernatant and the pellet washing. The pellets were dissolved I-. in 200 Al of3% (vol/vol) Triton X-100 and the tube was washed c 0.4[ /S,,^~~-- with the same volume of0.3 M sucrose. Solubilized pellet and m0 ,,a tube washings were used to determine bound [3H]TBZOH. Aqualuma was used as the scintillation fluid. In all experiments, 0.2 [3H]TBZOH was used at maximal specific activity (9,000 cpm/ pmol). -J 1 -- I I Protein was measured by the Lowry method. Dopamine / 5 10 15 20 25 30 hydroxylase and cytochrome c oxidase activities were assayed Free TBZOH, nMbi as in refs. 28 and 29, respectively. ATP-dependent norepinephrine uptake was measured as described (4, 30). RESULTS Binding Equilibrium. Tetrabenazine is rapidly metabolized when administered in vivo (31). In contrast, we found that neither bound nor free [3H]TBZOH was modified by incubation with purified chromaffin granule membrane, thus allowing binding studies. The time course of binding was exponential, about 2 hr being required to reach equilibrium at 1 nM [3H]TBZOH. Both specific and nonspecific binding were observed (Fig. 1A). Specific [3H]TBZOH binding sites were sat- 5 urated by either FLM unlabeled TBZOH or 10 ,uM tetraben- 0 azine. Nonspecific binding (linear with [3H]TBZOH concentration) occurred mainly on filters or on centrifugation tubes 0a at the membrane concentration used (Fig. 1). When the specific binding obtained with increasing [3H]TBZOH concentrations was analyzed by the method of Scatchard, linear plots were obtained (Fig. 1B), suggesting the presence ofone class ofbinding sites. From six independent measurements, a mean (± SD) Kd of3.1 ± 0.4 nM and a Bm,. of62 + 8 pmol/mg ofmembrane protein were derived. Such a Yd was only observed at site concentrations lower than the Kd (Fig. 1B Inset). The value ofBm. was confirmed by titration ofthe binding sites. When the spe- Bound, nM cific binding was measured as a function ofmembrane concentration, at a [3H]TBZOH concentration (20 nM) larger than Kd, FIG. 1. Bindingof [3HITBZOH to chromaffin granule membranes. an equivalent point of0.2 mg ofprotein per ml corresponding (A) Saturation isotherm. Membranes (14 ,g of protein per ml) were to the binding of 12 pmol of [3H]TBZOH/ml was determined, incubated with [3HJTBZOH (0.5-30 nM) in the absence (o) or presence (e) of 10 ,uM tetrabenazine for 2 hr in a final volume of 0.5 ml. Bound which indicated a Bma of 60 pmol/mg of protein. [3H]TBZOH was determined by filtration of 0.1-ml aliquots in dupli- Kinetics ofAssociation and Dissociation. [3H]TBZOH bind- cate. Similarresults were obtainedbycentrifugation. Free [3HITBZOH ing to chromaffin granule membrane was reversible. The dis- was measured by centrifugation of 0.2-ml aliquots. Each point is the sociation rate constant was measured directly by addition of 4 mean of four separate experiments. Nonspecific binding (e) was pro- ,uM tetrabenazine to membranes preincubated with 15 nM portional to free [3HITBZOH concentration with proportionality coef- [3H]TBZOH. Underconstant stirring, the dissociation was first- ficients of 0.0060 and 0.0165 for filtration and centrifugation experiments, respectively. Control filtration experiments indicated that 92% order for more than 30 min, characterized by k 1 = 1.8 x 10-3 of the nonspecific binding was associated with filters. Lines are the- s ' (r = 0.997). The association reaction was studied under con- oretical curves for total (solid line) or specific (broken line) binding. ditions such that <10% of the total [3H]TBZOH was bound. (B) Scatchard plot of the specific binding. Data obtained by filtration Under these conditions, free [3H]TBZOH concentration could have been used; from these, Bma, = 65 pmol/mg of protein and Kd be considered to be constant with time, and the association was = 2.7 nM were derived. Centrifugation experiments (data not shown) analyzed as a pseudo-first-order reaction (32) described by the gave B, = 45 pmol/mg of protein and Kd = 3.5 nM. The Hill coef- ficient was 1.0 in both cases. (Inset) Kd measurements as a function of equation membrane concentration. ln([Bej1 []) =-(k [L][L)+ t tion. When In ([Beq]- [B]) was plotted as a function of time, a straight line was obtained with a slope proportional to k, [L] in which [Beq] is the concentration of bound TBZOH at equi- + k-1. By measuring that slope at various [3H]TBZOH con- librium, [B] is the concentration of bound TBZOH at a given centrations (Fig. 2), k-1 and k, were found to be 0.7 x 10-3 S-I time t, [L] is the concentration of free TBZOH, k1 is the rate and 0.22 x 106 M-' s-1, respectively. The dissociation equi- constant of association, and k-1 is the rate constant of dissocia- librium constant calculated from kinetic data was 3.2 or 8.0 nM, Downloaded by guest on September 25, 2021 586 Neurobiology: Scherman et al. Proc. Natl. Acad. Sci. USA 80 (1983) from adrenal medulla and which contained mainly chromaffin granules and mitochondria. They were undetectable when a 0 similar pellet was prepared from adrenal cortex, where chromaffin granules were absent (data not shown). The localization 4 of the binding sites in chromaffin granule membranes was further demonstrated by centrifugation of these membranes on a linear sucrose gradient and analysis of the fractions for

C., 0 [3H]TBZOH binding (Fig. 3). The binding site distribution was coincident with that of dopamine /3-hydroxylase activity, a (0 marker of chromaffin granule membrane, and not with mitochondrial cytochrome c oxidase. 0 2 The relationship between catecholamine transport and TBZOH binding was then examined. A good correlation was observed between the fraction of [3H]TBZOH binding sites occupied and the inhibition by TBZOH of ATP-induced [3H]norepinephrine uptake (Fig. 4). Provided that TBZOH had the same affinity for the membrane as its tritiated derivative (an hypothesis which is verified by the displacement experi- 5 10 15 20 ment of Fig. 5), saturable [3H]TBZOH binding thus appeared TBZOH concentration nM to occur on the catecholamine carrier. It should be noted that, for this experiment, binding and uptake were measured under FIG. 2. Effect of free [3H]TBZOH concentration on kinetics of as- the same conditions-i.e., in presence of ATP and 5 ,uM nor- sociation. The time course of association between membranes (18 ,g of protein per ml) and [3H]TBZOH was studied over a ligand concen- epinephrine. Neither energization ofthe membrane by ATP nor tration range of 2.4 to 20 nM by the filtration technique. Association addition of 5 ,uM substrate affected [3H]TBZOH binding (see at equilibrium was determined after 2 hr of incubation; under the con- legends to Figs. 1 and 4; Table 1 and Fig. 5). ditions used, <20% of the total ligand was bound to membranes. For Pharmacology of [3H]TBZOH Binding. [3H]TBZOH was each ligand concentration, initial kinetics (0-180 sec) of TBZOH bind- displaced from its binding sites by the inhibitors ofmonoamine ing was linearized according to the equation of a pseudo-first-order uptake known to affect the carrier (Fig. 5). The Kd values of reaction, as explained in the text. The figure is a secondary plot of the slope of the straight lines thus obtained as a function of [3H]TBZOH these compounds were determined from their EC50 values and concentration. Rate constants k, and k-1 were derived from the slope compared with their ICs5 for ATP-induced norepinephrine up- and the y-axis intercept. Binding at equilibrium on the same prepa- take, measured at 5 gM norepinephrine (Table 1). The calcu- ration and under the same conditions gave Kd = 3.2 n]M and BmX = lated kd was of the same order as the IC50 for tetrabenazine, 56 pmol/mg of protein. dihydrotetrabenazine, haloperidol, and chlorpromazine. On the other hand, reserpine, which occupies the same qualitative depending on the k, measurement used (association or displace- position in the two sequences, was characterized by a Kd value ment experiment). These values agree reasonably well with 5 times higher than the IC50. Substrates of the uptake reaction, those determined by equilibrium measurements. such as serotonin or norepinephrine, also displaced [3H]TBZOH Relationship Between the [3H]TBZOH Binding Sites and from its binding sites. Under conditions of active transport- the Catecholamine Carrier. Saturable [3H]TBZOH binding i.e., in the presence ofATP and MgSO4-K4 values of240 and sites could be detected on a 1,000-27,000 X g pellet prepared 1,200 ,uM were derived from the EC,% for serotonin and nor-

0.1

0 !\ i ./ u .% !( x 0 i.

FIG. 3. Distribution of [3H]TBZOH

0 binding after isopycnic centrifugation of membranes on linear 0.45-1.45 M sucrose gradient. Chromaffin granule membranes (3 mg of protein) were centrifuged as in ref. 18. The binding of [3H]TBZOH (50 nM) was measured in the absence (a) or presence (o) of 5 uM tetrabenazine by the filtration technique, after incubation of 5-,lI aliquots in a final volume of 250 ,.dA Cytochrome c oxidase (o) and dopamine f3-hydroxylase (+) activities are

I , - markers of mitochondria and chromaffin 40 granules, respectively. Total collected ac- Fraction tivities have been normalized to unity. Downloaded by guest on September 25, 2021 Neurobiology: Scherman et aL Proc. NatL Acad. Sci. USA 80 (1983) 587 Table 1. Pharmacology of [3H]TBZOH binding Compounds EC5c, nM Kd, nM IC50 or K.* nM 0 Tetrabenazine 1.8 1.3 3.2 -a TBZOH 4.1 2.9 3.7 Reserpine 238 168 28 n. Haloperidol 1,071 750 550 CU Chlorproma- c _ 50 1 zine 1,890 1,330 2,500 Serotonin 345 x 103 240 x 103 (4.3 x 103) C / Norepinephrine 1,720 x 103 1,200 x 103 135 x 103t (12.5 x 103) EC50 values were obtained from Fig. 5. Kd values were calculated from EC50 as described in ref. 33. ICrc values for tetrabenazine, z / TBZOH, reserpine, haloperidol, and chlorpromazine were obtained from measurements of the inhibition of ATP-dependent norepineph- 0 50 100 rine uptake as described in the legend to Fig. 4. Uptake and displace- mentexperiments were performed on the same membrane preparation % maximal TBZOH binding and under the same conditions. * In parentheses are Km values measured at pH 8.5 (34). FIG. 4. Correlation between [3HITBZOH site occupancy and in- tK. measured at pH 7.5 (30). hibition of norepinephrine uptake. Membranes (11 /Ig of protein per ml) were preincubated for 2 hr at 200C with either 5 ,uM l-norepi- site is on monoamine carrier nephrine and various concentrations (1, 2, 3, 5, 8, 15, 30, and 60 nM) binding located the is supported of [3H]TBZOH (binding experiments) or the same concentrations of 1- by three lines of evidence: (i) [3H]TBZOH binds to extracts of [7,8-3H]norepinephrine and TBZOH (uptake experiments). ATP (2.5 medulla but not of cortex, and, in the medulla, binding is re- mM) and MgSO4 (1.25 mM) were then added. [3H]TBZOH binding stricted to chromaffin granule membrane (Fig. 3); (ii) [3H]TBZOH (mean of four measurements) was determined by the filtration tech- binding is clearly correlated with norepinephrine uptake inhi- nique after a further 30-min incubation. Kd and B. were 3.2 nM and bition (Fig. 4); and (iii) inhibitors and substrates of the uptake 60 pmol/mg of protein, respectively. Results were expressed as per- reaction its centage of occupied sites. The rate of 1-[7,8-3H]norepinephrine uptake displace [3H]TBZOH from binding site (Fig. 5). (mean of two measurements) was determined from uptakes at 0, 15, Their potency as displacers is qualitatively correlated with their and 30 min and was expressed as a percentage of the rate of transport IC50 or Km and for most ofthem the agreement is quantitatively in the absence of TBZOH (94 pmol/min per mg of protein). correct (Table 1). For norepinephrine and serotonin, the dif- ference observed between kd and Km might be explained by the epinephrine, respectively. Although these kd values differed hydrophobic nature of tetrabenazine which might attract the significantly from the reported Km (30, 34) it should be noted carrier in the lipidic bilayer, thus limiting the competition by that serotonin, the more potent displacer, is known to have a the more hydrophilic substrates. For reserpine, such an hy- Km 1/3 that of norepinephrine. pothesis cannot be proposed and we have no explanation for the discrepancy between IC50 and Kd. From a methodological point of view, [3H]TBZOH appears DISCUSSION to be a valuable tool because nonspecific binding is limited and The present study shows that the catecholamine carrier ofchro- affinity for the granule monoamine carrier is high. It compares maffin granule membrane can be reversibly labeled by the neu- favorably to [3H]imipramine, which binds to rat brain mem- roleptic derivative [3H]TBZOH. The contention that the drug- branes with a Kd of 2-8 nM, presumably on the serotonin up-

100

0EWU \t X0 FIG. 5. Displacement of [3H]- TBZOH. Membranes (7 ,ug of protein per ml, corresponding to 0.45 0~~~~~ nM [3H]TBZOH binding sites) were incubated for 60 min with 1 nM [3HITBZOH and various concentra- 60 tions of tetrabenazine (A), TBZOH CV (n), reserpine (e), haloperidol (v), C chlorpromazine (o), serotonin (o), or .0 l-norepinephrine (A). Incubations 40 with substrates (norepinephrine and z serotonin) also included 2.5 mM o \o ATP and 1.25 mM MgSO4. Bound N0 -A ligand was determined by filtration in duplicate. Maximal binding was \o \ A 0.13 nM. Nonspecificbinding(

Displacer concentration , log nM 1.0. Downloaded by guest on September 25, 2021 588 Neurobiology: Scherman et aL Proc. Nad Acad. Sci. USA 80 (1983) take system of neuronal membranes (35). Nevertheless, for 3. Taugner, G. (1971) Naunyn-Schmiedebergs Arch. Pharmakol. [3H]TBZOH binding experiments, care should be taken to 270, 392-406. perform Kd determinations at a site concentration lower than 4. Phillips, J. H. (1974) Biochem. J. 144, 311-318. Kd 5. Pollard, H. B., Zinder, O., Hoffman, A. G. & Nikodejevic, 0. (36). With chromaffin granule membranes, a protein con- (1976)J. Biol Chem. 251, 4544-4550. centration of 3-15 pug/ml (corresponding to 0.2-0.9 nM sites) 6. Casey, R. P., Njus, D., Radda, G. K. & Sehr, P. A. (1977) Bio- is dictated by the low Kd (3 nM) and more specifically by the chemistry 16, 972-977. high Bma originating from the use of purified membranes of 7. Flatmark, T. & Ingebretsen, 0. C. (1977) FEBS Lett. 78, 53-56. specialized organelles. Such a limitation in the concentration 8. Philipps, J. H. & Allison, Y. P. (1978) Biochem. J. 170, 661-672. of membrane to be used is not usually seen in crude systems 9. Johnson, R. G. & Scarpa, A. (1979) J. Biol Chem. 254, 3750- 3760. in which Bma is 2-3 orders of magnitude lower. For instance, 10. Scherman, D. & Henry, J. P. (1980) Biochim. Biophys. Acta 599, rat brain imipramine binding sites ofhorse striatum have a Bma 150-166. of 60-300 femtomol/mg of protein (35). 11. Holz, R. W. (1978) Proc. Nati Acad. Sci. USA 75, 5190-5194. The use of high concentrations of chromaffin granule mem- 12. Kanner, B. I., Sharon, I., Maron, R. & Schuldiner, S. (1980) branes resulted in imprecise determinations offree [3H]TBZOH. FEBS Lett. 111, 83-86. In addition, the presence of a radiolysis degradation product 13. Knoth, J., Handloser, K. & Njus, D. (1980) Biochemistry 19, 2938-2942. which did not bind to membranes (data not shown) resulted in 14. Scherman, D. & Henry, J. P. (1980) Biochim. Biophys. Acta 601, overestimating the free ligand concentration at high membrane 664-677. concentration (36, 37) and hence in overestimating Kd (Fig. 1B 15. Apps, D. K., Pryde, J. G. & Phillips, J. H. (1980) FEBS Lett. 111, Inset) (24). The use, in the present work, oflow membrane con- 386-390. centrations accounts for the low ICw values oftetrabenazine and 16. Pletscher, A. (1977) Br. J. Pharmacol 59, 419-424. TBZOH. Determinations performed at high membrane con- 17. Apps, D. K. & Schatz, G. (1979) Eur.J. Biochem. 100, 411-419. 18. Roisin, M. P., Scherman, D. & Henry, J. P. (1980) FEBS Lett. centrations (0.1-0.8 mg ofprotein per ml) are likely to be only 115, 143-147. titrations of the transporter drug binding sites. Thus, IC50 val- 19. Pletscher, A. (1976) BulL Schweiz. Akad. Med. Wiss. 32, 181-190. ues of 100 (23) and 150 nM (16) for tetrabenazine and of40 (16), 20. Isambert, M. F. & Henry, J. P. (1981) FEBS Lett. 136, 13-18. 66 (38), 100 (23), and about 200 nM (39) for reserpine have been 21. Maron, R., Fishkes, H., Kanner, B. I. & Schuldiner, S. (1979) reported. When IC50 is measured correctly, tetrabenazine is the Biochemistry 18, 4781-4785. more potent inhibitor of monoamine uptake by chromaffin 22. Isambert, M. F. & Henry, J. P. (1981) Biochimie 63, 211-219. 23. Scherman, D. & Henry, J. P. (1980) Biochem. Pharmacol 29, granules. 1883-1890. Some speculations can be made on the properties of the 24. Scherman, D., Jaudon, P. & Henry, J. P. (1981) C. R. Hebd. Se- monoamine carrier. By using a Vm. of2,100 pmol/min per mg ances Acad. Sci. Ser. D 293, 221-224. of protein for ATP-dependent uptake of norepinephrine by 25. Brossi, A., Chopard dit Jean, L. H. & Schnider, 0. (1958) Helv. chromaffin granule ghosts, a turnover number of35 molecules Chim. Acta 14, 1793-1800. ofnorepinephrine per min can be derived. This value is lower 26. Smith, A. D. & Winkler, H. (1967) Biochem. J. 103, 480-482. 27. Giraudat, J., Roisin, M. P. & Henry, J. P. (1981) Biochemistry than that reported for carriers of eukaryote organelles, such as 19, 4499-4505. the nucleotide carrier of mitochondria [1,600-2,000 (40)]. As- 28. Wallace, E. F., Krantz, M. J. & Lovenberg, W. (1973) Proc. Nati suming that 25% ofthe granule proteins are membrane bound Acad. Sci. USA 70, 2253-2255. and that 1 mg of granule protein contains 0.4 x 1012 granules 29. Smith, L. (1955) in Methods ofBiochemical Analysis, ed. Glick, [calculated from an internal volume of4.3 ,u1/mg ofprotein (8) D. (Wiley Interscience, New York), Vol. 2, pp. 427-436. and a mean diameter of270 nm a granule would have 22 30. Scherman, D. & Henry, J. P. (1981) Eur. J. Biochem. 116, 535- (41)], 539. molecules of catecholamine transporter. This value is of the 31. Schwartz, D. E., Bruderer, H., Rieder, J. & Brossi, A. (1966) same order ofmagnitude as that proposed for the ATPase com- Biochem. Pharmacol 15, 645-655. plex [10-20 (41)]. If a molecular weight of 30,000 is assumed 32. Kitabgi, P., Carraway, R., Van Rietschoten, J., Granier, C., for the carrier, similar to that of the mitochondrial nucleotide Morgat, J. L., Menez, A., Leeman, S. & Freychet, P. (1977) carrier (40), the transporter would represent 0.2% ofthe mem- Proc. Natl Acad. Sci. USA 74, 1846-1850. brane proteins. The carrier thus does not appear to be a major 33. Jacobs, S., Chang, K. J. & Cuatrecasas, P. (1975) Biochem. Bio- phys. Res. Commun. 66, 687-692. component of the membrane, even though this membrane is 34. Kanner, B. I., Fishkes, H., Maron, R., Sharon, I. & Schuldiner, highly specialized. S. (1979) FEBS Lett. 100, 175-178. 35. Palkovitz, M., Raisman, R., Briley, M. & Langer, S. Z. (1981) We are indebted to M. Dupuis from the slaughterhouse of Mantes Brain Res. 210, 493-498. (Yvelines) for collecting bovine adrenals. This work was supported by 36. Chang, K. J., Jacobs, S. & Cuatrecasas, P. (1975) Biochim. Bio- contracts from the Centre National de la Recherche Scientifique, the phys. Acta 406, 294-303. Delegation Generale A la Recherche Scientifique et Technique, and the 37. Builder, S. E. & Segel, I. H. (1978) AnaL Biochem. 85, 413-424. Institut National de la Sante et de la Recherche Medicale. 38. Jonasson, J., Rosengren, E. & Waldeck, B. (1964) Acta Physiol Scand. 60, 136-140. 1. Kirschner, N. (1962)J. Biol Chem. 237, 2311-2317. 39. Philipps, J. H. (1974) Biochem.J. 144, 319-325. 2. Carlsson, A., Hillarp, N. A. & Waldeck, B. (1962) Acta PhysioL 40. Vignais, P. V. (1976) Biochem. Biophys. Acta 456, 1-38. Scand. SuppL 59 215, 1-38. 41. Winkler, H. & Westhead, E. (1980) Neuroscience 15, 1803-1823. Downloaded by guest on September 25, 2021