Proc. Natl Acad. Sci. USAVol. 80, pp. 3508-3512, June 1983Neurobiology

Solubilization and characterization of high-affinity [3H]serotoninbinding sites from bovine cortical membranes

(receptor solubilization/central nervous system/Sephacryl S-300/glycerol gradient sedimentation)

SCOTT R. VANDENBERG*, ROBIN L. ALLGREN, RICHARD D. TODD, AND ROLAND D. CIARANELLOtLaboratory of Developmental Neurochemistry, Division of Child Psychiatry and Child Development, Department of Psychiatry and Behavioral Sciences, StanfordUniversity School of Medicine, Stanford, California 94305

Communicated by Richard F. Thompson, February 9, 1983

ABSTRACT High-affinity [3H]serotonin binding activity hasbeen solubilized from bovine cerebral cortical membranes by us-ing Triton X-100, Tween-80, and octyl-fi-D-glucopyranoside. Thismixture of detergents solubilizes the high-affinity [3H]serotoninbinding activity present in crude membrane preparations with re-tention of 75-90% specific binding. The detergent mixture waschosen because it can easily be removed from the solubilized frac-tion by dialysis and polystyrene bead adsorption, thus permittingfurther purification and isolation of the binding sites. Saturationanalysis reveals multiple components of high-affinity [3H]sero-tonin binding. In crude bovine cortical membranes, at least twobinding components are present. A higher-affinity binding com-ponent, as defined from curvilinear Scatchard plots, has a Kd for[3H]serotonin of 1-3 nM, whereas a lower-affinity component hasa Kd of 10-20 nM. In the solubilized preparation, only a singleclass of binding sites is apparent, with a Kd of 50-100 nM. Re-moval of detergents by dialysis and polystyrene bead adsorptionresults in restoration of the curvilinear Scatchard plot with ap-parent Kds similar to those observed in crude membrane prep-arations and with increased Bm.. values for each component. [3H]-Serotonin binding activity in the solubilized preparation is stableto Sephacryl S-300 column chromatography and to glycerol gra-dient sedimentation. Saturation analysis of the peak binding frac-tions from both these procedures once again yields curvilinearScatchard plots, indicating that the multiple high-affinity bindingcomponents are preserved and migrate together. The molecularweight, Stokes radius, and frictional coefficient of the binding site(s)have been calculated. After detergent removal the solubilized ma-terial shows many of the characteristics usually attributed to SIreceptors, such as high affinity for r3H]serotonin and its analogsand low affinity for serotonin antagonists.

Abundant evidence exists that serotonin is an important centralneurotransmitter involved in depressive disorders (1), pain per-ception (2), normal mood and affect, infantile autism (3), andsleep (4). Radioligand binding assays have been used to studythe receptors that may mediate these actions. According to Pe-routka et aL, serotonin binding sites may be classified as SI orS2 (5), based on their affinity for different classes of ligands.The S1 binding site has ahigh affinity for [3H]serotonin and [3H]-lysergic acid diethylamide (nanomolar Kd) and a low affinity forspiperone (micromolar Kd), is GTP-sensitive, and is thought tobe positively coupled to a serotonin-sensitive adenylate cyclase.The S2 receptor has high affinity for [3H]lysergic acid diethyl-amide and [3H]spiperone and a low affinity for serotonin.

Other studies have suggested that division into SI and S2 re-ceptors may be too limiting. Multiple serotonin receptor sub-types have been described with (i) different brain regional lo-

calizations (6); (ii) different physiologic functions (7, 8); (iii)ontogenetic variations (9); (iv) modulation by guanine nucleo-tides (10); and (v) induction of glycogenolysis (11). Thus, manyquestions regarding the role of various serotonin receptor sub-types, their mode of action, their physical characteristics, andtheir relationship to each other remain unanswered.The ability to solubilize and purify serotonin binding pro-

teins would greatly facilitate the study of these systems. Ilienet aL have reported that lysolecithin solubilizes a serotoninbinding site from rat cortex, which has high affinity for [3H]spi-perone and appears to have properties expected of an S2 bind-ing site (12-14). However, lysolecithin treatment failed to solu-bilize high-affinity binding sites for [3H]serotonin.

The present report describes the solubilization of high-af-finity [3H]serotonin binding activity from bovine frontal cor-tical membranes. This procedure, employing a mixture of de-tergents that can be removed from the solubilized preparation,gives a very high solubilization yield and preserves high spe-cific binding. Preliminary pharmacologic and physical char-acterizations are presented.

MATERIALS AND METHODSPreparation of Bovine Cerebral Cortical Membranes. Bo-

vine cerebral cortex was obtained fresh at a local slaughter-house. Tissues were dissected and packed in dry ice before transitto the laboratory. All homogenization, solubilization, and pu-rification steps were carried out at 0-4C unless otherwise stated.Frozen bovine frontal cortex was thawed in 0.32 M sucrosebuffered with 2.5mM Tris HCl at pH 7.4 and was homogenizedas a 10% (wt/vol) suspension in a Teflon glass grinder. The ho-mogenate was centrifuged at 580 x g for 10 min. The pellet wasdiscarded and the supernatant was centrifuged at 50,000 X gfor 20 min. washed in a small volume of 0.32 M sucrose, dilutedwith 25-28 vol of distilled water, mixed, and recentrifuged at50,000 X g for 20 min. The pellets were resuspended in a smallvolume of 0.32 M sucrose solution and incubated for 15 min at300C. Approximately 10-15 vol of cold distilled water were thenadded and the suspension was centrifuged at 40,000 X g for 25min. The opalescent grey-white pellet was resuspended in 3-4 vol of distilled water, sonicated (25 W, 5 sec), diluted withdistilled water, and centrifuged at 50,000 X g for 25 min. Thepellet was resuspended in distilled water, snap-frozen, and storedat -80°C. This fraction, which consisted primarily of lysed syn-aptosomal membranes, is designated the "crude" membranefraction.

Protein Determinations. Protein determinations were per-formed by using the Amidoschwartz stain technique (15) or by

* Present address: Div. of Neuropathology, Dept. of Pathology, Univ.of Virginia School of Medicine, Charlottesville, VA 22908.

t To whom reprint requests should be addressed.

3508

The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. NatL Acad. Sci. USA 80 (1983) 3509

the Lowry technique (16) after determining that the detergentsolution did not interfere with these assays.

3HiSerotonin Binding Assay in Crude Membranes. [3H]-Serotonin binding to crude cortical membranes was deter-mined by modification of the method of Peroutka et aL (10).The final volume of the binding assay was 200 1.l. Preincu-bation was carned out at 30WC for 12-15 min in 50 mM Tris HCl/6.56 mM CaC12, pH 7.2. Pargyline HCI (10 ,M final concen-tration) and the appropriate radiolabeled and unlabeled ligandswere then added. In initial experiments, the tubes were in-cubated at 300C for 20 min. In later experiments, assays wereperformed at 40C for 14-17 hr so that assay conditions in crudemembranes would be conducted under conditions comparableto those in the solubilized preparation (see below). Estimatesof equilibrium (Kds) and receptor site concentrations wereidentical under both assay conditions. After incubation, 5 ml ofcold binding buffer was then added, followed by vacuum-fil-tration onto Toyo GB1OOR glass filters. The filters were rinsedtwice with 5 ml of binding buffer and resuspended in Cytoscint(WestChem Products, San Diego, CA) for liquid scintillationspectrometry. Specific binding for [3H]serotonin varied from60% to 80% and was defined as that displaced by unlabeled 1.0-2.5 AiM serotonin. In all binding assays, triplicate determina-tions of total and nonspecific binding were made.Membrane Solubilization. Bovine cortical membranes were

centrifuged at 57,000 x g at 40C for 30 min. The pellet wasresuspended by addition of a solution of 4.0% (wt/vol) octyl-,B3D-glucopyranoside (Calbiochem-Behring)/3.0% Triton X-100(Sigma)/1.0% Tween 80 (Sigma) in 50 mM Tris HCl buffer andby sonication. The detergent suspension was incubated withperiodic agitation for 1 hr at 20TC, followed by incubation at OYCfor 45 min. The suspension was centrifuged at 100,000 X g (45-60 min) at 40C. The supernatant was considered as the solu-bilized fraction and was stored at -80°C.

Removal of Detergents from the Solubilized Fraction. Fordialysis, Spectapor tubing (Mr cutoff 12,000-14,000) was boiledin 0.5 M Na2EDTA and then thoroughly rinsed. The solubi-lized fraction was dialyzed at 40C overnight against 5 or 50 mMTris HCl buffer (pH 6.9-7.2). Residual detergent was then re-moved by batch treatment with Bio-Beads SM-2 at 4°C by using0.64 g of beads per ml of dialyzed preparation (17). After gentlemixing for 2 hr, an additional 0.50 g of beads per ml of dialyzedpreparation was added and was mixed for 1.5 hr. The dialyzed-adsorbed material was then stored at -80°C.

Assay for [3H]Serotonin Binding to Solubilized Membranes.The total binding assay volume was 200 ,l per tube. Aliquotsof the soluble fraction were placed into ice-cold binding buffer(50 mM Tris.HCI/6.56 mM CaC2/10 AM pargyline HC1, pH7.4) and the tubes were then supplemented with the appro-priate radiolabeled and unlabeled ligands. Each tube was flushedwith dry nitrogen, tightly stoppered, and incubated in the darkat 0°C for 14-17 hr. Association and dissociation kinetics werefirst determined and it was concluded that the system hadreached equilibrium under the assay conditions used. Specific[3H]serotonin binding in this fraction was 80-90%.

After incubation, 0.5 ml per tube of 25% (wt/vol) polyeth-ylene glycol in 50 mM Tris-HCl/6.56 mM CaCl2, pH 7.1, and0.2 ml of 0.1% (wt/vol) bovine gamma globulin (50mM Tris HClat pH 7.1) were added (18). The tubes were then incubated at0°C for 7-10 min before filtration and rinsing through ToyoGB100R glass filters. Specific binding was defined as describedfor crude membranes.

Sephacryl S-300 Chromatography. A column of SephacrylS-300 (80 x 1.6 cm) was packed and equilibrated at a flow rateof 24-30 ml/cm3 per hr by using the same buffer in which [3HJ-serotonin binding assays of the soluble fraction were con-

ducted. The buffer was supplemented with 0.20 M NaCl and0.2% (wt/vol) octyl-,BD-glucopyranoside. Protein standards ofcytochrome c (Mr 12,000), bovine serum albumin (Mr 66,000),bovine gamma globulin (Mr 169,000), catalase (Mr 244,000), andthyroglobulin (Mr 669,000) were used for column calibration.Three-milliliter fractions were assayed for [3H]serotonin bind-ing by using the technique described above for the solubilizedmembranes.

Glycerol Gradient Sedimentation. Samples of solubilizedmembranes (30-300 u1) were layered onto 5 ml of a 10-30%linear glycerol gradient containing solubilization buffer. Thesamples were centrifuged for 46 hr at 35,000 rpm in a BeckmanSW 39 rotor. Fractions were collected from the bottom of thetubes and assayed for [3H]serotonin binding. Sedimentationcoefficients were estimated by using bovine serum albumin asa sedimentation marker (assuming s2o,, = 4.6). There were noprotein concentration effects for the solubilized membranes overthe range of 0.1-1.0 mg of protein per gradient. Bovine serumalbumin had the same migration in the presence and absenceof detergents. Glycerol did not interfere significantly with [3H]-serotonin binding. Samples for saturation analysis were di-alyzed and treated with polystyrene beads as for solubilizedmembranes. Recoveries of [3H]serotonin binding activity priorto detergent removal were between 100% and 300% of thoseobserved in the solubilized membranes (assayed at 3-5nM [3H]-serotonin).

RESULTSDetergent Solubilization of Cortical Membranes. Before

selecting the combination used, several detergents were triedeither singly or in combination. These included digitonin, ly-solecithin, Triton X-100, Tween 80, Brij-58, NaDodSO4, andoctyl-,B-D-glucopyranoside. The intention was to select deter-gents that could be removed by dialysis or polystyrene beadadsorption, or both. The combination of Triton X-100, Tween80, and octyl-,-D-glucopyranoside gave a high degree of solu-bilization with the highest specific binding. This mixture sol-ubilized up to three times the number of high-affinity serotoninbinding sites (at 3 nM [3H]serotonin) as were measured in thecrude cortical membrane preparation, with 75-90% specificbinding (data not shown).

Binding Properties of [H]Serotonin in Crude Bovine Cor-tical Membranes. The binding of [3H]serotonin to crude bo-vine cortical membranes gives a curvilinear Scatchard plot, ashas been reported (19). Fig. 1 Top shows a typical curve of [3H]-serotonin binding to crude membranes. Assuming a two-bind-ing site model, replication of this curve (five determinations)gives ranges of Kd values for these sites of 1-3 and 10-20 nM,respectively. These values were determined by extrapolation ofthe tangents of the curvilinear Scatchard plots to their axes.Graphical computation by the method of Klotz and Hunston(20), again assuming a two-site model, was also performed. Cal-culated affinity constants were 1.4 and 28.3 nM for the twobinding components. Estimated Bm,,values for the two bindingsites were 0.27 pmol/mg of protein for the higher-affinity com-ponent and 2.35 pmol/mg of protein for the lower-affinitycomponent.

[3H]Serotonin Binding to Solubilized Membranes Before andAfter Detergent Removal. Saturation analysis of the solubi-lized fraction showed a shift to a monophasic curve in the pres-ence of detergents, with an apparent Kd of 50-100 nM (Fig.1 Middle). Removal of detergents by dialysis and polystyrenebead adsorption returns the binding kinetics of the solubilizedfraction to the curvilinear high-affinity activity observed in thecrude membranes (Fig. 1 Bottom). Binding parameters werecalculated to be: KdS = 1.3 nM and 22 nM and Bm,,,s = 0.78

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FIG. 1. Saturation analysis of [3H]serotonin binding. [3H]Sero-tonin binding was measured and plotted as a Scatchard curve. (Top)Crude membranes. (Middle) Soluble fraction. (Bottom) Soluble/deter-gent-firee fraction. Binding kinetics are described in the text. Points arethe average of triplicate determinations. Computer resolution of thecurvilinear Scatchard plots into high- and low-affinity components isshown by the dashed lines shown in Top andBottom. 5-HT, 5-hydroxy-tryptamine (serotonin).

and 7.29 pmol/mg of protein for the high- and lower-affinitybinding components, respectively. [3H]Spiperone at 0.25 nMfailed to demonstrate any specific binding to the detergent-freefraction, indicating that solubilization of an active S2 receptorpreparation did not occur. The high-affinity binding compo-nent (Kd = 1-3 nM) was labile to prolonged storage at -800C.After several weeks at this temperature, only the binding com-ponent with a Kd of 10-20 nM was observed.

Table 1 summarizes the [3H]serotonin binding characteris-tics of the solubilized and detergent-removed fractions. Theapparent number of binding sites in the solubilized and de-tergent-free fractions was consistently higher than the valuesfor the crude membrane preparation. The Scatchard analysesyielded estimates of this increase that were somewhat moreconservative than those obtained when binding assays wereperformed at a single [3H]serotonin concentration.

Competition Studies. Table 2 summarizes the results ofcompetition studies of [3H]serotonin against serotonin and 5-methoxytryptamine, two serotonin agonists, and cinanserin, aserotonin antagonist, for both crude membranes and the sol-ubilized/detergent-free fraction. There is little change in IC50values between the two fractions, indicating that the propertiesof the binding site(s) are retained after solubilization and de-tergent removal. The low affinity of cinanserin for the solu-bilized proteins and the apparent inability of [3H]spiperone tobind to the solubilized fraction indicates that this proceduremay not solubilize S2 binding activity. Alternatively, S2 bindingmay not be stable in this combination of detergents.

Sephacryl S-300 Chromatography. High-affinity [3H]sero-tonin binding activity was preserved after chromatography onSephacryl S-300. Fig. 2 shows the results of a representativecolumn run. A single major peak of binding activity emergesfrom the column with an apparent Mr of 98,000, as assessed bycochromatography with several globular-protein standards ofknown molecular weight. Saturation analysis of the major frac-tion after dialysis shows retention of the curvilinear bindingkinetics (Inset).

Glycerol Gradient Sedimentation and Calculated PhysicalProperties. High-affinity [3H]serotonin binding activity was alsopreserved after sedimentation in glycerol gradients. Fig. 3 isa representative [3H]serotonin binding profile from a sedi-mentation experiment showing that the solubilized [3H]sero-tonin binding activity sediments as a single symmetrical peakwith a sedimentation coefficient (mean ± SEM) of 3.5 ± 0.3(n = 7). The peak fractions were combined and the detergentswere removed. Saturation analysis of the detergent-free peakfraction gave a curvilinear Scatchard plot, as seen also in Figs.1 and 2. No specific binding of [3H]spiperone (at 0.25 nM) wasobserved. Sedimentation in Sephacryl S-300 column buffer gaveidentical results. However, sedimentation in no detergent gaves20,w of about 11.5 (data not shown).We employed the procedure of Siegel and Monty (21), in

which the gel-chromatographic and glycerol sedimentation dataare combined, to obtain a molecular weight corrected for par-ticle asymmetry. Our calculations assumed a single [3H]sero-tonin binding species. This is a reasonable approximation, be-cause even if more than one distinct binding species exist, theyare probably similar in size and shape, as they comigrate withtwo different physical separation procedures. The correctedphysical parameters for the high-affinity [3H]serotonin bindingsite were calculated to be: Mr, 58,000; Stokes radius (Ro), 3.9nm; frictional coefficient (f/fo), 1.53; and axial ratio, 10 (as-suming a prolate ellipsoid shape). The high frictional coefficientand axial ratio indicate that the binding site(s) has the propertiesof highly asymmetric proteins. Large frictional coefficients havebeen reported for another membrane-bound complex, the en-ergy-transducing mitochondrial ATPase, and may be due tobinding of detergents and phospholipids and the structuring ofsolvent molecules around hydrophobic sections of the proteins(22).

DISCUSSIONThe present study demonstrates the solubilization of high-af-finity [3H]serotonin binding activity from bovine cerebral cor-

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Table 1. [3H]Serotonin binding characteristics of crude membrane, solubilized, and solubilized/detergent-free fractions

Ligand TotalProtein, Be, bound, recovery, %

Fraction mg/ml pmol/mg pmol/ml pmol recovery Kd, nMCrude membrane 7.5 0.6 4.5 18.1 100 1-3

2.7 20.3 81.2 100 10-15Solubilized 2.8 34.7 97 388 - 75Solubilized/ 2.1 2.8 6.0 24.1 133 1-3

detergent-free 21.0 44.1 76.4 217 10-20

Fractions were prepared as described in Materials and Methods. The [3H]serotonin concentration wasvaried from 0.7 to 90 nM. Total recovery refers to recovery per 4-ml batch of initial membrane fraction.% recovery refers to yield of binding activity as compared to crude membranes. Values are the averagesof five experiments.

tical membranes. A mixture of the nonionic detergents TritonX-100, Tween 80, and octyl-p-D-glucopyranoside effectivelysolubilizes [3H]serotonin binding activity in high yield and withhigh specific binding. This detergent mixture is also conve-nient because it can be easily removed from the solubilizedfraction by dialysis and polystyrene bead adsorption, thus per-mitting further purification and characterization of the bindingsite(s).

Saturation analysis of crude bovine cortical membranes givescurvilinear Scatchard plots, indicating the probable existenceof multiple binding species. After solubilization, the Scatchardplot becomes monophasic and the Kd for [3H]serotonin in-creases. Removal of the detergents from this solubilized frac-tion results in the return of multiple-component high-affinitybinding activity.

Questions remain as to the relationship between these com-ponents of [3H]serotonin binding. At least four possibilities ex-ist:

(i) The binding sites may represent interconvertible forms ofa single protein moiety, as appears to be the case for some opi-ate and y-aminobutyric acid (GABA) receptors (23, 24). For thesereceptors, this interconversion is mediated by sodium ions. Ourpreliminary data indicate that addition of sodium to the assaymedium in concentrations as high as 200 mM does not cause asimilar type of interconversion.

(i) The components of high-affinity [3H]serotonin bindingmay represent different serotonin binding sites present on asingle protein. The different stabilities of the sites to storageand Bmax values argue against this possibility.

(iii) The multiple binding components may represent phys-ically independent sites. However, if physically distinct typesof high-affinity serotonin binding molecules exist, they are likelyto be of similar size and shape, because they comigrate duringexclusion chromatography and velocity sedimentation. Pre-vious studies have suggested the binding species may be dis-tinct and have implied that the two components can be partiallyseparated by subcellular fractionation (19).

Table 2. Displacement competition (ICro) of[3Hlserotonin binding

ICro, nMCrude Solubilized/

Compound membrane detergent-freeSerotonin 3 105-Methoxytryptamine 12 23Cinanserin 2,100 2,300

Binding assays were performed as described in Materials and Meth-ods. The [3H]serotonin concentration was 5-8 nM. Values are the av-erages of three experiments.

(iv) The receptors may exist in the membrane as an aggregateof identical receptor units that interact in a negatively coop-erative manner. Detergent solubilization may disaggregate thereceptors, resulting in a linear Scatchard plot with relativelylower affinity. Removal of detergent then may permit reag-gregation of the receptors and regeneration of the multiphasicScatchard curve.The physical characteristics of the solubilized [3H]serotonin

binding site(s) indicate a highly asymmetrical protein (axial ratioof 10) whose largest dimension is likely to be larger than its Stokesradius of 3.9 nm. This is certainly compatible with a mem-brane-bound molecule whose functions would include trans-membrane and intramembrane information transfer. However,the effects of detergent binding and solvent structuring on sol-ubilized membrane components have not been worked out andrequire further study.Our partially purified binding site preparation possesses

properties conventionally assigned to the SI receptor. Theseinclude high affinity for serotonin and its analogs and low af-finity for serotonin antagonists. A more detailed characteriza-

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FIG. 2. Gel filtration chromatography of soluble fraction. Chro-matography on Sephacryl S-300 was performed and 200-1.l aliquots ofeach fraction were assayed for [3Hlserotonin binding activity. The av-erage Mr (mean ± SEM) estimated for this peak fraction was 98,000 ±18,000 (n = 5 runs). (Inset) Fractions 27-33 were combined, and de-tergent was removed via dialysis and polystyrene bead adsorption.Binding assays were performed and the data were graphed as a Scat-chard plot. The data indicate return of the curvilinear Scatchard plotwhen the detergent is removed from the preparation. B, bound; B/F,bound/free.

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FIG. 3. Glycerol gradient sedimentation of solubiliGradient conditions were as described in the text and'fractions were assayed for specific [3H]serotonin bindtion is from right to left. Albuminsedimented in fracticchard curve for peak fractions. Fractions 7 and 8 were Iand treated with polystyrene beads. Sixty microgramused per binding assay. B and B/F, as in the legend

tion is necessary to fully identify this preparaticdone once higher degrees of purification are a

fication may also resolve the actual number ofponents and their relation to each other.

This study demonstrates that high affinitybinding activity can be solubilized from bovinebranes with high yield and high specific bindirpossible a number of biochemical, biophysicalcologic observations and is the first step in purterizing, and localizing serotonin receptors in t]vous system.

This study was funded by grants from the National I

tal Health (MH 25998) and the Scottish Rite Foundati

recipient of Neuropathology Traning Grant NS 07111. R. D.C. is a re-cipient of a Research Career Development Award (MH 00219). R.L.A.is the recipient of MSTP Training Grant GM 07365 from the NationalInstitutes of Health.

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2. Harvey, J. A. & Yeager, L. M. (1973) in Serotonin and Behavior,eds. Barchas, J. D. & Usdin, E. (Academic, New York), pp. 179-189.

3. Schain, R. J. & Freedman, D. X. (1961)J. Pediatr. 58, 315-320.4. Jouvet, M. (1973) in Serotonin and Behavior, eds. Barchas, J. D.

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212,827-829.6. Peroutka, S. J. & Snyder, S. H. (1981) Brain Res. 208, 339-347.7. Roberts, M. H. T. & Strangham, D. W. (1967)J. PhysioL (London)

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9. Nelson, D. L., Herbet, A., Enjalbert, A., Bockaert, J. & Har-

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PharmacoL 16, 700-708.ized membranes 11. Quach, T. T., Rose, C., Duchemin, A. M. & Schwartz, J. C. (1982)t0-1.d aliquots of Nature (London) 298, 373-375.

206. Sedimentaf

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L, and pharma- 21. Siegel, L. M. & Monty, K. J. (1966) Biochim. Biophys. Acta 112,

rifying, charac- 346-362.he central ner- 22. Todd, R. D., Griesenbeck, T. A. & Douglas, M. G. (1980)J. BioL

Chem. 255, 5461-5467.23. Lester, B. R., Miller, A. L. & Peck, E. J. (1981)J. Neurochem.

[nstitute of Men- 36, 154-164.ion. S.R.V. is the 24. Snyder, S. H. (1977) Sci. Am. 236 (3), 44-67.

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