- Email: [email protected]
[7] Photoaffinity labeling of vesicular acetylcholine transporter from electric organ of Torpedo
[7]
PHOTOLABELINGOF VAcChT
99
filters represents less than 1% of the total ACh added to the medium. Stocks (1000×) of L- or o-vesamicol (Research Biochemicals Inc., Natick, MA) are made up in water and stored at - 8 0 °. Bafilomycin A1 (LC Laboratories, Woburn, MA) is dissolved in DMSO as a 1000× stock. Homogenates from control PC-12 cells and hVAChT-expressing PC-12 cells are always analyzed together to assess the specific ACh uptake derived from endogenous rat VAChT, which is less than 2-fold over that seen at 4 ° or in the presence of 2/xM vesamicol. For [3H]vesamicol or [3H]TBZOH binding assay, aliquots (50/zl) of postnuclear homogenates containing 30/zg protein are mixed with uptake/ binding buffer and various concentrations of [3H]vesamicol (31 Ci/mmol, NEN Life Science Products) or 3H-TBZOH are added. Nonspecific binding is determined by incubating parallel samples in the presence of a 300fold excess of unlabeled L-vesamicol or tetrabenazine. The suspensions are warmed to 20° and incubated for 1 h. The uptake and binding reactions are stopped by placing and tubes in ice-cold water, and vacuum filtering the samples through GF/B glass fiber filters and washing with 5 ml ice-cold uptake/binding buffer. Radioactivity bound to the filters is solubilized in 1 ml of 1% SDS followed liquid scintillation counting. Kin, IC50, and Kd values are determined by nonlinear regression. Interassay variability is generally less than 10%.
[7]
Photoaffinity Labeling of Vesicular Acetylcholine T r a n s p o r t e r f r o m E l e c t r i c O r g a n o f Torpedo
By S T A N L E Y
M.
PARSONS, GARY
A.
ROGERS,
and LAWRENCE M.
GRACZ
Introduction Direct biochemical study of acetylcholine (AcCh) storage in test tubes became possible after methods were developed for the purification of purely cholinergic synaptic vesicles from the electric organ of the marine ray Torpedof -3 The purification has since been improved. 4 Vesicles of greater than 95% purity can be isolated in about 21 days. When the purified vesicles are suspended in magnesium ion and ATP, they internalize exogenous 1 A. Nagy, R. R. Baker, S. J. Morris, and V. P. Whittaker, Brain Res. 109, 285 (1976). 2 S. S. Carlson, J. A. Wagner, and R. B. Kelly, Biochemistry 17, 1188 (1978). 3 K. Ohsawa, G. H. C. Dowe, S. J. Morris, and V. P. Whittaker, Brain Res. 161, 447 (1979). 4 L. M. Gracz and S. M. Parsons, Biochim. Biophys. Acta 1292, 293 (1996).
METHODS IN ENZYMOLOGY,VOL. 296
Copyright © 1998by AcademicPress All rights of reproduction in any form reserved. 0076-6879/98$25.00
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AcCh via the vesicular AcCh transporter (VAcChT). 5 The availability of this assay combined with well-controlled work by Howard and colleagues in PC-12 (rat adrenal pheochromocytoma) cells led to characterization of the bioenergetics of AcCh storage. It has been shown that uptake is dependent on internal acidification of the vesicles by a V-type ATPase and that a separate VAcChT protein exchanges "luminal" protons for external (cytoplasmic) AcCh to effect storage of the neurotransmitter. 6-s Pharmacological characterization of the VAcChT began with the demonstration that it can be distinguished from other cholinergic proteins (such as the nicotinic and muscarinic receptors) by its relative insensitivity to known drugs. 9 The compound trans-2-(4-phenylpiperidino)cyclohexanol (then called AH5183 and now called vesamicol) was found to inhibit uptake noncompetitively l°,u by binding to an enantioselective site in the VAcChT. 12 These findings confirmed the hypothesis by Marshall 13 about the action of vesamicol based on physiologic experiments. Extensive structure-activity studies of AcCh 14'15and vesamicol. ~6-2° have been carried out utilizing synaptic vesicles purified from Torpedo. Synthesis of [3HlAzidoacetylcholine (I) and [3HlAzidoaminobenzovesamicol (II) The VAcChT has been photoaffinity labeled with analogs of AcCh and vesamicol called [3H]azidoAcCh (I) and [3H]azidoABV (II), respectively 5 S. M. Parsons and R. Koenigsberger, Proc. Natl. Acad. Sci. USA 77, 6234 (1980). 6 L. Toll and B. D. Howard, J. Biol. Chem. 255, 1787 (1980). 7 D. C. Anderson, S. C. King, and S. M. Parsons, Biochemistry 21, 3037 (1982). 8 B. W. Hicks and S. M. Parsons, J. Neurochem. 58, 1211 (1992). 9 D. C. Anderson, S. C. King, and S. M. Parsons, Molec. Pharmacol. 24, 48 (1983). 10 B. A. Bahr and S. M. Parsons, J. Neurochem. 46, 1214 (1986). n B. A. Bahr, E. D. Clarkson, G. A. Rogers, K. Noremberg, and S. M. Parsons, Biochemistry 31, 5752 (1992). 12 B. A. Bahr and S. M. Parsons, Proc. Natl. Acad. Sci. USA 83, 2267 (1986). 13 I. G. Marshall, Br. J. Pharmacol. 40, 68 (1970). 14 G. A. Rogers and S. M. Parsons, MoL Pharmacol. 36, 333 (1989). 15 E. D. Clarkson, G. A. Rogers, and S. M. Parsons, J. Neurochem. 59, 695 (1992). 16 G. m. Rogers, S. M. Parsons, D. C. Anderson, L. M. Nilsson, B. A. Bahr, W. D. Kornreich, R. Kaufman, R. S. Jacobs, and B. Kirtman, J. Med. Chem. 32, 1217 (1989). 17 G. A. Rogers, W. D. Kornreich, K. Hand, and S. M. Parsons, Mol. Pharmacol. 44, 633 (1993). 18 S. M. Efange, A. Khare, S. M. Parsons, R. Bau, and T. Metzenthin, J. Med. Chem. 36, 985 (1993). ,9 S. M. Efange, A. B. Khare, C. Foulon, S. K. Akella, and S. M. Parsons, J. Med. Chem. 37, 2574 (1994). 20 S. M. Efange, R. H. Mach, C. R. Smith, A. B. Khare, C. Foulon, S. K. Akella, S. R. Childers, and S. M. Parsons, Biochem. PharmacoL 49, 791 (1995).
[7]
PHOTOLABELINGOF VAcChT
101
O%c/OCH2--~ I
o
(i)
~
~
,~
o
3
N
? o
(,) Fro. 1. Chemical structures of azidoAcCh [compound (I)] and azidoABV [compound (II)].
(Fig. 1). The structure-activity studies mentioned above yielded the synthetic routes to chemical intermediates required to make radioactive arylazido compounds predicted to have relatively high affinity for the AcCh and vesamicol binding sites. All reagents needed for the syntheses of these compounds are available from standard commercial sources except as noted below. The reader should consult the original references for interpretation of the spectroscopic data used in the structure proofs.
ffH]Azidoacetylcholine
(I) 21
Cyclohexylmethyl Isonipecotate. Isonipecotic acid hydrochloride (2.1 g, 12.7 mmol) is suspended in 15 ml of thionyl chloride (SOCI2) and heated to 40° for 12 hr. Excess 8OC12 is removed on a rotary evaporator with the aid of dry CH2C12. The acid chloride is dissolved in dry CH2C12 and heated in a fume hood in order to remove the remaining SOCl 2. During several hours, aliquots of cyclohexylmethanol (a total of 3.5 equivalent) are added to the stirred solution, which is then refluxed overnight. The CHzCI2 solution is washed with cold aqueous carbonate and dried o v e r N a z S O 4 . Removal of the solvent results in an oil that is chromatographed on silica gel. Product ester is eluted with 5% (v/v) ethanol/CHCl3 in 65% yield as a colorless oil. The hydrochloride salt, which is produced by precipitating the product 21 G. A. Rogers and S. M. Parsons,
Biochemistry31, 5770 (1992).
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VESICULARCARRIERS
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dissolved in ether by bubbling in anhydrous HC1 gas, has melting point (mp) of 161.5-163 °.
Cyclohexylmethyl-cis-N- (4-azidophenacyl)-N-methyl Isonipecotate Bromide. Cyclohexylmethyl isonipecotate (9 rag, 40/~mol) and [3H]CH3I (0.65 tzmol, 10.8 Ci/mmol, Amersham Corp., Arlington Heights, IL) are combined in 0.8 ml of toluene in a 1-ml screw-cap vial. After 30 hr in the dark, succinic anhydride (5 mg) plus 80/zl of CC14 are added. The mixture is vortexed for 5 min to effect solution. This step facilitates separation of the product because succinic anhydride reacts with the large excess of unreacted cyclohexylmethyl isonipecotate (which is a secondary amine) but not with the methylated product (which is a tertiary amine). A large excess of cyclohexylmethyl isonipecotate is used to suppress double methylation. After 20 hr in the dark, the reaction mixture is chromatographed on silica gel. The product elutes with 5% methanol/CHCl~. Succinylated cyclohexylmethyl isonipecotate remains on the column. After the methanolic fractions are pooled and evaporated to dryness, the residue is dissolved in dry (C~H5)20 (200/zl). 4-Azidophenacyl bromide (5 mg, 21 /zmol) plus one drop of N,N-diisopropylethylamine are dissolved in the ethereal solution, which is stored in the dark for 2 days. Chromatography of the reaction mixture on silica gel provides product that elutes with methanol in 35% radiochemical yield. Fractions containing most of the radioactive peak are pooled, solvent is removed under vacuum, and the residue is taken up in 1 ml ethanol [yielding about 228 /zM (I)] for storage at - 2 0 °. The nonradioactive compound (FW 479) can be made similarly on a larger scale.
FH]AzidoABV (II) 22 N-(Trifluoroacetyl)-l-amino-5,8-dihydronaphthalene Oxide. A l-liter, three-necked, round-bottom flask is equipped with a large cold-finger condenser filled with solid CO2 and 2-propanol. To the flask 1-aminonaphthalene (79 g, 0.55 mol, carcinogenic!), (C2H5)20 (300 ml), ten-butanol (50 ml), and NH3 (200-300 ml) are added with stirring. Sodium (30 g, 1.3 mol, dried with paper towels just before weighing and returned to mineral oil after weighing until just before use) is added in portions over a 4-hr period, followed by an additional 50 ml of ten-butanol. After 1 hr, absolute ethanol (100 ml) is added slowly. This mixture is allowed to stir overnight and then is quenched by careful addition of NH4C1 (50 g) and H 2 0 (400 ml). The two liquid layers are separated, and the aqueous layer is extracted two times with ether. The ether extracts are combined with the organic layer, and this solution is extracted twice with water, once with a saturated NaC1 22G- A. Rogers and S. M. Parsons, Biochemistry 32, 8596 (1993).
[7]
PHOTOLABELINGoF VAcChT
103
solution, dried over Na2SO4, and finally filtered through MgSO4. Ether and most of the tert-butanol are removed at 65° on a rotary evaporator. This material may be used directly for the next step or colorless 1-amino5,8-dihydronaphthalene (mp 37-39 °) may be obtained in 97% yield after vacuum distillation. 1-Amino-5,8-dihydronaphthalene (60.1 g, 0.414 mol) is dissolved in 200 ml of stirred benzene and cooled to 0°. Trifluoroacetic anhydride (89 g, 0.42 tool) is added slowly because of the exothermicity of the reaction. The ammonium salt begins to precipitate almost immediately, but the reaction solution is homogeneous upon complete addition of the anhydride. This step is necessary to protect the amino group from oxidation during formation of the epoxide (below). The solution is maintained at 0° for 1 hr, after which benzene and trifluoroacetic acid are removed under reduced pressure. More benzene is added and again evaporated in order to promote the removal of trifluoroacetic acid. The resulting amide is dissolved in 300 ml of stirred (C2H5)20 to which is added 3-chloroperoxybenzoic acid (85.0 g, 80-85% pure). The solution is maintained near 10° throughout the addition and then allowed to warm to 23 ° and stirred for 5 hr. The product epoxide containing some 3-chlorobenzoic acid is collected by filtration and washed with ether. The solid is resuspended in 400 ml of (C2H5)20, thoroughly washed by agitation, and recollected by filtration to yield 89.0 g of epoxide (mp 174-178.5°). After extraction of 3-chlorobenzoic acid from the ethereal mother liquor with aqueous carbonate, an additional 2.1 g of product can be isolated by evaporation of the ether for an overall yield of 85%. Purification of the epoxide by crystallization from CHC13 with a wash of cold (C2H5)20 raises the melting point to 179.5-181 °.
(+_)-trans-5-Amino-2-hydroxy-3-(4-phenylpiperidino)tetralin [(+_)-ABV, FW 322]. N-(Trifluoroacetyl)-l-amino-5,8-dihydronaphthalene oxide (1.9 g, 7.4 mmol) is dissolved in 25 ml of absolute EtOH to which is added 4phenylpiperidine (3.0 g, 19 mmol). The solution is maintained at 45 ° for 17 hr and then refluxed for 3 hr. After 7 hr at 23° a crystalline solid is collected by filtration and set aside. The solid is a positional isomer of (_+)ABV. The mother liquor is evaporated to an oil that is taken up in CC14. This process is repeated in order to remove most of the EtOH. When the oil is again dissolved in CC14, crystallization of 4-phenylpiperidinium trifluoroacetate commences. The remaining mother liquor is chromatographed on silica gel where N-(trifluoroacetyl)-4-phenylpiperidine elutes with CC14 and (_+)-ABV with CC14/CHC13 (0.84 g, 35% yield). (_+)-ABV is crystallized from CHC13/ethanol (mp 174-175°C). 16 (-)-N-Glycyl-4-aminobenzovesamicol [(-)-GlyABV]. (_+)-ABV and N-tert-butoxycarbonylglycine p-nitrophenyl ester (1 equivalent) are reacted
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VESICULARCARRIERS
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in acetic acid in a closed vial overnight at 23°, and the acetic acid is removed in vacuo. The condensation product is taken up in 50% (v/v) trifluoroacetic acid/CHzCl2 and allowed to deblock in a sealed vial overnight at 45 ° to yield (_+)-GlyABV trifluoroacetate after removal of solvent in vacuo. After dissolving the trifluoroacetate in CH2C12 and washing it with aqueous carbonate, neutral (_+)-GIyABV is purified by chromatography on silica gel and then resolved into its enantiomers by chromatography on a Chiralpak AD semipreparative column (Daicel Chemical Industries, Exton, PA) in 20% 2-propanol/hexane. 17 The elution order of the enantiomers is extremely sensitive to the composition of the solvent and age of the column; thus, the optical rotations of the separated enantiomers should be taken to confirm their identities.
(-)-N-(4'-Azidobenzoylglycyl)-4-aminobenzovesamicol ([3H]azidoABV). N-[benzoate-3,5-3Hz]Succinimidyl 4-azidobenzoate (5.09 nmol, 49.1 Ci/mmol, NEN Division of DuPont, Inc., Boston, MA) is combined with 10.7 ~g (28.2 nmol) of (-)-GlyABV in 100 tzl of 2-propanol and placed in the dark. After 4 days the sample is diluted with 100/xl of ethanol. The product is purified by high-performance liquid chromatography on a semipreparative Chiralpak AD (Daicel Chemical Ind.) column (25% ethanol/hexane at 4 ml/min) with collection of fractions. It is important to turn the UV detector off as soon as elution of (II) begins at about 24 min. Unreacted (-)-GlyABV elutes at about 16 min. 22 Fractions containing most of the radioactive peak are pooled, solvent is removed under vacuum, and the residue is taken up in 1 ml ethanol [yielding about 3.26/xM (II)] for storage at -20 °. About 160 ~Ci of (II) in 64% radiochemical yield is obtained. The nonradioactive compound (FW 524) can be made similarly on a larger scale. Isolation of Cholinergic Synaptic Vesicles from Electric Organ of Torpedo
Torpedo are obtained live from a commercial fisherman or a commercial supplier of marine animals. Different species of Torpedo are found in substantial abundance in most of the major oceans of the world, and they often are caught when feeding on commercially exploited fish stocks. Torpedo californica commonly weigh 20-40 pounds, and about 15% of the body weight is electric organ. The fish are rested in a filtered well-aerated tank of seawater for 3-4 days after delivery. They are stunned by a blow to the back of the head and pithed with a scalpel, and the nerve bundles innervating the electric organs are severed. Because the fiat cartilaginous skull efficiently delivers the blow to the brain, the rays are immediately rendered unconscious and do not suffer as far as can be determined. The
[71
PHOTOLABELINGOF VAcChT
105
electric organs, which fill most of the wings, are readily recognized and removed by blunt dissection after the overlying skin is peeled back. They are frozen immediately in liquid nitrogen, after which they are fractured into chunks that are packaged into 800-g portions and stored at -100 ° until use. 4 The deep-frozen electric organ (800 g) is shaved very finely with a rapidly spinning blade to produce a "snow." The yield of vesicles decreases greatly if a very fine snow is not produced, and a very sharp blade seems to be the most important factor in obtaining favorable yields. A simple, commercially available machine used to make flavored "snow cones" at carnivals works well. The snow is allowed to warm slowly to - 2 ° with occasional mixing. The slush is suspended in 800 ml of ice-cold buffer composed of 0.76 M glycine containing 0.05 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES), 0.01 M ethylene glycol bis(/3aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 0.001 M ethylenediaminetetraacetic acid (EDTA), 0.02% NAN3, and 2 /~M 2,6-bis(1,1-dimethylethyl)-4-methylphenol (BHT) adjusted to pH 7.2 with 0.80 M NaOH. All subsequent steps in the vesicle purification are conducted at 4°. The slush is divided into portions that are slowly sheared with a blender for 20 sec, after which they are recombined. Approximately 300 ml is homogenized with five slow strokes using a tightly fitting, spinning Teflon pestle in a 350-ml glass mortar of the Potter-Elvehjem type. A drill press can be adapted to drive the pestle. The tissue homogenate is centrifuged in six 500-ml polycarbonate bottles at 17,700g for 1 hr to remove membrane debris. Twenty 70-ml polycarbonate centrifuge tubes having O ring screw caps are filled with supernatant to within 1 cm of the neck. Using a cannulated syringe, 6 ml of isosmotic "cushion" composed of 650 mM sucrose, 7.5% (w/w) Ficoll 400 (Sigma, St. Louis, MO), 10 mM HEPES, 1 mM EDTA, and 1 mM EGTA (adjusted to pH 7.0 with 0.80 M KOH) having a density of 1.117 g/ml is injected under the supernatant onto the bottom of each tube. Ficoll 400 is used to obtain increased density beyond that provided by isosmotic sucrose because it contributes little to the osmotic pressure and its high viscosity stabilizes the interface between the supernatant and the cushion. After balancing them with additional supernatant, the tubes are centrifuged at 49,000g for 16-21 hr in two Beckman Type 21 rotors. The membranes focused on top of the cushions are removed with a cannulated syringe and pooled to yield about 160 ml of crude vesicles. The vesicular pool is mixed with sufficient fresh cushion solution to increase the density of the mixture enough to layer under 0.40 M sucrose (see proceeding text). To accomplish this, 15 ml of fresh cushion solution is added to the pool, which then is mixed. A drop of the mixed pool is
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placed onto 0.40 M sucrose in a test tube to determine whether it falls to the bottom. Additional fresh cushion in 3 ml increments is added to the pool with mixing and the mixture is tested until it does fall. About 20-40 ml of cushion is required. Isosmotic density gradient solutions (0.5 liter each) composed of 0.01 M HEPES, 0.001 M EDTA, 0.001 M EGTA, and 0.02% (w/v) NAN3, adjusted to pH 7.0 with 0.8 M KOH and containing either 0.80 M glycine or 0.65 M sucrose, are prepared. An isosmotic solution containing 0.40 M sucrose is prepared by mixing 50 ml of the 0 M sucrose solution with 80 ml of the 0.65 M sucrose solution. A typical density gradient is formed from 0 M sucrose (7 ml), 0.40 M sucrose (8 ml), pooled vesicles containing additional cushion (22 ml), and 0.65 M sucrose (4 ml). The solutions are applied to the bottom of a 1 × 3.5-inch dome-top polyallomer quick-seal tube (Beckman Coulter Instruments, Inc., Fullerton, CA) in the order stated with a cannulated syringe. The eight required tubes are heat sealed and centrifuged at 196,200g for 3.5 hr in a Beckman VTi 50 vertical rotor. They are opened by carefully slicing the tops off with a single-edge razor blade. In initial work, 20 fractions of 1.5 ml each are obtained from each gradient by bottom puncture. The fractions are assayed for ATP content using the luciferase assay.23 Mature cholinergic synaptic vesicles isolated with this procedure (so-called VP1 vesicles) band at 1.055 g/ml and contain ATP as well as AcCh. The densities of the fractions can be estimated from the refractive indices,z4 The vesicles are found in a faint milky layer near the top, and after experience is gained, they can be withdrawn rapidly from the opened centrifuge tube by use of a cannulated syringe and visual inspection. Vesicles in the pool (50 ml) are further purified by size-exclusion chromatography on a Sephacryl S-1000 column (150 cm × 1.5 cm) equilibrated with buffer containing 820 mM glycine, 5 mM HEPES, 1 mM EDTA, 1 mM EGTA, and 0.02% NaN3 (adjusted to pH 7.0 with 0.80 M KOH). The column effluent is monitored by apparent absorbance at 350 nm. The vesicles elute in the middle of the resolving volume as the second peak of apparent absorbance (due to light scattering). The fractions containing vesicles are pooled and concentrated with a Centricon centrifugal ultrafiltration device (Amicon, Danvers, MA, 10 kDa cutoff) to 1-2 mg protein/ ml if desired. About 5 mg of vesicular protein is obtained as determined by the Bradford 25 dye assay. The VAcChT in these vesicles is stable for weeks when stored in a closed vessel at 4°. 23 G. A. Kimmich, J. Randles, and J. S. Brand, A n a l Biochem. 69, 187 (1975). 24 p. Sheeler, "Centrifugation in Biology and Medical Science," pp. 246-247. Wiley, New York, 1981. 25 M. M. Bradford, A n a l Biochem. 72, 248-254 (1976).
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PHOTOLABELINGOF VAcChT
107
Assay of Ligand Binding or Transport by Purified Synaptic Vesicles Binding and transport assays are done similarly to each other. Purified vesicles at about 0.075 to 0.2 mg protein/ml are incubated with a radioactive compound in buffer containing 0.10 M HEPES, 0.70 M glycine, 0.001 M EDTA, and 0.001 M EGTA adjusted to pH 7.80 with 0.80 M KOH (TB). This pH is optimal for both the AcCh and vesamicol families of ligands in both transport and binding assays. The buffer also includes components such as paraoxon to inhibit AcCh esterase and MgATP to drive transport as needed. Samples are prepared in the absence and presence of an excess of nonradioactive ligand that blocks the specific binding or transport of the radioactive ligand. Samples are mixed well by use of a vortex device after addition of reagents. Glass-fiber filters (Whatman, Clifton, N J, GF/F, 1.3 cm) that have been pretreated with polyethyleneimine are mounted on a 10-place filter manifold/collection box (Hoefer Scientific Instruments, San Francisco, CA, model FH 225V). To prepare them, 400 filters are soaked 3 hr in 400 ml of 0.5% (w/v) polyethyleneimine in water. They are poured gently into a Btichner funnel of 8 inch diameter and rinsed generously with water by filtration. The funnel with the filters is placed overnight in an oven at 37° for drying. Dry filters are removed carefully from the funnel with tweezers and placed in a plastic food baggy with zipper after which they can be stored at room temperature in the dark. After incubation of vesicles with a radioactive ligand, a portion (typically 50-100/xl) is applied with suction assistance onto a filter prewetted just before with ice-cold TB. The filter then is immediately and rapidly washed by 3-4 1-ml portions of ice-cold TB with suction assistance. The radioactivity trapped on the filter is determined by liquid scintillation spectroscopy. Filters must be extracted overnight by the scintillation cocktail (a type compatible with aqueous solutions) for accurate and full determination of bound radioactivity. AcCh becomes soluble in cocktail containing 10% water better than it does in dry scintillation cocktail. The decrease in bound radioactivity due to the presence of excess nonradioactive inhibitor is the specific binding or transport by the vesicles.
Reversible Binding of Compound (I) Before irreversible photoaffinity labeling is attempted, it is important to demonstrate that a potential label binds in a well-behaved manner to the expected site. As compound (I) is stable to normal indoor lighting, its interaction with cholinergic synaptic vesicles can be studied under reversible conditions. Although the effect of sunlight on the compound has not been determined, it should be avoided by working in an inner room or in a
108
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room with shaded windows. These comments apply to compound (If) also (see below). All TB contains 0.15 mM paraoxon. Purified synaptic vesicles at about 2 mg protein/ml in TB are incubated at 23 ° for 30 rain to allow full inactivation of acetylcholinesterase. Two microliters of compound (1) in ethanol prepared as above is dried in v a c u o and dissolved in 40 p,1 TB to yield about 10 ~M of compound (I). The exact concentration of compound (I) is determined by measuring the radioactivity in a 5 p,l portion. An approximately isosmotic solution of AcCh in which 0.35 M AcCh chloride replaces the 0.70 M glycine in TB is prepared by dissolving 150 mg of preweighed AcCh chloride (Sigma) in 2.36 ml of TB lacking glycine. The pH of this solution should be checked and adjusted to 7.80 if necessary. Serial dilutions of this solution are made with TB to prepare additional isosmotic solutions containing 35, 3.5, and 0.35 mM AcCh. Also, 0.35 M AcCh chloride containing vesamicol is prepared by dissolving 150 mg of preweighed AcCh chloride in 2.36 ml of TB lacking glycine but containing 60 /~M (-)-vesamicol hydrochloride (FW 295.5, Research Biochemicals International, Inc.). Vesamicol hydrochloride is soluble to at least 1 mM in aqueous solutions. Vesamicol is used to measure the extent of nonspecific binding of (I) because at equilibrium it is a competitive inhibitor of the binding of AcCh analogs, u Because they spontaneously degrade slowly, aqueous solutions of AcCh must be prepared shortly before use. Eightysix microliters from each stock solution (including TB) is dispensed into individual tubes, and 4 /~1 compound (I) in TB is added and mixed in. Vesicular suspension (10/A) is added and allowed to equilibrate for 15 min at 23 °. Bound compound (1) is determined by filtration and washing of 90 /~1 of the suspension as described above. The results of a typical binding experiment are shown in Fig. 2. Because the concentration of the binding site (about 40 nM) is comparable to the dissociation constant for (I), the concentration of free (I) has to be calculated iteratively at each concentration of AcCh. Hence, for each total concentration of (I), the free concentration in solution is calculated from Eqs. (1) and (2): (I)F = (|)T -- (I)B (I)B = [Bmax X (I)v X KIA]/(1)F X KIA + A c C h x K(I) + K(I) x KIA]
(1)
(2)
where (I)F is the free concentration of (I), (I)T is the total concentration of (I), (I)B is the bound concentration of (I), K(I ) is the dissociation constant for the specific VAcChT- (I) complex (240 nM 21), KIA is the dissociation constant for the specific VAcChT • AcCh complex, and AcCh is the concentration of AcCh. Regression analysis is done with the program Scientist
[7]
PHOTOLABELINGOF V A c C h T
109
240 200
___//
•
,.C 160 rO ¢.)
~0 120 "U
"~ ~'~
80
40 0
0
Z j 10,~
10~-~
i0'-~
10'-1
16o
:
[AeCh](M)
FIG. 2. Inhibition of the reversible binding of compound (I) by AcCh. Final concentrations of the vesicular protein and (I) were 0.2 mg/ml and 440 nM, respectively. Bound (I) was determined by filtration as described in the text. Nonspecifically bound (I) was estimated in the presence of 60 I~M vesamicol (plus 300 mM AcCh) and was subtracted from the total amount bound. The estimated KTA for AcCh from regression analysis is 17 _~ 3 mM using Eqs. (1) and (2) in the text. (Reprinted with permission from G. A. Rogers and S. M. Parsons. Biochemistry 31, 5770 (1992). Copyright 1992 American Chemical Society.)
( M i c r o M a t h Scientific Software, Salt L a k e City, U T ) , which is available only for personal computers. N o n r a d i o a c t i v e A c C h inhibited the binding of c o m p o u n d (I) with a dissociation constant of 17 ± 3 m M in the experiment of Fig. 2. T h e highest c o n c e n t r a t i o n of A c C h investigated displaced essentially all (I), as vesamicol displaced only a small additional amount. T h e equilibrium dissociation constant o b s e r v e d for A c C h is relatively high and thus possibly suspect as not arising f r o m the correct A c C h binding site. H o w e v e r , the a p p a r e n t dissociation constant for A c C h binding to this site decreases to a b o u t 0.3 m M u n d e r active transport conditions. T h e latter value is less than the estimated c o n c e n t r a t i o n of A c C h in cholinergic nerve terminals. 26 T h e a p p a r e n t increase in affinity of A c C h u n d e r active transport conditions occurs because of a large kinetic c o m p o n e n t in the Michaelis constant. 1~ Thus, analog (I) binds to the site for A c C h transport. This result d e m o n strates why it was necessary to carry out a structure-activity study before choosing an affinity label analog of A c C h for this system. A c C h and its close analogs bind to the V A c C h T so weakly that they would be unlikely to provide specific labeling of the transporter sufficient to identify it. 26S. M. Parsons, C. Prior, and I. G. Marshall, in "International Review of Neurobiology,'" Vol. 35 (R. J. Bradley and R. A. Harris, Eds.), pp. 279-390. Academic Press, New York, 1993.
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VESICULAR CARRIERS
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Reversible Binding of Compound (II) Compound (If) has moderately high affinity for the VAcChT, and for this reason the high-affinity ligand ABV 17 is used in this experiment to block specific binding of compound (II). Synaptic vesicles from the center of the vesicular peak (having about 30/zg protein/ml) in the size exclusion chromatography step of the purification are used without concentration. Vesicles are diluted 60-fold into TB to about 0.5/~g protein/ml. All aqueous solutions of compound (II) and ABV must be handled in glass containers coated with Sigmacote (Sigma Chemical Co.) or the compounds will be lost through adsorption to the vessel surface. Plastic containers are unsuitable. Compound (If) in ethanol (1.5/zl) prepared as above is dried in vacuo and taken up in 50/zl TB to yield a solution that is about 100 nM in (|I). Serial dilutions with TB to about 1 nM of (If) are prepared on a 50/zl scale with retention of all of the dilutions. (+)-ABV (0.322 mg) is dissolved in 10 ml ethanol to yield 100/zM, and 1/zl is added to each of 6 tubes. The ethanol is evaporated from the tubes. Vesicles (100/zl) are added to these tubes and to 6 additional ones. After incubation for 30 min, two 10-~1 portions of each dilution of (II) are added to two samples of vesicles, one containing (+)-ABV and one not. After incubation for 11 hr in sealed tubes, the final total concentration of (If) in each sample is determined by measuring the radioactivity in a 10-/zl portion with liquid scintillation spectroscopy. The bound radioactivity then is determined by filtration and washing of a 90~1 portion as described above. Figure 3 shows the results of a typical experiment. Equations (3) and (4) are fit simultaneously to the data sets for total and nonspecific binding, respectively, using Scientist. (|I)T = Bmax X (II)/[(II) + K(II)] + S x (If) + C (II)N = S X (II) + C
(3) (4)
where (II)T is the total amount of bound (II), Bmax is the concentration of specific binding sites, (II) is the concentration of free (II) [assumed equal to all (II) in solution], K(II) is the dissociation constant for the specific VAcChT- (II) complex, (II)N is the amount of nonspecifically bound (II), S is the slope of the nonspecific binding of (II), and C is the intercept of the nonspecific binding of (II). The dissociation constant for specifically bound (II) was 2.1 + 0.4 nM and the Bmaxwas 0.29 + 0.03 nM (corresponding to 580 + 80 pmol/mg) in the experiment of Fig. 3. Photoaffinity Labeling and Visualization of Acetylcholine-binding Protein Photolysis of Compound (I) Bound to Vesicles. All TB contains 0.15 mM paraoxon. Synaptic vesicles (0.8 ml containing 0.8 mg protein) are
PHOTOLABELING ov VAcChT
[71
111
0.400
z~
0 ma00
",C O
'~ o.~oo
Z
,~ 0.100 o~
0.000
• 0.0
I 2.0 Cone.
I 4.0
I 0.0
of [all] AzidoABV
I 8.0
10.
(nM)
FIG. 3. Saturation of the reversible binding of compound (II). Bound (II) was determined by filtration as described in the text in the absence (0) or presence (m) of 800 nM (_+)-ABV to yield data sets representing the amount of total and nonspecificbinding at the indicated concentrations of compound (II), respectively.A simultaneous fit of Eqs. (3) and (4) in the text to the two sets of data yielded K(II)equal to 2.1 _+0.4 nM and Bmaxequal to 0.29 -+ 0.03 nM (corresponding to 580 + 80 pmol/mg) for specific binding. (Reprinted with permission from G. A. Rogers and S. M. Parsons, Biochemistry 32, 8596 (1993). Copyright 1993American Chemical Society.) incubated in TB for at least 30 min. Seven portions of 100/xl each are placed into separate tubes. Three microliters of compound (|) in ethanol prepared as above is dried in v a c u o and dissolved in 60/xl TB to yield about 10 txM of (I). The exact concentration of compound (I) is determined by measuring the radioactivity in a 5-txl portion. Nonradioactive (1) (0.479 mg) is dissolved in 1 ml of TB [yielding 1 m M (|)]. ( - )-Vesamicol hydrochloride is made up to 1 mM in TB by dissolving 0.296 mg in i ml. Concentrated M g A T P in TB is adjusted to p H 7.80 with K O H and diluted to 100 m M with TB. AcCh is made up to 350 m M as above. Samples A and B (lanes 2 and 4 in Fig. 4) receive 145/xl of TB; sample C (lane 3) receives 25/xl of nonradioactive (I) and 120 ixl of TB; sample D (lane 6) receives 25/xl of nonradioactive (l), 25/xl of MgATP, and 95/xl of TB; sample E (lane 8) receives 71.4 txl AcCh and 73.6/xl of TB; sample F (lane 7) receives 2.14 txl AcCh and 142.9/xl of TB; and sample G (lane 9) receives 0.25/xl vesamicol and 144.75 b~l of TB. All samples receive 5 txl radioactive (I). Because of a high level of nonspecific binding, a concentration of (I) slightly below its dissociation constant value is used in this experiment. This yields an optimal compromise between good specific and excessive nonspecific labeling. Samples destined for lanes 3-9 (Fig. 4) are irradiated simultaneously using a handheld 18-watt lamp (Blak-Ray, UVP, Inc., San Gabriel, CA) that emits light in a band of wavelengths centered at 366 nm. Irradiation is carried out in four periods of 2 min each with mixing after each period.
112
VESICULAR CARRIERS 1
2
3
4
5
6
7
[71 8
9
116-97-66--
45--
29--
Fia. 4. Photoaffinity labeling and visualization of the AcCh-binding protein by compound ([). Vesicles were labeled in the presence or absence of the indicated reagents and then subjected to SDS-PAGE and autofluorography as described in the text. The figure is a composite showing Coomassie Blue staining, a short-term film exposure to the dye front region of the gel, and a long-term exposure to the stacking and resolving regions of the gel. Lane 5 contained standard proteins with their masses (kDa) indicated to the left. Lanes 1 and 4 are the Coomassie staining and autofiuorographic patterns, respectively, of the same sample of vesicles. AzidoAcCh refers to compound (I). Lanes 2 and 3 were controls that received no irradiation or 100/xM of nonradioactive (I), respectively. Lanes 7 and 8 contained 3 or 100 mM AcCh, respectively, and lane 9 contained 1 ~M vesamicol. Lane 6 contained 100 /xM of nonradioactive (I) and 10 mM of MgATP. (Reprinted with permission from G. A. Rogers and S. M. Parsons, Biochemistry 31, 5770 (1992). Copyright 1992 American Chemical Society.)
U n d e r similar conditions, the half-life for photolysis of the azido g r o u p is a b o u t 1 m i n as d e t e r m i n e d by u l t r a v i o l e t spectroscopy. A f t e r photolysis, a n a l i q u o t of 20/~1 is t a k e n f r o m each s a m p l e a n d filtered. T h e filtered samples are w a s h e d r e p e a t e d l y with T B at 23 °, a n d b o u n d t r i t i u m is determ i n e d . V e s a m i c o l (1 ~ M ) blocks 45 p m o l of label i n c o r p o r a t i o n / m g of p r o t e i n . This c o r r e s p o n d s to a b o u t 9% of the specific b i n d i n g assessed s e p a r a t e l y with 2 5 0 / z M [3H]vesamicol ( N E N division of D u P o n t , Inc.).
[71
PHOTOLABELINGOF VAcChT
113
The remaining volume of each sample is centrifuged at 160,000g and 23 ° in a Beckman Airfuge for 60 min in order to pellet the vesicles, which are immediately analyzed by SDS-PAGE. SDS-PA GE and Autofluorography of Vesicles Photolabeled with Compound (I). Photolabeled and pelleted synaptic vesicles are subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 10% resolving gel, a 3% stacking gel, and the discontinuous buffer system of Laemmli. 27 For this purpose, after removal of the supernatant the pelleted vesicles are dissociated in 80/zl of a treatment buffer made by dissolving 4.8 g urea in 4 ml of 10% SDS and diluting with 5 ml of 0.75 M Tris-HC1 (pH 8.8). The samples are vortexed repeatedly during a 50min period with the addition of 12/xl of 2-mercaptoethanol to each after 20 min. Finally, all samples are heated to 90° for 2 min. One-half of each sample is stored at - 2 0 ° for possible reanalysis and the other half is subjected to electrophoresis. After electrophoresis, the gel is stained with Coomassie Brilliant Blue R in order to visualize proteins and then treated with Intensify (Du Pont, Inc.) as instructed by the manufacturer before being dried onto filter paper under vacuum. The dried gel is autofluorographed with XAR-5 film (Kodak, Rochester, NY) at - 8 0 °. A continuous region from about 50 kDa to the top of the resolving gel is heavily labeled by photolyzed (I) (Fig. 4, lane 4). The protein stain (lane 1) of the same gel lane demonstrates that the diffuseness of the fluorogram does not arise from poorly focused proteins. No protein corresponding to the diffuse pattern of radiolabeling is stained clearly. An absence of extensive nonspecific aggregation is indicated by the lack of protein staining or radioactivity in the stacking gel. Lane 2 (Fig. 4) demonstrates that photolysis is required for labeling, and lanes 3 and 6 show that excess nonradioactive (I) blocks labeling of virtually all proteins, whether or not vesicles are energized by ATP. Lanes 7 and 8 (Fig. 4) show increasing levels of protection from labeling by 3 and 100 mM ACh, respectively. The rather modest amount of protection obtained with 3 mM AcCh is consistent with the KIA value of 17 mM. Lane 9 (Fig. 4) shows that vesamicol at only 1 tzM nearly completely blocks the diffuse labeling seen in lane 4. In summary, the protection pattern seen in Fig. 4 is fully consistent with specific labeling of the VAcChT at the AcCh binding site. Photoaffinity Labeling and Visualization of Vesamicol-Binding Protein
Photolysis of Compound (II) Bound to Vesicles. Four glass test tubes treated with Sigmacote each receive 4.6/.d of the ethanolic solution of (II) 27U. K. Laemmli, Nature 227, 680 (1970).
114
VESICULARCARRIERS
[71
prepared above. Five microliters of a 1 mM solution of (-)-vesamicol hydrochloride in ethanol is added to the second tube (final concentration to be 50/zM (-)-vesamicol), and 1 /zl of a 100 ~M ethanolic solution of (_+)-ABV is added to the third tube [final concentration to be 1/zM (_+)ABV]. A fifth treated tube receives 1.1 pA of the ethanolic (_+)-ABV. The ethanol in all tubes is evaporated. Next, 110 p~l of synaptic vesicles that are 0.65 mg of protein/ml TB is added to the fifth tube and incubated 1 hr to prebind ABV. Volumes of 100/~1 of the fresh vesicular suspension are added to the first three test tubes, and 100/zl of the vesicular suspension prebound to (_+)-ABV is added to the fourth test tube. All four suspensions are incubated for 10 min with occasional mixing and then illuminated with an 18-W UV lamp (Ultra-Violet Products Inc., San Gabriel, CA) that emits light in a band of wavelengths centered at 254 nm. For 10 min the tubes are alternately illuminated and vortexed to yield a total illumination time of about 5 rain each. Following the photolysis, 5 /zl of the solution is removed from each tube and the radioactivity measured.
SDS-PA GE and Autofiuorography of Vesicles Photolabeled with Compound (II). Fifty microliters is removed from each sample of photolabeled vesicles from above and diluted with 100/zl TB. The remaining portion of each sample is stored at -20 ° for possible reanalysis. The diluted vesicles are pelleted in an Airfuge (Beckman Instrument Co.) at 73,000 rpm for 70 min. The supernatant is carefully removed, and the pellets are covered with 20/zl of treatment buffer that contains 10% sodium dodecyl sulfate (SDS) and 20% glycerol in 0.125 M Tris-HC1 (pH 6.8). The samples are vortexed occasionally over a 6-hr period at 23 °. Finally, each is diluted with 20/zl of a 10% (w/v) solution of dithiothreitol (DTT) and heated in a 90° bath for 3 min. Molecular weight standards are treated with the same solutions but are given only a brief incubation prior to addition of the DTT. The dissociated samples are subjected to SDS-PAGE using a 5-15% linear gradient resolving gel, a 3% stacking gel, and the discontinuous buffer system of Laemmli.27 Electrophoresis is halted when the tracking dye is about 1 cm from the bottom of the gel. After electrophoresis, the gel is stained with Coomassie Brilliant Blue R in order to visualize proteins and then treated with Intensify (DuPont) before being dried onto filter paper under vacuum. The dried gel is autofluorographed with XAR-5 film (Kodak) at -80 ° for 3 days. The amount of radioactivity in each sample after photolysis should be similar and correspond to about 150 nM compound (II). Because the concentration of binding sites (about 450 nM as determined by dilution of a small volume of the untreated vesicles into 250 nM [3H]vesamicol) is in excess of both this concentration and the dissociation constant for compound (I|) (see above), almost all of (II) present in the solution will be
[71
PHOTOLABELINGOF VAcChT
115
specifically bound in the absence of competing ligand. Protein staining of the SDS-PAGE gel should reveal well-focused bands throughout the molecular mass range of 12-200 kDa. Autofluorography of the unprotected sample (Fig. 5, sample 1) demonstrates heavy labeling of a continuous region from about 50 to 200 kDa. Quantitation of the absorbance indicates that 94% of the total labeling of proteins (exclusive of the labeling at the dye front) is in this region. In addition, four polypeptides with Mr values of about 22,600, 33,000, 35,100, and 37,500 are specifically labeled. The 33kDa band is somewhat diffuse, and correlation with the Coomassie image
200 116 97 77 42.7
!
66
55.5
30 17.2
--
E
E
E
E
FIG. 5. Photoaffinity labeling and visualization of the vesamicol-binding protein with compound (I|). Vesicles were labeled in the absence or presence of competing ligands and then subjected to S D S - P A G E and autofluorography as described in the text. The figure is a composite showing Coomassie Blue staining (the lane called Protein) of Sample 1 and the autofluorograph of all four gel lanes. Sample 1 is the autofluorograph for nonblocked vesicles. Samples 2 and 3 show labeling when vesamicol or 1/xM (+_)-ABV was added simultaneously with the addition of compound (II), respectively, and sample 4 shows labeling when 1 /zM (+_)-ABV was added 1 hr prior to the addition of (ll). The masses (kDa) and positions of standard proteins are indicated to the left. Specifically labeled bands at 22.6, 33, 35.1, and 37.5 kDa in Sample 1 were visible in the original film, but may not be visible in the reproduction. (Reprinted with permission from G. A. Rogers and S. M. Parsons, Biochemistry 32, 8596 (1993). Copyright 1993 American Chemical Society.)
116
VESICULARCARRIERS
[81
suggests that the diffuse appearance arises as the result of a doublet of closely spaced bands at 32.4 and 33.8 kDa. Addition of 50/~M vesamicol coincident with compound (II) completely blocks incorporation of radioactivity into components with Mr greater than 12 kDa (sample 2). At the lower concentration of 1/zM, the higher affinity (+)-ABV protected nearly all sites above 12 kDa from being labeled, whether it was applied coincidentally (sample 3) or prior (sample 4) to exposure of the vesicles to compound (II). Neither vesamicol nor (_+)-ABV produced any visible change in the protein staining pattern (data not shown). Whether the specifically labeled species of Mr 22-38 kDa are proteolytic fragments of the VAcChT is not known. Overall, the protection pattern seen in Fig. 5 is fully consistent with specific labeling of the VAcChT at the vesamicol binding site. The different electrophoretic patterns for specific labeling in Figs. 4 and 5 are due to the use of different types of resolving gels. Vesicles labeled with compounds (I) and (II) yield similar patterns of specific labeling after SDS-PAGE utilizing the same gel conditions. The extreme streaking of the specifically labeled species is not due to inadequate sample preparation, as far as can be determined. Changes in the sample dissociation conditions, such as use of higher concentrations of SDS, urea, low temperature, and different reducing agents, do not affect the results. Thus, the AcCh and vesamicol binding sites reside in the same protein as judged by SDS-PAGE, and this protein exhibits poor staining and migration behavior in SDSPAGE. Possibly the VAcChT in synaptic vesicles of the electric organ is extraordinarily heavily glycosylated, or even high concentrations of SDS do not succeed in dissociating the protein fully.
[8] B i o e n e r g e t i c C h a r a c t e r i z a t i o n o f y - A m i n o b u t y r i c Acid Transporter of Synaptic Vesicles By
JOHANNES W.
HELL
and
REINHARD
JAHN
Introduction Neurotransmitters are stored in synaptic vesicles and, on depolarization and subsequent calcium influx, released from nerve terminals by exocytosis. The main inhibitory neurotransmitter in the mammalian brain is y-aminobutyric acid (GABA), whereas glycine is the prevailing inhibitory neurotransmitter in the spinal cord. The GABA uptake into synaptic vesicles is mediated by the vesicular GABA transporter, which probably works as an electrogenic GABA proton antiporter in contrast to the sodium-driven
METHODS IN ENZYMOLOGY, VOL. 296
Copyright © 1998 by Academic Press All rights of reproduction in any form reserved. 0076-6879/98 $25.00
PHOTOLABELINGOF VAcChT
99
filters represents less than 1% of the total ACh added to the medium. Stocks (1000×) of L- or o-vesamicol (Research Biochemicals Inc., Natick, MA) are made up in water and stored at - 8 0 °. Bafilomycin A1 (LC Laboratories, Woburn, MA) is dissolved in DMSO as a 1000× stock. Homogenates from control PC-12 cells and hVAChT-expressing PC-12 cells are always analyzed together to assess the specific ACh uptake derived from endogenous rat VAChT, which is less than 2-fold over that seen at 4 ° or in the presence of 2/xM vesamicol. For [3H]vesamicol or [3H]TBZOH binding assay, aliquots (50/zl) of postnuclear homogenates containing 30/zg protein are mixed with uptake/ binding buffer and various concentrations of [3H]vesamicol (31 Ci/mmol, NEN Life Science Products) or 3H-TBZOH are added. Nonspecific binding is determined by incubating parallel samples in the presence of a 300fold excess of unlabeled L-vesamicol or tetrabenazine. The suspensions are warmed to 20° and incubated for 1 h. The uptake and binding reactions are stopped by placing and tubes in ice-cold water, and vacuum filtering the samples through GF/B glass fiber filters and washing with 5 ml ice-cold uptake/binding buffer. Radioactivity bound to the filters is solubilized in 1 ml of 1% SDS followed liquid scintillation counting. Kin, IC50, and Kd values are determined by nonlinear regression. Interassay variability is generally less than 10%.
[7]
Photoaffinity Labeling of Vesicular Acetylcholine T r a n s p o r t e r f r o m E l e c t r i c O r g a n o f Torpedo
By S T A N L E Y
M.
PARSONS, GARY
A.
ROGERS,
and LAWRENCE M.
GRACZ
Introduction Direct biochemical study of acetylcholine (AcCh) storage in test tubes became possible after methods were developed for the purification of purely cholinergic synaptic vesicles from the electric organ of the marine ray Torpedof -3 The purification has since been improved. 4 Vesicles of greater than 95% purity can be isolated in about 21 days. When the purified vesicles are suspended in magnesium ion and ATP, they internalize exogenous 1 A. Nagy, R. R. Baker, S. J. Morris, and V. P. Whittaker, Brain Res. 109, 285 (1976). 2 S. S. Carlson, J. A. Wagner, and R. B. Kelly, Biochemistry 17, 1188 (1978). 3 K. Ohsawa, G. H. C. Dowe, S. J. Morris, and V. P. Whittaker, Brain Res. 161, 447 (1979). 4 L. M. Gracz and S. M. Parsons, Biochim. Biophys. Acta 1292, 293 (1996).
METHODS IN ENZYMOLOGY,VOL. 296
Copyright © 1998by AcademicPress All rights of reproduction in any form reserved. 0076-6879/98$25.00
100
VESICULAR CARRIERS
[7]
AcCh via the vesicular AcCh transporter (VAcChT). 5 The availability of this assay combined with well-controlled work by Howard and colleagues in PC-12 (rat adrenal pheochromocytoma) cells led to characterization of the bioenergetics of AcCh storage. It has been shown that uptake is dependent on internal acidification of the vesicles by a V-type ATPase and that a separate VAcChT protein exchanges "luminal" protons for external (cytoplasmic) AcCh to effect storage of the neurotransmitter. 6-s Pharmacological characterization of the VAcChT began with the demonstration that it can be distinguished from other cholinergic proteins (such as the nicotinic and muscarinic receptors) by its relative insensitivity to known drugs. 9 The compound trans-2-(4-phenylpiperidino)cyclohexanol (then called AH5183 and now called vesamicol) was found to inhibit uptake noncompetitively l°,u by binding to an enantioselective site in the VAcChT. 12 These findings confirmed the hypothesis by Marshall 13 about the action of vesamicol based on physiologic experiments. Extensive structure-activity studies of AcCh 14'15and vesamicol. ~6-2° have been carried out utilizing synaptic vesicles purified from Torpedo. Synthesis of [3HlAzidoacetylcholine (I) and [3HlAzidoaminobenzovesamicol (II) The VAcChT has been photoaffinity labeled with analogs of AcCh and vesamicol called [3H]azidoAcCh (I) and [3H]azidoABV (II), respectively 5 S. M. Parsons and R. Koenigsberger, Proc. Natl. Acad. Sci. USA 77, 6234 (1980). 6 L. Toll and B. D. Howard, J. Biol. Chem. 255, 1787 (1980). 7 D. C. Anderson, S. C. King, and S. M. Parsons, Biochemistry 21, 3037 (1982). 8 B. W. Hicks and S. M. Parsons, J. Neurochem. 58, 1211 (1992). 9 D. C. Anderson, S. C. King, and S. M. Parsons, Molec. Pharmacol. 24, 48 (1983). 10 B. A. Bahr and S. M. Parsons, J. Neurochem. 46, 1214 (1986). n B. A. Bahr, E. D. Clarkson, G. A. Rogers, K. Noremberg, and S. M. Parsons, Biochemistry 31, 5752 (1992). 12 B. A. Bahr and S. M. Parsons, Proc. Natl. Acad. Sci. USA 83, 2267 (1986). 13 I. G. Marshall, Br. J. Pharmacol. 40, 68 (1970). 14 G. A. Rogers and S. M. Parsons, MoL Pharmacol. 36, 333 (1989). 15 E. D. Clarkson, G. A. Rogers, and S. M. Parsons, J. Neurochem. 59, 695 (1992). 16 G. m. Rogers, S. M. Parsons, D. C. Anderson, L. M. Nilsson, B. A. Bahr, W. D. Kornreich, R. Kaufman, R. S. Jacobs, and B. Kirtman, J. Med. Chem. 32, 1217 (1989). 17 G. A. Rogers, W. D. Kornreich, K. Hand, and S. M. Parsons, Mol. Pharmacol. 44, 633 (1993). 18 S. M. Efange, A. Khare, S. M. Parsons, R. Bau, and T. Metzenthin, J. Med. Chem. 36, 985 (1993). ,9 S. M. Efange, A. B. Khare, C. Foulon, S. K. Akella, and S. M. Parsons, J. Med. Chem. 37, 2574 (1994). 20 S. M. Efange, R. H. Mach, C. R. Smith, A. B. Khare, C. Foulon, S. K. Akella, S. R. Childers, and S. M. Parsons, Biochem. PharmacoL 49, 791 (1995).
[7]
PHOTOLABELINGOF VAcChT
101
O%c/OCH2--~ I
o
(i)
~
~
,~
o
3
N
? o
(,) Fro. 1. Chemical structures of azidoAcCh [compound (I)] and azidoABV [compound (II)].
(Fig. 1). The structure-activity studies mentioned above yielded the synthetic routes to chemical intermediates required to make radioactive arylazido compounds predicted to have relatively high affinity for the AcCh and vesamicol binding sites. All reagents needed for the syntheses of these compounds are available from standard commercial sources except as noted below. The reader should consult the original references for interpretation of the spectroscopic data used in the structure proofs.
ffH]Azidoacetylcholine
(I) 21
Cyclohexylmethyl Isonipecotate. Isonipecotic acid hydrochloride (2.1 g, 12.7 mmol) is suspended in 15 ml of thionyl chloride (SOCI2) and heated to 40° for 12 hr. Excess 8OC12 is removed on a rotary evaporator with the aid of dry CH2C12. The acid chloride is dissolved in dry CH2C12 and heated in a fume hood in order to remove the remaining SOCl 2. During several hours, aliquots of cyclohexylmethanol (a total of 3.5 equivalent) are added to the stirred solution, which is then refluxed overnight. The CHzCI2 solution is washed with cold aqueous carbonate and dried o v e r N a z S O 4 . Removal of the solvent results in an oil that is chromatographed on silica gel. Product ester is eluted with 5% (v/v) ethanol/CHCl3 in 65% yield as a colorless oil. The hydrochloride salt, which is produced by precipitating the product 21 G. A. Rogers and S. M. Parsons,
Biochemistry31, 5770 (1992).
102
VESICULARCARRIERS
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dissolved in ether by bubbling in anhydrous HC1 gas, has melting point (mp) of 161.5-163 °.
Cyclohexylmethyl-cis-N- (4-azidophenacyl)-N-methyl Isonipecotate Bromide. Cyclohexylmethyl isonipecotate (9 rag, 40/~mol) and [3H]CH3I (0.65 tzmol, 10.8 Ci/mmol, Amersham Corp., Arlington Heights, IL) are combined in 0.8 ml of toluene in a 1-ml screw-cap vial. After 30 hr in the dark, succinic anhydride (5 mg) plus 80/zl of CC14 are added. The mixture is vortexed for 5 min to effect solution. This step facilitates separation of the product because succinic anhydride reacts with the large excess of unreacted cyclohexylmethyl isonipecotate (which is a secondary amine) but not with the methylated product (which is a tertiary amine). A large excess of cyclohexylmethyl isonipecotate is used to suppress double methylation. After 20 hr in the dark, the reaction mixture is chromatographed on silica gel. The product elutes with 5% methanol/CHCl~. Succinylated cyclohexylmethyl isonipecotate remains on the column. After the methanolic fractions are pooled and evaporated to dryness, the residue is dissolved in dry (C~H5)20 (200/zl). 4-Azidophenacyl bromide (5 mg, 21 /zmol) plus one drop of N,N-diisopropylethylamine are dissolved in the ethereal solution, which is stored in the dark for 2 days. Chromatography of the reaction mixture on silica gel provides product that elutes with methanol in 35% radiochemical yield. Fractions containing most of the radioactive peak are pooled, solvent is removed under vacuum, and the residue is taken up in 1 ml ethanol [yielding about 228 /zM (I)] for storage at - 2 0 °. The nonradioactive compound (FW 479) can be made similarly on a larger scale.
FH]AzidoABV (II) 22 N-(Trifluoroacetyl)-l-amino-5,8-dihydronaphthalene Oxide. A l-liter, three-necked, round-bottom flask is equipped with a large cold-finger condenser filled with solid CO2 and 2-propanol. To the flask 1-aminonaphthalene (79 g, 0.55 mol, carcinogenic!), (C2H5)20 (300 ml), ten-butanol (50 ml), and NH3 (200-300 ml) are added with stirring. Sodium (30 g, 1.3 mol, dried with paper towels just before weighing and returned to mineral oil after weighing until just before use) is added in portions over a 4-hr period, followed by an additional 50 ml of ten-butanol. After 1 hr, absolute ethanol (100 ml) is added slowly. This mixture is allowed to stir overnight and then is quenched by careful addition of NH4C1 (50 g) and H 2 0 (400 ml). The two liquid layers are separated, and the aqueous layer is extracted two times with ether. The ether extracts are combined with the organic layer, and this solution is extracted twice with water, once with a saturated NaC1 22G- A. Rogers and S. M. Parsons, Biochemistry 32, 8596 (1993).
[7]
PHOTOLABELINGoF VAcChT
103
solution, dried over Na2SO4, and finally filtered through MgSO4. Ether and most of the tert-butanol are removed at 65° on a rotary evaporator. This material may be used directly for the next step or colorless 1-amino5,8-dihydronaphthalene (mp 37-39 °) may be obtained in 97% yield after vacuum distillation. 1-Amino-5,8-dihydronaphthalene (60.1 g, 0.414 mol) is dissolved in 200 ml of stirred benzene and cooled to 0°. Trifluoroacetic anhydride (89 g, 0.42 tool) is added slowly because of the exothermicity of the reaction. The ammonium salt begins to precipitate almost immediately, but the reaction solution is homogeneous upon complete addition of the anhydride. This step is necessary to protect the amino group from oxidation during formation of the epoxide (below). The solution is maintained at 0° for 1 hr, after which benzene and trifluoroacetic acid are removed under reduced pressure. More benzene is added and again evaporated in order to promote the removal of trifluoroacetic acid. The resulting amide is dissolved in 300 ml of stirred (C2H5)20 to which is added 3-chloroperoxybenzoic acid (85.0 g, 80-85% pure). The solution is maintained near 10° throughout the addition and then allowed to warm to 23 ° and stirred for 5 hr. The product epoxide containing some 3-chlorobenzoic acid is collected by filtration and washed with ether. The solid is resuspended in 400 ml of (C2H5)20, thoroughly washed by agitation, and recollected by filtration to yield 89.0 g of epoxide (mp 174-178.5°). After extraction of 3-chlorobenzoic acid from the ethereal mother liquor with aqueous carbonate, an additional 2.1 g of product can be isolated by evaporation of the ether for an overall yield of 85%. Purification of the epoxide by crystallization from CHC13 with a wash of cold (C2H5)20 raises the melting point to 179.5-181 °.
(+_)-trans-5-Amino-2-hydroxy-3-(4-phenylpiperidino)tetralin [(+_)-ABV, FW 322]. N-(Trifluoroacetyl)-l-amino-5,8-dihydronaphthalene oxide (1.9 g, 7.4 mmol) is dissolved in 25 ml of absolute EtOH to which is added 4phenylpiperidine (3.0 g, 19 mmol). The solution is maintained at 45 ° for 17 hr and then refluxed for 3 hr. After 7 hr at 23° a crystalline solid is collected by filtration and set aside. The solid is a positional isomer of (_+)ABV. The mother liquor is evaporated to an oil that is taken up in CC14. This process is repeated in order to remove most of the EtOH. When the oil is again dissolved in CC14, crystallization of 4-phenylpiperidinium trifluoroacetate commences. The remaining mother liquor is chromatographed on silica gel where N-(trifluoroacetyl)-4-phenylpiperidine elutes with CC14 and (_+)-ABV with CC14/CHC13 (0.84 g, 35% yield). (_+)-ABV is crystallized from CHC13/ethanol (mp 174-175°C). 16 (-)-N-Glycyl-4-aminobenzovesamicol [(-)-GlyABV]. (_+)-ABV and N-tert-butoxycarbonylglycine p-nitrophenyl ester (1 equivalent) are reacted
104
VESICULARCARRIERS
[7]
in acetic acid in a closed vial overnight at 23°, and the acetic acid is removed in vacuo. The condensation product is taken up in 50% (v/v) trifluoroacetic acid/CHzCl2 and allowed to deblock in a sealed vial overnight at 45 ° to yield (_+)-GlyABV trifluoroacetate after removal of solvent in vacuo. After dissolving the trifluoroacetate in CH2C12 and washing it with aqueous carbonate, neutral (_+)-GIyABV is purified by chromatography on silica gel and then resolved into its enantiomers by chromatography on a Chiralpak AD semipreparative column (Daicel Chemical Industries, Exton, PA) in 20% 2-propanol/hexane. 17 The elution order of the enantiomers is extremely sensitive to the composition of the solvent and age of the column; thus, the optical rotations of the separated enantiomers should be taken to confirm their identities.
(-)-N-(4'-Azidobenzoylglycyl)-4-aminobenzovesamicol ([3H]azidoABV). N-[benzoate-3,5-3Hz]Succinimidyl 4-azidobenzoate (5.09 nmol, 49.1 Ci/mmol, NEN Division of DuPont, Inc., Boston, MA) is combined with 10.7 ~g (28.2 nmol) of (-)-GlyABV in 100 tzl of 2-propanol and placed in the dark. After 4 days the sample is diluted with 100/xl of ethanol. The product is purified by high-performance liquid chromatography on a semipreparative Chiralpak AD (Daicel Chemical Ind.) column (25% ethanol/hexane at 4 ml/min) with collection of fractions. It is important to turn the UV detector off as soon as elution of (II) begins at about 24 min. Unreacted (-)-GlyABV elutes at about 16 min. 22 Fractions containing most of the radioactive peak are pooled, solvent is removed under vacuum, and the residue is taken up in 1 ml ethanol [yielding about 3.26/xM (II)] for storage at -20 °. About 160 ~Ci of (II) in 64% radiochemical yield is obtained. The nonradioactive compound (FW 524) can be made similarly on a larger scale. Isolation of Cholinergic Synaptic Vesicles from Electric Organ of Torpedo
Torpedo are obtained live from a commercial fisherman or a commercial supplier of marine animals. Different species of Torpedo are found in substantial abundance in most of the major oceans of the world, and they often are caught when feeding on commercially exploited fish stocks. Torpedo californica commonly weigh 20-40 pounds, and about 15% of the body weight is electric organ. The fish are rested in a filtered well-aerated tank of seawater for 3-4 days after delivery. They are stunned by a blow to the back of the head and pithed with a scalpel, and the nerve bundles innervating the electric organs are severed. Because the fiat cartilaginous skull efficiently delivers the blow to the brain, the rays are immediately rendered unconscious and do not suffer as far as can be determined. The
[71
PHOTOLABELINGOF VAcChT
105
electric organs, which fill most of the wings, are readily recognized and removed by blunt dissection after the overlying skin is peeled back. They are frozen immediately in liquid nitrogen, after which they are fractured into chunks that are packaged into 800-g portions and stored at -100 ° until use. 4 The deep-frozen electric organ (800 g) is shaved very finely with a rapidly spinning blade to produce a "snow." The yield of vesicles decreases greatly if a very fine snow is not produced, and a very sharp blade seems to be the most important factor in obtaining favorable yields. A simple, commercially available machine used to make flavored "snow cones" at carnivals works well. The snow is allowed to warm slowly to - 2 ° with occasional mixing. The slush is suspended in 800 ml of ice-cold buffer composed of 0.76 M glycine containing 0.05 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES), 0.01 M ethylene glycol bis(/3aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 0.001 M ethylenediaminetetraacetic acid (EDTA), 0.02% NAN3, and 2 /~M 2,6-bis(1,1-dimethylethyl)-4-methylphenol (BHT) adjusted to pH 7.2 with 0.80 M NaOH. All subsequent steps in the vesicle purification are conducted at 4°. The slush is divided into portions that are slowly sheared with a blender for 20 sec, after which they are recombined. Approximately 300 ml is homogenized with five slow strokes using a tightly fitting, spinning Teflon pestle in a 350-ml glass mortar of the Potter-Elvehjem type. A drill press can be adapted to drive the pestle. The tissue homogenate is centrifuged in six 500-ml polycarbonate bottles at 17,700g for 1 hr to remove membrane debris. Twenty 70-ml polycarbonate centrifuge tubes having O ring screw caps are filled with supernatant to within 1 cm of the neck. Using a cannulated syringe, 6 ml of isosmotic "cushion" composed of 650 mM sucrose, 7.5% (w/w) Ficoll 400 (Sigma, St. Louis, MO), 10 mM HEPES, 1 mM EDTA, and 1 mM EGTA (adjusted to pH 7.0 with 0.80 M KOH) having a density of 1.117 g/ml is injected under the supernatant onto the bottom of each tube. Ficoll 400 is used to obtain increased density beyond that provided by isosmotic sucrose because it contributes little to the osmotic pressure and its high viscosity stabilizes the interface between the supernatant and the cushion. After balancing them with additional supernatant, the tubes are centrifuged at 49,000g for 16-21 hr in two Beckman Type 21 rotors. The membranes focused on top of the cushions are removed with a cannulated syringe and pooled to yield about 160 ml of crude vesicles. The vesicular pool is mixed with sufficient fresh cushion solution to increase the density of the mixture enough to layer under 0.40 M sucrose (see proceeding text). To accomplish this, 15 ml of fresh cushion solution is added to the pool, which then is mixed. A drop of the mixed pool is
106
VESICULAR CARRIERS
[7]
placed onto 0.40 M sucrose in a test tube to determine whether it falls to the bottom. Additional fresh cushion in 3 ml increments is added to the pool with mixing and the mixture is tested until it does fall. About 20-40 ml of cushion is required. Isosmotic density gradient solutions (0.5 liter each) composed of 0.01 M HEPES, 0.001 M EDTA, 0.001 M EGTA, and 0.02% (w/v) NAN3, adjusted to pH 7.0 with 0.8 M KOH and containing either 0.80 M glycine or 0.65 M sucrose, are prepared. An isosmotic solution containing 0.40 M sucrose is prepared by mixing 50 ml of the 0 M sucrose solution with 80 ml of the 0.65 M sucrose solution. A typical density gradient is formed from 0 M sucrose (7 ml), 0.40 M sucrose (8 ml), pooled vesicles containing additional cushion (22 ml), and 0.65 M sucrose (4 ml). The solutions are applied to the bottom of a 1 × 3.5-inch dome-top polyallomer quick-seal tube (Beckman Coulter Instruments, Inc., Fullerton, CA) in the order stated with a cannulated syringe. The eight required tubes are heat sealed and centrifuged at 196,200g for 3.5 hr in a Beckman VTi 50 vertical rotor. They are opened by carefully slicing the tops off with a single-edge razor blade. In initial work, 20 fractions of 1.5 ml each are obtained from each gradient by bottom puncture. The fractions are assayed for ATP content using the luciferase assay.23 Mature cholinergic synaptic vesicles isolated with this procedure (so-called VP1 vesicles) band at 1.055 g/ml and contain ATP as well as AcCh. The densities of the fractions can be estimated from the refractive indices,z4 The vesicles are found in a faint milky layer near the top, and after experience is gained, they can be withdrawn rapidly from the opened centrifuge tube by use of a cannulated syringe and visual inspection. Vesicles in the pool (50 ml) are further purified by size-exclusion chromatography on a Sephacryl S-1000 column (150 cm × 1.5 cm) equilibrated with buffer containing 820 mM glycine, 5 mM HEPES, 1 mM EDTA, 1 mM EGTA, and 0.02% NaN3 (adjusted to pH 7.0 with 0.80 M KOH). The column effluent is monitored by apparent absorbance at 350 nm. The vesicles elute in the middle of the resolving volume as the second peak of apparent absorbance (due to light scattering). The fractions containing vesicles are pooled and concentrated with a Centricon centrifugal ultrafiltration device (Amicon, Danvers, MA, 10 kDa cutoff) to 1-2 mg protein/ ml if desired. About 5 mg of vesicular protein is obtained as determined by the Bradford 25 dye assay. The VAcChT in these vesicles is stable for weeks when stored in a closed vessel at 4°. 23 G. A. Kimmich, J. Randles, and J. S. Brand, A n a l Biochem. 69, 187 (1975). 24 p. Sheeler, "Centrifugation in Biology and Medical Science," pp. 246-247. Wiley, New York, 1981. 25 M. M. Bradford, A n a l Biochem. 72, 248-254 (1976).
[7]
PHOTOLABELINGOF VAcChT
107
Assay of Ligand Binding or Transport by Purified Synaptic Vesicles Binding and transport assays are done similarly to each other. Purified vesicles at about 0.075 to 0.2 mg protein/ml are incubated with a radioactive compound in buffer containing 0.10 M HEPES, 0.70 M glycine, 0.001 M EDTA, and 0.001 M EGTA adjusted to pH 7.80 with 0.80 M KOH (TB). This pH is optimal for both the AcCh and vesamicol families of ligands in both transport and binding assays. The buffer also includes components such as paraoxon to inhibit AcCh esterase and MgATP to drive transport as needed. Samples are prepared in the absence and presence of an excess of nonradioactive ligand that blocks the specific binding or transport of the radioactive ligand. Samples are mixed well by use of a vortex device after addition of reagents. Glass-fiber filters (Whatman, Clifton, N J, GF/F, 1.3 cm) that have been pretreated with polyethyleneimine are mounted on a 10-place filter manifold/collection box (Hoefer Scientific Instruments, San Francisco, CA, model FH 225V). To prepare them, 400 filters are soaked 3 hr in 400 ml of 0.5% (w/v) polyethyleneimine in water. They are poured gently into a Btichner funnel of 8 inch diameter and rinsed generously with water by filtration. The funnel with the filters is placed overnight in an oven at 37° for drying. Dry filters are removed carefully from the funnel with tweezers and placed in a plastic food baggy with zipper after which they can be stored at room temperature in the dark. After incubation of vesicles with a radioactive ligand, a portion (typically 50-100/xl) is applied with suction assistance onto a filter prewetted just before with ice-cold TB. The filter then is immediately and rapidly washed by 3-4 1-ml portions of ice-cold TB with suction assistance. The radioactivity trapped on the filter is determined by liquid scintillation spectroscopy. Filters must be extracted overnight by the scintillation cocktail (a type compatible with aqueous solutions) for accurate and full determination of bound radioactivity. AcCh becomes soluble in cocktail containing 10% water better than it does in dry scintillation cocktail. The decrease in bound radioactivity due to the presence of excess nonradioactive inhibitor is the specific binding or transport by the vesicles.
Reversible Binding of Compound (I) Before irreversible photoaffinity labeling is attempted, it is important to demonstrate that a potential label binds in a well-behaved manner to the expected site. As compound (I) is stable to normal indoor lighting, its interaction with cholinergic synaptic vesicles can be studied under reversible conditions. Although the effect of sunlight on the compound has not been determined, it should be avoided by working in an inner room or in a
108
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room with shaded windows. These comments apply to compound (If) also (see below). All TB contains 0.15 mM paraoxon. Purified synaptic vesicles at about 2 mg protein/ml in TB are incubated at 23 ° for 30 rain to allow full inactivation of acetylcholinesterase. Two microliters of compound (1) in ethanol prepared as above is dried in v a c u o and dissolved in 40 p,1 TB to yield about 10 ~M of compound (I). The exact concentration of compound (I) is determined by measuring the radioactivity in a 5 p,l portion. An approximately isosmotic solution of AcCh in which 0.35 M AcCh chloride replaces the 0.70 M glycine in TB is prepared by dissolving 150 mg of preweighed AcCh chloride (Sigma) in 2.36 ml of TB lacking glycine. The pH of this solution should be checked and adjusted to 7.80 if necessary. Serial dilutions of this solution are made with TB to prepare additional isosmotic solutions containing 35, 3.5, and 0.35 mM AcCh. Also, 0.35 M AcCh chloride containing vesamicol is prepared by dissolving 150 mg of preweighed AcCh chloride in 2.36 ml of TB lacking glycine but containing 60 /~M (-)-vesamicol hydrochloride (FW 295.5, Research Biochemicals International, Inc.). Vesamicol hydrochloride is soluble to at least 1 mM in aqueous solutions. Vesamicol is used to measure the extent of nonspecific binding of (I) because at equilibrium it is a competitive inhibitor of the binding of AcCh analogs, u Because they spontaneously degrade slowly, aqueous solutions of AcCh must be prepared shortly before use. Eightysix microliters from each stock solution (including TB) is dispensed into individual tubes, and 4 /~1 compound (I) in TB is added and mixed in. Vesicular suspension (10/A) is added and allowed to equilibrate for 15 min at 23 °. Bound compound (1) is determined by filtration and washing of 90 /~1 of the suspension as described above. The results of a typical binding experiment are shown in Fig. 2. Because the concentration of the binding site (about 40 nM) is comparable to the dissociation constant for (I), the concentration of free (I) has to be calculated iteratively at each concentration of AcCh. Hence, for each total concentration of (I), the free concentration in solution is calculated from Eqs. (1) and (2): (I)F = (|)T -- (I)B (I)B = [Bmax X (I)v X KIA]/(1)F X KIA + A c C h x K(I) + K(I) x KIA]
(1)
(2)
where (I)F is the free concentration of (I), (I)T is the total concentration of (I), (I)B is the bound concentration of (I), K(I ) is the dissociation constant for the specific VAcChT- (I) complex (240 nM 21), KIA is the dissociation constant for the specific VAcChT • AcCh complex, and AcCh is the concentration of AcCh. Regression analysis is done with the program Scientist
[7]
PHOTOLABELINGOF V A c C h T
109
240 200
___//
•
,.C 160 rO ¢.)
~0 120 "U
"~ ~'~
80
40 0
0
Z j 10,~
10~-~
i0'-~
10'-1
16o
:
[AeCh](M)
FIG. 2. Inhibition of the reversible binding of compound (I) by AcCh. Final concentrations of the vesicular protein and (I) were 0.2 mg/ml and 440 nM, respectively. Bound (I) was determined by filtration as described in the text. Nonspecifically bound (I) was estimated in the presence of 60 I~M vesamicol (plus 300 mM AcCh) and was subtracted from the total amount bound. The estimated KTA for AcCh from regression analysis is 17 _~ 3 mM using Eqs. (1) and (2) in the text. (Reprinted with permission from G. A. Rogers and S. M. Parsons. Biochemistry 31, 5770 (1992). Copyright 1992 American Chemical Society.)
( M i c r o M a t h Scientific Software, Salt L a k e City, U T ) , which is available only for personal computers. N o n r a d i o a c t i v e A c C h inhibited the binding of c o m p o u n d (I) with a dissociation constant of 17 ± 3 m M in the experiment of Fig. 2. T h e highest c o n c e n t r a t i o n of A c C h investigated displaced essentially all (I), as vesamicol displaced only a small additional amount. T h e equilibrium dissociation constant o b s e r v e d for A c C h is relatively high and thus possibly suspect as not arising f r o m the correct A c C h binding site. H o w e v e r , the a p p a r e n t dissociation constant for A c C h binding to this site decreases to a b o u t 0.3 m M u n d e r active transport conditions. T h e latter value is less than the estimated c o n c e n t r a t i o n of A c C h in cholinergic nerve terminals. 26 T h e a p p a r e n t increase in affinity of A c C h u n d e r active transport conditions occurs because of a large kinetic c o m p o n e n t in the Michaelis constant. 1~ Thus, analog (I) binds to the site for A c C h transport. This result d e m o n strates why it was necessary to carry out a structure-activity study before choosing an affinity label analog of A c C h for this system. A c C h and its close analogs bind to the V A c C h T so weakly that they would be unlikely to provide specific labeling of the transporter sufficient to identify it. 26S. M. Parsons, C. Prior, and I. G. Marshall, in "International Review of Neurobiology,'" Vol. 35 (R. J. Bradley and R. A. Harris, Eds.), pp. 279-390. Academic Press, New York, 1993.
110
VESICULAR CARRIERS
[71
Reversible Binding of Compound (II) Compound (If) has moderately high affinity for the VAcChT, and for this reason the high-affinity ligand ABV 17 is used in this experiment to block specific binding of compound (II). Synaptic vesicles from the center of the vesicular peak (having about 30/zg protein/ml) in the size exclusion chromatography step of the purification are used without concentration. Vesicles are diluted 60-fold into TB to about 0.5/~g protein/ml. All aqueous solutions of compound (II) and ABV must be handled in glass containers coated with Sigmacote (Sigma Chemical Co.) or the compounds will be lost through adsorption to the vessel surface. Plastic containers are unsuitable. Compound (If) in ethanol (1.5/zl) prepared as above is dried in vacuo and taken up in 50/zl TB to yield a solution that is about 100 nM in (|I). Serial dilutions with TB to about 1 nM of (If) are prepared on a 50/zl scale with retention of all of the dilutions. (+)-ABV (0.322 mg) is dissolved in 10 ml ethanol to yield 100/zM, and 1/zl is added to each of 6 tubes. The ethanol is evaporated from the tubes. Vesicles (100/zl) are added to these tubes and to 6 additional ones. After incubation for 30 min, two 10-~1 portions of each dilution of (II) are added to two samples of vesicles, one containing (+)-ABV and one not. After incubation for 11 hr in sealed tubes, the final total concentration of (If) in each sample is determined by measuring the radioactivity in a 10-/zl portion with liquid scintillation spectroscopy. The bound radioactivity then is determined by filtration and washing of a 90~1 portion as described above. Figure 3 shows the results of a typical experiment. Equations (3) and (4) are fit simultaneously to the data sets for total and nonspecific binding, respectively, using Scientist. (|I)T = Bmax X (II)/[(II) + K(II)] + S x (If) + C (II)N = S X (II) + C
(3) (4)
where (II)T is the total amount of bound (II), Bmax is the concentration of specific binding sites, (II) is the concentration of free (II) [assumed equal to all (II) in solution], K(II) is the dissociation constant for the specific VAcChT- (II) complex, (II)N is the amount of nonspecifically bound (II), S is the slope of the nonspecific binding of (II), and C is the intercept of the nonspecific binding of (II). The dissociation constant for specifically bound (II) was 2.1 + 0.4 nM and the Bmaxwas 0.29 + 0.03 nM (corresponding to 580 + 80 pmol/mg) in the experiment of Fig. 3. Photoaffinity Labeling and Visualization of Acetylcholine-binding Protein Photolysis of Compound (I) Bound to Vesicles. All TB contains 0.15 mM paraoxon. Synaptic vesicles (0.8 ml containing 0.8 mg protein) are
PHOTOLABELING ov VAcChT
[71
111
0.400
z~
0 ma00
",C O
'~ o.~oo
Z
,~ 0.100 o~
0.000
• 0.0
I 2.0 Cone.
I 4.0
I 0.0
of [all] AzidoABV
I 8.0
10.
(nM)
FIG. 3. Saturation of the reversible binding of compound (II). Bound (II) was determined by filtration as described in the text in the absence (0) or presence (m) of 800 nM (_+)-ABV to yield data sets representing the amount of total and nonspecificbinding at the indicated concentrations of compound (II), respectively.A simultaneous fit of Eqs. (3) and (4) in the text to the two sets of data yielded K(II)equal to 2.1 _+0.4 nM and Bmaxequal to 0.29 -+ 0.03 nM (corresponding to 580 + 80 pmol/mg) for specific binding. (Reprinted with permission from G. A. Rogers and S. M. Parsons, Biochemistry 32, 8596 (1993). Copyright 1993American Chemical Society.) incubated in TB for at least 30 min. Seven portions of 100/xl each are placed into separate tubes. Three microliters of compound (|) in ethanol prepared as above is dried in v a c u o and dissolved in 60/xl TB to yield about 10 txM of (I). The exact concentration of compound (I) is determined by measuring the radioactivity in a 5-txl portion. Nonradioactive (1) (0.479 mg) is dissolved in 1 ml of TB [yielding 1 m M (|)]. ( - )-Vesamicol hydrochloride is made up to 1 mM in TB by dissolving 0.296 mg in i ml. Concentrated M g A T P in TB is adjusted to p H 7.80 with K O H and diluted to 100 m M with TB. AcCh is made up to 350 m M as above. Samples A and B (lanes 2 and 4 in Fig. 4) receive 145/xl of TB; sample C (lane 3) receives 25/xl of nonradioactive (I) and 120 ixl of TB; sample D (lane 6) receives 25/xl of nonradioactive (l), 25/xl of MgATP, and 95/xl of TB; sample E (lane 8) receives 71.4 txl AcCh and 73.6/xl of TB; sample F (lane 7) receives 2.14 txl AcCh and 142.9/xl of TB; and sample G (lane 9) receives 0.25/xl vesamicol and 144.75 b~l of TB. All samples receive 5 txl radioactive (I). Because of a high level of nonspecific binding, a concentration of (I) slightly below its dissociation constant value is used in this experiment. This yields an optimal compromise between good specific and excessive nonspecific labeling. Samples destined for lanes 3-9 (Fig. 4) are irradiated simultaneously using a handheld 18-watt lamp (Blak-Ray, UVP, Inc., San Gabriel, CA) that emits light in a band of wavelengths centered at 366 nm. Irradiation is carried out in four periods of 2 min each with mixing after each period.
112
VESICULAR CARRIERS 1
2
3
4
5
6
7
[71 8
9
116-97-66--
45--
29--
Fia. 4. Photoaffinity labeling and visualization of the AcCh-binding protein by compound ([). Vesicles were labeled in the presence or absence of the indicated reagents and then subjected to SDS-PAGE and autofluorography as described in the text. The figure is a composite showing Coomassie Blue staining, a short-term film exposure to the dye front region of the gel, and a long-term exposure to the stacking and resolving regions of the gel. Lane 5 contained standard proteins with their masses (kDa) indicated to the left. Lanes 1 and 4 are the Coomassie staining and autofiuorographic patterns, respectively, of the same sample of vesicles. AzidoAcCh refers to compound (I). Lanes 2 and 3 were controls that received no irradiation or 100/xM of nonradioactive (I), respectively. Lanes 7 and 8 contained 3 or 100 mM AcCh, respectively, and lane 9 contained 1 ~M vesamicol. Lane 6 contained 100 /xM of nonradioactive (I) and 10 mM of MgATP. (Reprinted with permission from G. A. Rogers and S. M. Parsons, Biochemistry 31, 5770 (1992). Copyright 1992 American Chemical Society.)
U n d e r similar conditions, the half-life for photolysis of the azido g r o u p is a b o u t 1 m i n as d e t e r m i n e d by u l t r a v i o l e t spectroscopy. A f t e r photolysis, a n a l i q u o t of 20/~1 is t a k e n f r o m each s a m p l e a n d filtered. T h e filtered samples are w a s h e d r e p e a t e d l y with T B at 23 °, a n d b o u n d t r i t i u m is determ i n e d . V e s a m i c o l (1 ~ M ) blocks 45 p m o l of label i n c o r p o r a t i o n / m g of p r o t e i n . This c o r r e s p o n d s to a b o u t 9% of the specific b i n d i n g assessed s e p a r a t e l y with 2 5 0 / z M [3H]vesamicol ( N E N division of D u P o n t , Inc.).
[71
PHOTOLABELINGOF VAcChT
113
The remaining volume of each sample is centrifuged at 160,000g and 23 ° in a Beckman Airfuge for 60 min in order to pellet the vesicles, which are immediately analyzed by SDS-PAGE. SDS-PA GE and Autofluorography of Vesicles Photolabeled with Compound (I). Photolabeled and pelleted synaptic vesicles are subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 10% resolving gel, a 3% stacking gel, and the discontinuous buffer system of Laemmli. 27 For this purpose, after removal of the supernatant the pelleted vesicles are dissociated in 80/zl of a treatment buffer made by dissolving 4.8 g urea in 4 ml of 10% SDS and diluting with 5 ml of 0.75 M Tris-HC1 (pH 8.8). The samples are vortexed repeatedly during a 50min period with the addition of 12/xl of 2-mercaptoethanol to each after 20 min. Finally, all samples are heated to 90° for 2 min. One-half of each sample is stored at - 2 0 ° for possible reanalysis and the other half is subjected to electrophoresis. After electrophoresis, the gel is stained with Coomassie Brilliant Blue R in order to visualize proteins and then treated with Intensify (Du Pont, Inc.) as instructed by the manufacturer before being dried onto filter paper under vacuum. The dried gel is autofluorographed with XAR-5 film (Kodak, Rochester, NY) at - 8 0 °. A continuous region from about 50 kDa to the top of the resolving gel is heavily labeled by photolyzed (I) (Fig. 4, lane 4). The protein stain (lane 1) of the same gel lane demonstrates that the diffuseness of the fluorogram does not arise from poorly focused proteins. No protein corresponding to the diffuse pattern of radiolabeling is stained clearly. An absence of extensive nonspecific aggregation is indicated by the lack of protein staining or radioactivity in the stacking gel. Lane 2 (Fig. 4) demonstrates that photolysis is required for labeling, and lanes 3 and 6 show that excess nonradioactive (I) blocks labeling of virtually all proteins, whether or not vesicles are energized by ATP. Lanes 7 and 8 (Fig. 4) show increasing levels of protection from labeling by 3 and 100 mM ACh, respectively. The rather modest amount of protection obtained with 3 mM AcCh is consistent with the KIA value of 17 mM. Lane 9 (Fig. 4) shows that vesamicol at only 1 tzM nearly completely blocks the diffuse labeling seen in lane 4. In summary, the protection pattern seen in Fig. 4 is fully consistent with specific labeling of the VAcChT at the AcCh binding site. Photoaffinity Labeling and Visualization of Vesamicol-Binding Protein
Photolysis of Compound (II) Bound to Vesicles. Four glass test tubes treated with Sigmacote each receive 4.6/.d of the ethanolic solution of (II) 27U. K. Laemmli, Nature 227, 680 (1970).
114
VESICULARCARRIERS
[71
prepared above. Five microliters of a 1 mM solution of (-)-vesamicol hydrochloride in ethanol is added to the second tube (final concentration to be 50/zM (-)-vesamicol), and 1 /zl of a 100 ~M ethanolic solution of (_+)-ABV is added to the third tube [final concentration to be 1/zM (_+)ABV]. A fifth treated tube receives 1.1 pA of the ethanolic (_+)-ABV. The ethanol in all tubes is evaporated. Next, 110 p~l of synaptic vesicles that are 0.65 mg of protein/ml TB is added to the fifth tube and incubated 1 hr to prebind ABV. Volumes of 100/~1 of the fresh vesicular suspension are added to the first three test tubes, and 100/zl of the vesicular suspension prebound to (_+)-ABV is added to the fourth test tube. All four suspensions are incubated for 10 min with occasional mixing and then illuminated with an 18-W UV lamp (Ultra-Violet Products Inc., San Gabriel, CA) that emits light in a band of wavelengths centered at 254 nm. For 10 min the tubes are alternately illuminated and vortexed to yield a total illumination time of about 5 rain each. Following the photolysis, 5 /zl of the solution is removed from each tube and the radioactivity measured.
SDS-PA GE and Autofiuorography of Vesicles Photolabeled with Compound (II). Fifty microliters is removed from each sample of photolabeled vesicles from above and diluted with 100/zl TB. The remaining portion of each sample is stored at -20 ° for possible reanalysis. The diluted vesicles are pelleted in an Airfuge (Beckman Instrument Co.) at 73,000 rpm for 70 min. The supernatant is carefully removed, and the pellets are covered with 20/zl of treatment buffer that contains 10% sodium dodecyl sulfate (SDS) and 20% glycerol in 0.125 M Tris-HC1 (pH 6.8). The samples are vortexed occasionally over a 6-hr period at 23 °. Finally, each is diluted with 20/zl of a 10% (w/v) solution of dithiothreitol (DTT) and heated in a 90° bath for 3 min. Molecular weight standards are treated with the same solutions but are given only a brief incubation prior to addition of the DTT. The dissociated samples are subjected to SDS-PAGE using a 5-15% linear gradient resolving gel, a 3% stacking gel, and the discontinuous buffer system of Laemmli.27 Electrophoresis is halted when the tracking dye is about 1 cm from the bottom of the gel. After electrophoresis, the gel is stained with Coomassie Brilliant Blue R in order to visualize proteins and then treated with Intensify (DuPont) before being dried onto filter paper under vacuum. The dried gel is autofluorographed with XAR-5 film (Kodak) at -80 ° for 3 days. The amount of radioactivity in each sample after photolysis should be similar and correspond to about 150 nM compound (II). Because the concentration of binding sites (about 450 nM as determined by dilution of a small volume of the untreated vesicles into 250 nM [3H]vesamicol) is in excess of both this concentration and the dissociation constant for compound (I|) (see above), almost all of (II) present in the solution will be
[71
PHOTOLABELINGOF VAcChT
115
specifically bound in the absence of competing ligand. Protein staining of the SDS-PAGE gel should reveal well-focused bands throughout the molecular mass range of 12-200 kDa. Autofluorography of the unprotected sample (Fig. 5, sample 1) demonstrates heavy labeling of a continuous region from about 50 to 200 kDa. Quantitation of the absorbance indicates that 94% of the total labeling of proteins (exclusive of the labeling at the dye front) is in this region. In addition, four polypeptides with Mr values of about 22,600, 33,000, 35,100, and 37,500 are specifically labeled. The 33kDa band is somewhat diffuse, and correlation with the Coomassie image
200 116 97 77 42.7
!
66
55.5
30 17.2
--
E
E
E
E
FIG. 5. Photoaffinity labeling and visualization of the vesamicol-binding protein with compound (I|). Vesicles were labeled in the absence or presence of competing ligands and then subjected to S D S - P A G E and autofluorography as described in the text. The figure is a composite showing Coomassie Blue staining (the lane called Protein) of Sample 1 and the autofluorograph of all four gel lanes. Sample 1 is the autofluorograph for nonblocked vesicles. Samples 2 and 3 show labeling when vesamicol or 1/xM (+_)-ABV was added simultaneously with the addition of compound (II), respectively, and sample 4 shows labeling when 1 /zM (+_)-ABV was added 1 hr prior to the addition of (ll). The masses (kDa) and positions of standard proteins are indicated to the left. Specifically labeled bands at 22.6, 33, 35.1, and 37.5 kDa in Sample 1 were visible in the original film, but may not be visible in the reproduction. (Reprinted with permission from G. A. Rogers and S. M. Parsons, Biochemistry 32, 8596 (1993). Copyright 1993 American Chemical Society.)
116
VESICULARCARRIERS
[81
suggests that the diffuse appearance arises as the result of a doublet of closely spaced bands at 32.4 and 33.8 kDa. Addition of 50/~M vesamicol coincident with compound (II) completely blocks incorporation of radioactivity into components with Mr greater than 12 kDa (sample 2). At the lower concentration of 1/zM, the higher affinity (+)-ABV protected nearly all sites above 12 kDa from being labeled, whether it was applied coincidentally (sample 3) or prior (sample 4) to exposure of the vesicles to compound (II). Neither vesamicol nor (_+)-ABV produced any visible change in the protein staining pattern (data not shown). Whether the specifically labeled species of Mr 22-38 kDa are proteolytic fragments of the VAcChT is not known. Overall, the protection pattern seen in Fig. 5 is fully consistent with specific labeling of the VAcChT at the vesamicol binding site. The different electrophoretic patterns for specific labeling in Figs. 4 and 5 are due to the use of different types of resolving gels. Vesicles labeled with compounds (I) and (II) yield similar patterns of specific labeling after SDS-PAGE utilizing the same gel conditions. The extreme streaking of the specifically labeled species is not due to inadequate sample preparation, as far as can be determined. Changes in the sample dissociation conditions, such as use of higher concentrations of SDS, urea, low temperature, and different reducing agents, do not affect the results. Thus, the AcCh and vesamicol binding sites reside in the same protein as judged by SDS-PAGE, and this protein exhibits poor staining and migration behavior in SDSPAGE. Possibly the VAcChT in synaptic vesicles of the electric organ is extraordinarily heavily glycosylated, or even high concentrations of SDS do not succeed in dissociating the protein fully.
[8] B i o e n e r g e t i c C h a r a c t e r i z a t i o n o f y - A m i n o b u t y r i c Acid Transporter of Synaptic Vesicles By
JOHANNES W.
HELL
and
REINHARD
JAHN
Introduction Neurotransmitters are stored in synaptic vesicles and, on depolarization and subsequent calcium influx, released from nerve terminals by exocytosis. The main inhibitory neurotransmitter in the mammalian brain is y-aminobutyric acid (GABA), whereas glycine is the prevailing inhibitory neurotransmitter in the spinal cord. The GABA uptake into synaptic vesicles is mediated by the vesicular GABA transporter, which probably works as an electrogenic GABA proton antiporter in contrast to the sodium-driven
METHODS IN ENZYMOLOGY, VOL. 296
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