- Email: [email protected]
[5] Noncovalent and covalent labeling of vesicular monoamine transporter with tetrabenazine and ketanserin derivatives: Purification of photolabeled protein
[5]
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[5] N o n c o v a l e n t a n d C o v a l e n t L a b e l i n g o f Vesicular Monoamine Transporter with Tetrabenazine and Ketanserin Derivatives; Purification of Photolabeled Protein By
J E A N - P I E R R E H E N R Y , C O R I N N E SAGNI~,
M A R I E - F R A N ~ O I S E ISAMBERT,
and B R U N O G A S N I E R
Introduction The vesicular monoamine transporter (VMAT) accumulates monoamines inside secretory granules or synaptic vesicles by catalyzing a H+/ monoamine antiport. Monoamine uptake is coupled to the generation of a proton electrochemical gradient by an ATP-dependent H + pump of the vacuolar type. The uptake is extremely efficient, and in the adrenal medulla chromaffin cells, the monoamine gradient between the cytosol and the secretory granules has been estimated to five orders of magnitude. 1'2 The transporter has a low specificity and will accept catecholamines, such as adrenaline, noradrenaline, or dopamine, the indoleamine serotonin (5-HT), histamine, and nonnatural substrates such as m-iodobenzylguanidine (MIBG) or the neurotoxin 1-methyl-4-phenylpyridinium (MPP +). The vesicular monoamine transporter from various origins (rat, human, and bovine) has been cloned and expressed in various cell types. 1,2 Two isoforms, VMAT1 and VMAT2, have been identified which have different localizations and slightly different properties. However, most of our knowledge regarding VMAT has been obtained on the protein from bovine chromaffin granules, where the major expressed isoform is VMAT2. An interesting pharmacology has been developed from this preparation. 3 Two different binding sites have been identified. The first one binds reserpine, whereas the second one binds tetrabenazine (TBZ) and ketanserin (KET). The H + electrochemical gradient generated in the presence of ATP greatly accelerates reserpine binding, but has no effect on TBZ and KET binding. As for other transporters, the enzymology of VMAT is complicated by the fact that VMAT catalyzes a vectorial activity, which has to be coupled to a proton gradient generating system. It is possible to reconstitute the purified protein in liposomes and to impose a pH gradient on the vesii S. Schuldiner, A. Shirvan, and M. Linial, Physiol. Rev. 75, 369 (1995). 2 y . Liu and R. H. Edwards, Am. Rev. Neurosc. 20, 125 (1997). 3 j. p. H e n r y and D. Scherman, Biochem. Pharmacol. 38, 2395 (1989).
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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cles. 4 However, it is also possible to use nonvectorial ligand binding to assay VMAT. Because TBZ and KET derivative binding is not affected by the H ÷ electrochemical gradient, these compounds are very useful ligands. Photosensitive derivatives of TBZ and KET can furthermore be used to label VMAT by photoaffinity techniques. An interesting aspect of covalent labeling is that the protein can be purified irrespective of its activity. During such purification studies, we introduced a new technique based on the selective aggregation of VMAT solubilized in sodium dodecyl sulfate (SDS) that results from the polytopic structure of the transporter. Consequently, it might be useful for the purification of other such transporters. TBZ and KET Binding to Vesicular Monoamine Transporter
TBZ Binding Various tritiated derivatives of TBZ have been used. The first one was a-[2-3H]dihydrotetrabenazine (2-hydroxy-3-isobutyl-9,10-dimethoxy-l,2,3, 4,6,7-hexahydro-llbH-benzo[a]quinolizine, [2-3H]TBZOH), obtained by reduction of TBZ with KB3H4 . This compound had a specific activity of about 15 Ci/mmol (0.5 TBq/mmol). 5 More recently, another tritiated derivative of a-dihydrotetrabenazine (a[O-methyl-3H]dihydrotetrabena zine) has been commercialized that has a higher specific activity (4.44-6.48 TBq/mmol, 120-175 Ci/mmol, Amersham, Buckinghamshire, U.K.). aDihydrotetrabenazine contains two enantiomers and the binding is stereospecific, the (+) isomer being the active one. 6 [O-methyl-3H]Tetrabenazine has also been tested successfully as a high specific activity ligand. 7 For Scatchard analysis of a-[O-methyl-3H]TBZOH binding to membrane preparations, various concentrations of the ligand at full specific activity (265-385 dpm/fmol) are incubated with membranes suspended in 0.2 ml of isosmotic medium pH 7.4 (saline or sucrose). When purified chromaffin granule membranes are used, 8,9 the membrane concentration is limited to about 10/xg of protein/ml to keep the site concentration lower than the equilibrium dissociation constant Ka.5 Usually, duplicate incubations are performed for each concentration, and nonspecific binding is 4 R. Yelin and S. Schuldiner, M e & o d s EnzymoL 296, [4], (1998) (this volume). 5 D. Scherman, P. Jaudon, and J. P. Henry, Proc. Natl. Acad. Sci. USA 80, 584 (1983). 6 M. R. Kilbourn, L. C. Lee, T. M. Vander Borght, D. M. Jewett, and K. A. Frey, Eur. J. PharmacoL 278, 249 (1995). 7 T. M. Vander Borght, A. A. F. Sima, M. R. Kilbourn, T. J. Desmond, D. E. Kuhl, and K. A. Frey, Neuroscience 68, 955 (1995). 8 A. D. Smith and H. Winkler, Biochem. J. 103, 480 (1967). 9 C. Sagn6, M. F. Isambert, J. P. Henry, and B. Gasnier, Biochem. J. 316, 825 (1996).
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measured by adding 2/zM TBZ to a third series of incubations. The mixtures are incubated for at least 2 hr at 25 °. Bound ligand is separated by filtration on H A W P filters (Millipore, Bedford, MA), pretreated with 0.3% PEI (polyethyleneimine) to decrease the background. The samples are diluted with 2 ml of ice-cold buffer containing 10/zM TBZ before filtration, and the radioactivity on the filter is measured by liquid scintillation counting in Ready Protein Plus mixture (Beckman, Fullerton, CA) or equivalent solutions. With purified chromaffin granule membranes, the signal-to-noise ratio at a ligand concentration equivalent to Kd is about 30. In all cases tested, corresponding essentially to VMAT2 from various origins, the equilibrium dissociation constant Kd is in the 1-10 nM concentration range. With purified chromaffin granule membranes, 8,9 K d and Bmax (maximal concentration of bound ligand, corresponding to the density of sites) are 3 nM and 60 pmol/mg of protein, respectively. The assay of VMAT density in a given membrane preparation can be performed by measuring B . . . . or more simply, if Ka is known, by measuring the bound ligand at a concentration equivalent to Kd. This protocol can be applied to VMAT solubilized by cholate or octyl /3-glucoside. Originally, the protein incubated with [3H]TBZOH was precipitated with polyethylene glycol 6000 before filtration. 1° However, this is not necessary since the solubilized transporter is adsorbed on GF/B glass fiber filters (Whatman, Clifton, N J) that have been pretreated by incubation for at least 3 hr with 0.3% PEI. 11 The incubation mixture is filtered under reduced pressure on these filters and the radioactivity is measured as previously. [3H]TBZOH binding ha.s also been measured on bovine chromaffin cells in culture. 12 [3H]TBZOH is a permeant molecule and specific binding can be measured on intact cells. The assay is performed in four plastic wells, each containing 500,000 cells. The culture medium is pipetted off and 3 nM [3H]TBZOH is added in i ml of Locke medium. For nonspecific binding determination, 2/xM TBZ is added to four other wells. After 1 hr incubation at room temperature, the medium is removed and the cells are washed three times by incubating for 3 min with 2 ml of Locke solution. Finally, the cells are collected in 0.2% Triton X-100 for counting radioactivity. Under the conditions described and for 500,000 cells, specifically and nonspecifically bound [3H]TBZOH are, respectively, 30-50 and 15 fmol. Specific binding does not change with the age of the culture, whereas nonspecific binding increases with the proliferation of nonchromaffin cells. The density 10 D. Scherman and J. P. Henry, Biochemistry 22, 2805 (1983). 11 M. S. Vincent and J. A. Near, Molec. Pharmacol. 40, 889 (1991). 17 C. Desnos, M. P. Laran, and D. Scherman, J. Neurochem. g9, 2105 (1992).
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of [3H]TBZOH binding sites, derived from Bmax, is 1 pmol/mg of protein (Ka = 16 nM), which should be compared to the value obtained on adrenal medulla homogenates (Bmax = 3 pmoles/mg of protein, Kd = 5 nM). One difficulty in using this method is compromising the rate of dissociation of the ligand from its site during the washing of intact cells. An alternative would be to perform the assay on cells with membranes permeabilized by the bacterial toxin streptolysin O. In this case, cells are placed in a 2-cm diameter plastic well and are resuspended in a medium containing 150 mM potassium glutamate/0.5 mM EGTA/5 mM magnesium acetate/ 10 mM PIPES, pH 7.2 (KG medium) containing 50 U/ml streptolysin O (Biomerieux, Lyon, France)/2 mM dithiothreitol (DTT), and 0.1% bovine serum albumin (BSA). After incubation in this medium for 15 min at room temperature, the solution is pipetted off. Then the cells are incubated with 3 nM [3H]TBZOH in KG medium for at least 1 hr at room temperature. At the end of the incubation, cells are rapidly washed two times with icecold KG and 0.3 ml of 0.1 N NaOH is added. After 15 min, the radioactivity of the suspension is counted by liquid scintillation. Nonspecific binding is measured by similar experiments to which 0.5/zM TBZ is added to the KG medium.
K E T Binding [3H]KET, which is principally a 5-HTzA receptor ligand, is obtained commercially with a high specific activity (40-90 Ci/mmol, 1.5-3.33 Tbq/ mmol), from NEN Life Science Products (Boston, MA). Binding to VMAT can be performed under the conditions used for [3H]TBZOH, taking two precautions.13 The first is to perform the filtrations rapidly in ice-cold buffer, because the rate of dissociation of [3H]KET is extremely rapid (half-life of 40 sec at 0°). The incubation mixture is diluted 10 times in ice-cold buffer, rapidly filtered on GF/C glass filters (Whatman), and washed with 1 ml of ice-cold buffer. Binding is measured at 30° (Kd of 45 nM) or, more easily at 0° (Kd of 6 nM). The second is to control pharmacologically the binding of [3H]KET to VMAT and not to 5-HT2A receptors. [3H]KET specifically bound to VMAT is obtained by subtracting the radioactivity measured in assays containing 2/xM TBZ, whereas that bound to 5-HT2A receptor was obtained by subtracting the radioactivity of incubations performed in the presence of 3/zM methysergide. In bovine chromaffin granule membranes, no 5-HT2A receptor binding can be detected. Photoaffinity Labeling of Vesicular Monoamine Transporter using 7-azido[8-125I]Iodoketanserin. 7-Azido[8-125I]iodoketanserin (AZIK) can a3F. Darchen, D. Scherman, P. M. Laduron, and J. P. Henry, Mol. Pharmacol.33, 672 (1988).
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be obtained from Amersham, but at the present time custom preparation is required. To decrease the cost, it is possible to order 7-amino[8-125I]iodo ketanserin and to use it in a one-step synthesis of AZIK. TM The carrier-free amino derivative solution (100/zCi) is dried under a flux of argon in the original vial to which an efflux active charcoal guard has been added. The compound is resuspended in 50/xl of 1 N HC1. Sodium nitrite (2 /xl, 2.5 M) is added with a Hamilton syringe under magnetic stirring at 4°. The solution is stirred at 4° in the dark for 30 min. Sodium azide (2/z, 2.5 M) is added at 4° and the mixture stirred for another 30 min in the dark. The AZIK formed is separated from the mixture by high-performance liquid chromatography (HPLC) on a C18 /xBondapak column (Waters, Milford, MA) with H20-CH3OH-trifluoroacetic acid (TFA) (50:50:0.1) as the solvent and a UV detector operating at 315 nm. The active fractions are concentrated to dryness, resuspended in ethanol, and kept at -20 °. The yield is 80%. Photolabeling with [t25I]AZIK. For analytical purposes, 15 membrane samples with a concentration of about 3 nM of KET/TBZ binding sites are incubated with a one- to two-order-of-magnitude lower concentration of AZIK, in order to bind a large fraction of the probe. For instance, 50/xg of protein/ml of purified bovine chromaffin granule membranes 8,9 is incubated with 40 pM [125I]AZIK (0.07 tzCi/ml, 0.16 x 10 6 dpm/ml) in 5 ml of 0.3 M sucrose/10 mM HEPES, pH 7.5, for 1 hr at 0°, in a 3.5-cm diameter well of a cell culture plate. The incubated samples are irradiated for 10 min with a UV lamp operating at 350 nm at an average distance of 6 cm, under magnetic stirring. The conditions for irradiation are selected to flatten the 240 nm absorbance peak of 7-azidoketanserin and to minimize the labeling of phospholipids at 254 nm. After irradiation, the membranes are washed twice by 4-fold dilution in sucrose buffer followed by centrifugation for 20 min at 140,000g at 4°. Samples (50-20 tzg of protein) are analyzed by SDS-PAGE, using an acrylamide concentration of 10% (w/v) and a bisacrylamide:acrylamide ratio of 0.8 : 30 (w/w). Proteins are stained with silver nitrate, using sodium thiosulfate to sensitize the gels] 6 or Coomassie blue. Autoradiography is obtained by exposure of the dried gel to Kodak X-OMAT AR films with Lumix MR 800 screens (Agfa Gevaert, Rueil-Malmaison, France) for 2-30 days at - 8 0 °, or by using a Phosphorimager apparatus (Molecular Dynamics, Sunnyvale, CA). When quantitative data are required, the stained gel is cut into 2-mm slices and the radioactivity of the slices is measured with 14 M. F. Isambert, B. Gasnier, D. Botton, and J. P. Henry, Biochemistry 31, 1980 (1992). 15 M. F. Isarnbert, B. Gasnier, P. M. Laduron, and J. P. Henry, Biochemistry 28, 2265 (1989). 16 T. Rabilloud, G. Carpentier, and P. Tarroux, Electrophoresis 9, 288 (1988).
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a y-counter (Fig. 1). As described for [3H]KET binding, the specificity of the labeling, i.e., labeling on VMAT and not on 5-HT2A receptor, is tested pharmacologically: VMAT labeling is inhibited in the presence of 2/zM TBZ, whereas 2 tzM methysergide inhibits 5-HTeA receptor labeling. In bovine chromaffin granules membranes, VMAT is labeled as a broad diffuse band with an apparent molecular mass centered at 73 kDa when 10% acrylamide concentration is used for SDS-PAGE. Increasing the acrylamide concentration increases the apparent molecular mass. In addition to the broad diffuse band corresponding to VMAT, another component is often visible as a sharp band with an apparent molecular mass of 40 kDa. This component is thought to be nonspecifically labeled since the corresponding band is not suppressed in the presence of TBZ. In some experiments, cytochrome b-561 (27 kDa), which is the major membrane protein, is also labeled. Bovine VMAT2 expressed in COS cells is labeled by the same technique as a component with an apparent molecular mass of 80 kDa. [125I]AZIK has also been used to photolabel a large stock of membranes, 14 for the purification of VMAT. Membranes (50 mg of protein) are thawed and suspended (2 mg/ml, final concentration) in 10 mM Tris-HC1 buffer (pH 7.5) containing 1 mM EDTA, 6 /xg/ml leupeptin, 5 /xg/ml aprotinin, 10 /zg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride (PMSF), at 0 °. [125I]AZIK(80/xCi; 145 × 106 cpm) is added and incubated for 1 hr in the dark at 0°. The incubation medium is positioned at 6 cm from a 350-nm UV light source and irradiated for 12 min at 0°. During these experiments, it has been noted that a radiolabeled pho1000
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slice number FiG. 1. Photoaffinity labeling of VMAT from purified bovine chromaffin granule membranes, using [125I]AZIK. In this experiment, 60/xg of photolabeled protein, corresponding to 95,000 and 50,000 cpm for, respectively, the samples incubated in the absence (filled squares) and in the presence (open squares) of 0.2/zM TBZ, were analyzed by SDS-PAGE.
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toproduct not covalently bound has a high affinity for the TBZ binding site, thus leading to a large overestimation of the protein covalently labeled (see Fig. 1). To minimize this effect, TMTBZ is added to a final concentration of 2/xM after irradiation and the mixture is incubated for 2 hr at room temperature. After centrifugation for 20 min at 140,000g at 4 °, the pellet is resuspended in about the same volume of TBZ-containing buffer and centrifuged again under the same conditions. The pellet is then resuspended in 5 ml of water containing leupeptin/aprotinin/pepstatin/PMSF at the concentration indicated above. The labeled membranes are stored in aliquots at - 8 0 °. The amount of photolabeled VMAT is determined by SDSPAGE, gel slices, and y counting of the 73-kDa peak. The specific activity of VMAT (typically 1000-2000 cpm/pmol) is calculated by dividing this amount by the [3H]TBZOH binding site density of the membrane preparation. Purification of Photolabeled VMAT VMAT from bovine chromaffin granule membranes has been purified in an active or inactive form. In all cases, ligand binding has been used to monitor the purification, but this has been done in different ways. [3H]TBZOH binding to the solubilized protein has allowed purification of an active protein in a 5-step protocol, u In a second procedure, 4 [3H]RES (reserpine) is bound to membranes, and because RES dissociation is extremely slow, the protein can be solubilized without release of the ligand. 3 Because the photolabeling of VMAT by AZIK is very specific, it is possible to purify the protein by simply measuring the specific activity of the 125I-labeled material. Because the covalently modified protein is only a minor species, these measurements can be made to monitor the purification of not only the inactive but also the active protein. The inactive protein is purified by classical chromatographic techniques. 14 However, a new purification step, based on selective SDS-resistant thermal aggregation of VMAT, has been introduced 9 that may be of general use for the purification of polytopic membrane proteins. Two different procedures using this step will be described. First Protocol
Bovine chromaffin granule VMAT partially purified by DEAE-cellulose chromatography at acid pH is used. TM All steps are carried out at room temperature. Membranes (40 mg of protein) and photolabeled membranes (4 mg of protein) are suspended together in 10 ml of 10 mM sodium acetate/ 30 mM NaC1 (pH 4.5) containing 1 mM EDTA, 6 tzg/ml leupeptin, 5 txg/ ml aprotonin, 10/xg/ml pepstatin, and 1 mM PMSF. They are solubilized
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by sequential addition of sulfobetaine 3-12 and Nonidet P-40, each at 3.7% final concentration (w/v). The mixture is incubated with stirring for 15 min at 30°, diluted with the same buffer to lower the detergent concentration to 1%, and centrifuged for 40 min at 215,000gmaxat 4 °. Using a peristaltic pump operating at a flow rate of 1.5 ml/min the supernatant is applied to a MemSep 1000 cartridge (Millipore, Bedford, MA), previously equilibrated in the acetate/NaC1 buffer containing 0.5% sulfobetaine 3-12 and 0.5% Nonidet P-40. The cartridge is washed with the same buffer, at 1 ml/min, until the radioactivity of the effluent is constant (about 20 ml). This radioactivity is mainly associated with phospholipids. The labeled protein is eluted by adding 200 mM NaC1 to the same buffer. A 2.7-ml fraction is collected in a tube containing 10/xg/ml pepstatin (final concentration). PMSF is added to 1 mM final concentration, and the eluate is neutralized by addition of 2 M Tris buffer (pH 8.8). Detergents are removed by adsorption at a flow rate of 0.4 ml/min on a 2-ml Extracti-Gel Column (Pierce, Rockford, IL), previously equilibrated in water. The labeled fractions (4 ml) are concentrated 20-fold by ultrafiltration on a Centricon 30 device (Amicon, Danvers, MA). The buffer in the concentrate is subsequently changed by a 10-fold dilution with 100 mM Tris-HC1 (pH 6.8), and the sample is recentrifuged using the Centricon 30. More than 90% of the radioactivity is recovered in the concentrate (about 200/xl). The concentration of Tris-HC1 (pH 6.8) in the sample is raised to 150 mM, and SDS and 2-mercaptoethanol are added at final concentrations of 2% (w/v) and 5% (v/v), respectively. After these steps, the protein is purified 10-fold with a 30% yield (Table I), as judged by SDS-PAGE, slicing of the gel, and summation of the radioactivity associated with the
TABLE I PURIFICATION OF PHOTOLABELED V M A T ~
Purification step
Protein (mg)
10 -3 × Labeled transporter (cpm)
Membranes DEAE-cellulose eluate First H P L C eluate Second H P L C eluate
43.3 1.47 0.060 0.0041
103 35.9 23.8 2.7
Yield (%)
Purification factor
100 35 23 2.6
1 10.2 166 274
a T h e results are for a purification starting with bovine chromaffin granule m e m b r a n e s , s'9 obtained from 100 g of adrenal medulla. [Adapted from C. Sagn6, M. F. Isambert, J. P. Henry, and B. Gasnier, Biochem. J. 316, 825 (1996) with permission.]
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73-kDa peak. At this step, protein concentration is measured by the technique of Schaffner and Weissman. 17 The sample (300/xl) containing the proteins in SDS-2-mercaptoethanol is then heated at 100° for 10 min in a closed vial. Aggregates consisting of VMAT and only a limited number of proteins in the extract are formed. The aggregates are isolated by size-exclusion HPLC. The heated sample is filtered on a Durapore 0.45 /xm (Millipore) and injected onto a 7.8 × 300 mm Protein-Pak 300 SW 10-/xm column (Waters) using prefiltered 0.1% SDS, 200 mM sodium phosphate, pH 6.9, as eluent with a flow rate of 0.2 ml/min. The fractions collected (0.2 ml) are analyzed by y counting and S D S - P A G E (Fig. 2). The photolabeled aggregates elute in the void volume (5.5 ml). This step is efficient, with a purification factor of 17 and a yield of 65% (Table I). From its specific activity, the protein can be considered as 50% pure. However, on S D S - P A G E it remains as an aggregate which ~ does not enter the separating gel. The next step is the dissociation of the aggregates, using the trifluoroacetic acid (TFA) treatment described by Hennessey and Scarborough. 18 Salts in the pooled fractions are diluted about 100-fold in three cycles of dilution with filtered (0.45 txm) water and centrifuged on a Centricon 30. The concentrate (about 200 /xl) is freeze-dried in 1.5 ml polypropylene Microfuge tubes with a SpeedVac concentrator (Savant, Farmingdale, NY) and resuspended in 100/xl of anhydrous trifluoroacetic acid (TFA) under a chemical hood. Anhydrous TFA (sequencing grade) is purchased ampuled under nitrogen (Pierce). The TFA is then evaporated under a stream of argon. In order to achieve maximal removal of TFA, the tube is inclined and rotated manually during this operation. After evaporation of the TFA, the dried film on the walls of the tube is resuspended in 250 txl of prefiltered 150 mM sodium phosphate, 2% (w/v) SDS, 5% (w/v) 2-mercaptoethanol, and fractionated by size-exclusion HPLC under the conditions used for isolation of the aggregates. Two radioactive peaks are eluted at 5.5 and 6.3 ml, which correspond to residual aggregates and to the purified disaggregated transporter, respectively, as assessed by SDS-PAGE. The disaggregation yield, defined as the percentage of monomeric VMAT in the total specific radioactivity, is estimated from the areas of the major peaks. A mean value of 70% + 12% (n = 14) has been calculated, with variations between 47 and 93%. The various fractions are analyzed by SDS-PAGE. Careful observation of the stained proteins shows that the monomeric transporter, which appears as a homogeneous broad band, elutes from the size-exclusion column just 17 W. Schaffner and C. Weissmann, A n a l Biochem. 56, 502 (1973). is j. p. Hennessey and G. A. Scarborough, Anal. Biochem. 176, 284 (1989).
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A *
"It
*
,It
28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 --i
--97 --66 11 - - 4 5
- --31
__22
B 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 --i
--97 --66 --45
--31
__22 --f FIG. 2. SDS-PAGE analysis of the aggregated material fractionated by size exclusion HPLC. (A) Silver-stained gel; (B) autoradiogram. The partially purified [a25I]AZIK-labeled transporter, monitored by its radioactivity (B), is quantitatively aggregated by heating at 100° in SDS/2-mercaptoethanol. The aggregate is separated from the bulk of proteins by sizeexclusion HPLC. [Reproduced from C. Sagn6, M. F. Isambert, J. P. Henry, and B. Gasnier, Biochem. J. 316, 825 (1996) with permission.]
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after a 70-kDa doublet, which has been identified by immunoblotting as dopamine /3-monooxygenase. Only fractions devoid of dopamine /3-monooxygenase are pooled. The corresponding overall purification factor is 275, consistent with a purification of the transporter to homogeneity (Table I). However, the yield drops severely at the last step (about 10%), because of the variability of the disaggregation efficiency and of the overlap of VMAT and dopamine/3-monooxygenase peaks. Second Protocol
Purified aggregated VMAT can be useful for some purposes, such as protein sequencing a9 or antibody preparation. In this case, the protocol can be modified by adding another purification step, affinity chromatography on immobilized wheat germ agglutinin (WGA),TM after D E A E chromatography and omitting the disaggregation step, which decreases the yield of the purification. The neutralized eluate of the DEAE-MemSep cartridge is incubated with Sepharose 6MB-bound W G A (1 ml of wet resin prepared from a water suspension, Pharmacia, Piscataway, N J), with gentle mechanical stirring for 2 hr in a 10-ml closed plastic chromatographic column. The column is centrifuged at low speed, and the supernatant is discarded. The resin is washed at flow rate of 1 ml/min by 12 volumes of 0.1% Nonidet P-40 and then 25 volumes of 200 mM NaC1/0.05% SDS/10 mM Tris-HC1 (pH 6.8). The labeled protein is then eluted by incubation with 10 volumes of the last buffer containing 200 mM N-acetylglucosamine. The W G A eluate is concentrated about 50-fold by centrifugation on Centriprep 30 and Centricon 30 devices (Amicon). During this step, the buffer is exchanged for 100 mM Tris-HC1 (pH 6.8) containing 0.05% SDS. The SDS and Tris-HC1 (pH 6.8) in the concentrates are raised to 2% and 150 mM, respectively, and 2mercaptoethanol is added to a 5% final concentration. The sample (700/xl) is heated for 10 min at 100 ° in a closed vial and injected onto a size-exclusion HPLC column as before. The pooled radioactive fractions are concentrated, and their salt concentration is diluted 100fold with water by centrifugation on Centricon 30 devices. The total yield of the three-step procedure is 20%. The purity of the preparation is checked by S D S - P A G E on an aliquot previously disaggregated by treatment with anhydrous TFA, as previously described. VMAT is the major component of the preparation as shown by the fact that N-terminal sequence analysis indicated only one component with the sequence of bVMAT2. 19 C. Sagn6, M. F. Isambert, J. Vandekerckhove, J. P. Henry, and B. Gasnier, Biochemistry 36, 3345 (1997).
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[5] N o n c o v a l e n t a n d C o v a l e n t L a b e l i n g o f Vesicular Monoamine Transporter with Tetrabenazine and Ketanserin Derivatives; Purification of Photolabeled Protein By
J E A N - P I E R R E H E N R Y , C O R I N N E SAGNI~,
M A R I E - F R A N ~ O I S E ISAMBERT,
and B R U N O G A S N I E R
Introduction The vesicular monoamine transporter (VMAT) accumulates monoamines inside secretory granules or synaptic vesicles by catalyzing a H+/ monoamine antiport. Monoamine uptake is coupled to the generation of a proton electrochemical gradient by an ATP-dependent H + pump of the vacuolar type. The uptake is extremely efficient, and in the adrenal medulla chromaffin cells, the monoamine gradient between the cytosol and the secretory granules has been estimated to five orders of magnitude. 1'2 The transporter has a low specificity and will accept catecholamines, such as adrenaline, noradrenaline, or dopamine, the indoleamine serotonin (5-HT), histamine, and nonnatural substrates such as m-iodobenzylguanidine (MIBG) or the neurotoxin 1-methyl-4-phenylpyridinium (MPP +). The vesicular monoamine transporter from various origins (rat, human, and bovine) has been cloned and expressed in various cell types. 1,2 Two isoforms, VMAT1 and VMAT2, have been identified which have different localizations and slightly different properties. However, most of our knowledge regarding VMAT has been obtained on the protein from bovine chromaffin granules, where the major expressed isoform is VMAT2. An interesting pharmacology has been developed from this preparation. 3 Two different binding sites have been identified. The first one binds reserpine, whereas the second one binds tetrabenazine (TBZ) and ketanserin (KET). The H + electrochemical gradient generated in the presence of ATP greatly accelerates reserpine binding, but has no effect on TBZ and KET binding. As for other transporters, the enzymology of VMAT is complicated by the fact that VMAT catalyzes a vectorial activity, which has to be coupled to a proton gradient generating system. It is possible to reconstitute the purified protein in liposomes and to impose a pH gradient on the vesii S. Schuldiner, A. Shirvan, and M. Linial, Physiol. Rev. 75, 369 (1995). 2 y . Liu and R. H. Edwards, Am. Rev. Neurosc. 20, 125 (1997). 3 j. p. H e n r y and D. Scherman, Biochem. Pharmacol. 38, 2395 (1989).
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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cles. 4 However, it is also possible to use nonvectorial ligand binding to assay VMAT. Because TBZ and KET derivative binding is not affected by the H ÷ electrochemical gradient, these compounds are very useful ligands. Photosensitive derivatives of TBZ and KET can furthermore be used to label VMAT by photoaffinity techniques. An interesting aspect of covalent labeling is that the protein can be purified irrespective of its activity. During such purification studies, we introduced a new technique based on the selective aggregation of VMAT solubilized in sodium dodecyl sulfate (SDS) that results from the polytopic structure of the transporter. Consequently, it might be useful for the purification of other such transporters. TBZ and KET Binding to Vesicular Monoamine Transporter
TBZ Binding Various tritiated derivatives of TBZ have been used. The first one was a-[2-3H]dihydrotetrabenazine (2-hydroxy-3-isobutyl-9,10-dimethoxy-l,2,3, 4,6,7-hexahydro-llbH-benzo[a]quinolizine, [2-3H]TBZOH), obtained by reduction of TBZ with KB3H4 . This compound had a specific activity of about 15 Ci/mmol (0.5 TBq/mmol). 5 More recently, another tritiated derivative of a-dihydrotetrabenazine (a[O-methyl-3H]dihydrotetrabena zine) has been commercialized that has a higher specific activity (4.44-6.48 TBq/mmol, 120-175 Ci/mmol, Amersham, Buckinghamshire, U.K.). aDihydrotetrabenazine contains two enantiomers and the binding is stereospecific, the (+) isomer being the active one. 6 [O-methyl-3H]Tetrabenazine has also been tested successfully as a high specific activity ligand. 7 For Scatchard analysis of a-[O-methyl-3H]TBZOH binding to membrane preparations, various concentrations of the ligand at full specific activity (265-385 dpm/fmol) are incubated with membranes suspended in 0.2 ml of isosmotic medium pH 7.4 (saline or sucrose). When purified chromaffin granule membranes are used, 8,9 the membrane concentration is limited to about 10/xg of protein/ml to keep the site concentration lower than the equilibrium dissociation constant Ka.5 Usually, duplicate incubations are performed for each concentration, and nonspecific binding is 4 R. Yelin and S. Schuldiner, M e & o d s EnzymoL 296, [4], (1998) (this volume). 5 D. Scherman, P. Jaudon, and J. P. Henry, Proc. Natl. Acad. Sci. USA 80, 584 (1983). 6 M. R. Kilbourn, L. C. Lee, T. M. Vander Borght, D. M. Jewett, and K. A. Frey, Eur. J. PharmacoL 278, 249 (1995). 7 T. M. Vander Borght, A. A. F. Sima, M. R. Kilbourn, T. J. Desmond, D. E. Kuhl, and K. A. Frey, Neuroscience 68, 955 (1995). 8 A. D. Smith and H. Winkler, Biochem. J. 103, 480 (1967). 9 C. Sagn6, M. F. Isambert, J. P. Henry, and B. Gasnier, Biochem. J. 316, 825 (1996).
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measured by adding 2/zM TBZ to a third series of incubations. The mixtures are incubated for at least 2 hr at 25 °. Bound ligand is separated by filtration on H A W P filters (Millipore, Bedford, MA), pretreated with 0.3% PEI (polyethyleneimine) to decrease the background. The samples are diluted with 2 ml of ice-cold buffer containing 10/zM TBZ before filtration, and the radioactivity on the filter is measured by liquid scintillation counting in Ready Protein Plus mixture (Beckman, Fullerton, CA) or equivalent solutions. With purified chromaffin granule membranes, the signal-to-noise ratio at a ligand concentration equivalent to Kd is about 30. In all cases tested, corresponding essentially to VMAT2 from various origins, the equilibrium dissociation constant Kd is in the 1-10 nM concentration range. With purified chromaffin granule membranes, 8,9 K d and Bmax (maximal concentration of bound ligand, corresponding to the density of sites) are 3 nM and 60 pmol/mg of protein, respectively. The assay of VMAT density in a given membrane preparation can be performed by measuring B . . . . or more simply, if Ka is known, by measuring the bound ligand at a concentration equivalent to Kd. This protocol can be applied to VMAT solubilized by cholate or octyl /3-glucoside. Originally, the protein incubated with [3H]TBZOH was precipitated with polyethylene glycol 6000 before filtration. 1° However, this is not necessary since the solubilized transporter is adsorbed on GF/B glass fiber filters (Whatman, Clifton, N J) that have been pretreated by incubation for at least 3 hr with 0.3% PEI. 11 The incubation mixture is filtered under reduced pressure on these filters and the radioactivity is measured as previously. [3H]TBZOH binding ha.s also been measured on bovine chromaffin cells in culture. 12 [3H]TBZOH is a permeant molecule and specific binding can be measured on intact cells. The assay is performed in four plastic wells, each containing 500,000 cells. The culture medium is pipetted off and 3 nM [3H]TBZOH is added in i ml of Locke medium. For nonspecific binding determination, 2/xM TBZ is added to four other wells. After 1 hr incubation at room temperature, the medium is removed and the cells are washed three times by incubating for 3 min with 2 ml of Locke solution. Finally, the cells are collected in 0.2% Triton X-100 for counting radioactivity. Under the conditions described and for 500,000 cells, specifically and nonspecifically bound [3H]TBZOH are, respectively, 30-50 and 15 fmol. Specific binding does not change with the age of the culture, whereas nonspecific binding increases with the proliferation of nonchromaffin cells. The density 10 D. Scherman and J. P. Henry, Biochemistry 22, 2805 (1983). 11 M. S. Vincent and J. A. Near, Molec. Pharmacol. 40, 889 (1991). 17 C. Desnos, M. P. Laran, and D. Scherman, J. Neurochem. g9, 2105 (1992).
76
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of [3H]TBZOH binding sites, derived from Bmax, is 1 pmol/mg of protein (Ka = 16 nM), which should be compared to the value obtained on adrenal medulla homogenates (Bmax = 3 pmoles/mg of protein, Kd = 5 nM). One difficulty in using this method is compromising the rate of dissociation of the ligand from its site during the washing of intact cells. An alternative would be to perform the assay on cells with membranes permeabilized by the bacterial toxin streptolysin O. In this case, cells are placed in a 2-cm diameter plastic well and are resuspended in a medium containing 150 mM potassium glutamate/0.5 mM EGTA/5 mM magnesium acetate/ 10 mM PIPES, pH 7.2 (KG medium) containing 50 U/ml streptolysin O (Biomerieux, Lyon, France)/2 mM dithiothreitol (DTT), and 0.1% bovine serum albumin (BSA). After incubation in this medium for 15 min at room temperature, the solution is pipetted off. Then the cells are incubated with 3 nM [3H]TBZOH in KG medium for at least 1 hr at room temperature. At the end of the incubation, cells are rapidly washed two times with icecold KG and 0.3 ml of 0.1 N NaOH is added. After 15 min, the radioactivity of the suspension is counted by liquid scintillation. Nonspecific binding is measured by similar experiments to which 0.5/zM TBZ is added to the KG medium.
K E T Binding [3H]KET, which is principally a 5-HTzA receptor ligand, is obtained commercially with a high specific activity (40-90 Ci/mmol, 1.5-3.33 Tbq/ mmol), from NEN Life Science Products (Boston, MA). Binding to VMAT can be performed under the conditions used for [3H]TBZOH, taking two precautions.13 The first is to perform the filtrations rapidly in ice-cold buffer, because the rate of dissociation of [3H]KET is extremely rapid (half-life of 40 sec at 0°). The incubation mixture is diluted 10 times in ice-cold buffer, rapidly filtered on GF/C glass filters (Whatman), and washed with 1 ml of ice-cold buffer. Binding is measured at 30° (Kd of 45 nM) or, more easily at 0° (Kd of 6 nM). The second is to control pharmacologically the binding of [3H]KET to VMAT and not to 5-HT2A receptors. [3H]KET specifically bound to VMAT is obtained by subtracting the radioactivity measured in assays containing 2/xM TBZ, whereas that bound to 5-HT2A receptor was obtained by subtracting the radioactivity of incubations performed in the presence of 3/zM methysergide. In bovine chromaffin granule membranes, no 5-HT2A receptor binding can be detected. Photoaffinity Labeling of Vesicular Monoamine Transporter using 7-azido[8-125I]Iodoketanserin. 7-Azido[8-125I]iodoketanserin (AZIK) can a3F. Darchen, D. Scherman, P. M. Laduron, and J. P. Henry, Mol. Pharmacol.33, 672 (1988).
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be obtained from Amersham, but at the present time custom preparation is required. To decrease the cost, it is possible to order 7-amino[8-125I]iodo ketanserin and to use it in a one-step synthesis of AZIK. TM The carrier-free amino derivative solution (100/zCi) is dried under a flux of argon in the original vial to which an efflux active charcoal guard has been added. The compound is resuspended in 50/xl of 1 N HC1. Sodium nitrite (2 /xl, 2.5 M) is added with a Hamilton syringe under magnetic stirring at 4°. The solution is stirred at 4° in the dark for 30 min. Sodium azide (2/z, 2.5 M) is added at 4° and the mixture stirred for another 30 min in the dark. The AZIK formed is separated from the mixture by high-performance liquid chromatography (HPLC) on a C18 /xBondapak column (Waters, Milford, MA) with H20-CH3OH-trifluoroacetic acid (TFA) (50:50:0.1) as the solvent and a UV detector operating at 315 nm. The active fractions are concentrated to dryness, resuspended in ethanol, and kept at -20 °. The yield is 80%. Photolabeling with [t25I]AZIK. For analytical purposes, 15 membrane samples with a concentration of about 3 nM of KET/TBZ binding sites are incubated with a one- to two-order-of-magnitude lower concentration of AZIK, in order to bind a large fraction of the probe. For instance, 50/xg of protein/ml of purified bovine chromaffin granule membranes 8,9 is incubated with 40 pM [125I]AZIK (0.07 tzCi/ml, 0.16 x 10 6 dpm/ml) in 5 ml of 0.3 M sucrose/10 mM HEPES, pH 7.5, for 1 hr at 0°, in a 3.5-cm diameter well of a cell culture plate. The incubated samples are irradiated for 10 min with a UV lamp operating at 350 nm at an average distance of 6 cm, under magnetic stirring. The conditions for irradiation are selected to flatten the 240 nm absorbance peak of 7-azidoketanserin and to minimize the labeling of phospholipids at 254 nm. After irradiation, the membranes are washed twice by 4-fold dilution in sucrose buffer followed by centrifugation for 20 min at 140,000g at 4°. Samples (50-20 tzg of protein) are analyzed by SDS-PAGE, using an acrylamide concentration of 10% (w/v) and a bisacrylamide:acrylamide ratio of 0.8 : 30 (w/w). Proteins are stained with silver nitrate, using sodium thiosulfate to sensitize the gels] 6 or Coomassie blue. Autoradiography is obtained by exposure of the dried gel to Kodak X-OMAT AR films with Lumix MR 800 screens (Agfa Gevaert, Rueil-Malmaison, France) for 2-30 days at - 8 0 °, or by using a Phosphorimager apparatus (Molecular Dynamics, Sunnyvale, CA). When quantitative data are required, the stained gel is cut into 2-mm slices and the radioactivity of the slices is measured with 14 M. F. Isambert, B. Gasnier, D. Botton, and J. P. Henry, Biochemistry 31, 1980 (1992). 15 M. F. Isarnbert, B. Gasnier, P. M. Laduron, and J. P. Henry, Biochemistry 28, 2265 (1989). 16 T. Rabilloud, G. Carpentier, and P. Tarroux, Electrophoresis 9, 288 (1988).
78
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a y-counter (Fig. 1). As described for [3H]KET binding, the specificity of the labeling, i.e., labeling on VMAT and not on 5-HT2A receptor, is tested pharmacologically: VMAT labeling is inhibited in the presence of 2/zM TBZ, whereas 2 tzM methysergide inhibits 5-HTeA receptor labeling. In bovine chromaffin granules membranes, VMAT is labeled as a broad diffuse band with an apparent molecular mass centered at 73 kDa when 10% acrylamide concentration is used for SDS-PAGE. Increasing the acrylamide concentration increases the apparent molecular mass. In addition to the broad diffuse band corresponding to VMAT, another component is often visible as a sharp band with an apparent molecular mass of 40 kDa. This component is thought to be nonspecifically labeled since the corresponding band is not suppressed in the presence of TBZ. In some experiments, cytochrome b-561 (27 kDa), which is the major membrane protein, is also labeled. Bovine VMAT2 expressed in COS cells is labeled by the same technique as a component with an apparent molecular mass of 80 kDa. [125I]AZIK has also been used to photolabel a large stock of membranes, 14 for the purification of VMAT. Membranes (50 mg of protein) are thawed and suspended (2 mg/ml, final concentration) in 10 mM Tris-HC1 buffer (pH 7.5) containing 1 mM EDTA, 6 /xg/ml leupeptin, 5 /xg/ml aprotinin, 10 /zg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride (PMSF), at 0 °. [125I]AZIK(80/xCi; 145 × 106 cpm) is added and incubated for 1 hr in the dark at 0°. The incubation medium is positioned at 6 cm from a 350-nm UV light source and irradiated for 12 min at 0°. During these experiments, it has been noted that a radiolabeled pho1000
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slice number FiG. 1. Photoaffinity labeling of VMAT from purified bovine chromaffin granule membranes, using [125I]AZIK. In this experiment, 60/xg of photolabeled protein, corresponding to 95,000 and 50,000 cpm for, respectively, the samples incubated in the absence (filled squares) and in the presence (open squares) of 0.2/zM TBZ, were analyzed by SDS-PAGE.
[5]
LABELING AND PURIFICATION OF V M A T
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toproduct not covalently bound has a high affinity for the TBZ binding site, thus leading to a large overestimation of the protein covalently labeled (see Fig. 1). To minimize this effect, TMTBZ is added to a final concentration of 2/xM after irradiation and the mixture is incubated for 2 hr at room temperature. After centrifugation for 20 min at 140,000g at 4 °, the pellet is resuspended in about the same volume of TBZ-containing buffer and centrifuged again under the same conditions. The pellet is then resuspended in 5 ml of water containing leupeptin/aprotinin/pepstatin/PMSF at the concentration indicated above. The labeled membranes are stored in aliquots at - 8 0 °. The amount of photolabeled VMAT is determined by SDSPAGE, gel slices, and y counting of the 73-kDa peak. The specific activity of VMAT (typically 1000-2000 cpm/pmol) is calculated by dividing this amount by the [3H]TBZOH binding site density of the membrane preparation. Purification of Photolabeled VMAT VMAT from bovine chromaffin granule membranes has been purified in an active or inactive form. In all cases, ligand binding has been used to monitor the purification, but this has been done in different ways. [3H]TBZOH binding to the solubilized protein has allowed purification of an active protein in a 5-step protocol, u In a second procedure, 4 [3H]RES (reserpine) is bound to membranes, and because RES dissociation is extremely slow, the protein can be solubilized without release of the ligand. 3 Because the photolabeling of VMAT by AZIK is very specific, it is possible to purify the protein by simply measuring the specific activity of the 125I-labeled material. Because the covalently modified protein is only a minor species, these measurements can be made to monitor the purification of not only the inactive but also the active protein. The inactive protein is purified by classical chromatographic techniques. 14 However, a new purification step, based on selective SDS-resistant thermal aggregation of VMAT, has been introduced 9 that may be of general use for the purification of polytopic membrane proteins. Two different procedures using this step will be described. First Protocol
Bovine chromaffin granule VMAT partially purified by DEAE-cellulose chromatography at acid pH is used. TM All steps are carried out at room temperature. Membranes (40 mg of protein) and photolabeled membranes (4 mg of protein) are suspended together in 10 ml of 10 mM sodium acetate/ 30 mM NaC1 (pH 4.5) containing 1 mM EDTA, 6 tzg/ml leupeptin, 5 txg/ ml aprotonin, 10/xg/ml pepstatin, and 1 mM PMSF. They are solubilized
80
VESICULAR CARRIERS
[51
by sequential addition of sulfobetaine 3-12 and Nonidet P-40, each at 3.7% final concentration (w/v). The mixture is incubated with stirring for 15 min at 30°, diluted with the same buffer to lower the detergent concentration to 1%, and centrifuged for 40 min at 215,000gmaxat 4 °. Using a peristaltic pump operating at a flow rate of 1.5 ml/min the supernatant is applied to a MemSep 1000 cartridge (Millipore, Bedford, MA), previously equilibrated in the acetate/NaC1 buffer containing 0.5% sulfobetaine 3-12 and 0.5% Nonidet P-40. The cartridge is washed with the same buffer, at 1 ml/min, until the radioactivity of the effluent is constant (about 20 ml). This radioactivity is mainly associated with phospholipids. The labeled protein is eluted by adding 200 mM NaC1 to the same buffer. A 2.7-ml fraction is collected in a tube containing 10/xg/ml pepstatin (final concentration). PMSF is added to 1 mM final concentration, and the eluate is neutralized by addition of 2 M Tris buffer (pH 8.8). Detergents are removed by adsorption at a flow rate of 0.4 ml/min on a 2-ml Extracti-Gel Column (Pierce, Rockford, IL), previously equilibrated in water. The labeled fractions (4 ml) are concentrated 20-fold by ultrafiltration on a Centricon 30 device (Amicon, Danvers, MA). The buffer in the concentrate is subsequently changed by a 10-fold dilution with 100 mM Tris-HC1 (pH 6.8), and the sample is recentrifuged using the Centricon 30. More than 90% of the radioactivity is recovered in the concentrate (about 200/xl). The concentration of Tris-HC1 (pH 6.8) in the sample is raised to 150 mM, and SDS and 2-mercaptoethanol are added at final concentrations of 2% (w/v) and 5% (v/v), respectively. After these steps, the protein is purified 10-fold with a 30% yield (Table I), as judged by SDS-PAGE, slicing of the gel, and summation of the radioactivity associated with the
TABLE I PURIFICATION OF PHOTOLABELED V M A T ~
Purification step
Protein (mg)
10 -3 × Labeled transporter (cpm)
Membranes DEAE-cellulose eluate First H P L C eluate Second H P L C eluate
43.3 1.47 0.060 0.0041
103 35.9 23.8 2.7
Yield (%)
Purification factor
100 35 23 2.6
1 10.2 166 274
a T h e results are for a purification starting with bovine chromaffin granule m e m b r a n e s , s'9 obtained from 100 g of adrenal medulla. [Adapted from C. Sagn6, M. F. Isambert, J. P. Henry, and B. Gasnier, Biochem. J. 316, 825 (1996) with permission.]
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73-kDa peak. At this step, protein concentration is measured by the technique of Schaffner and Weissman. 17 The sample (300/xl) containing the proteins in SDS-2-mercaptoethanol is then heated at 100° for 10 min in a closed vial. Aggregates consisting of VMAT and only a limited number of proteins in the extract are formed. The aggregates are isolated by size-exclusion HPLC. The heated sample is filtered on a Durapore 0.45 /xm (Millipore) and injected onto a 7.8 × 300 mm Protein-Pak 300 SW 10-/xm column (Waters) using prefiltered 0.1% SDS, 200 mM sodium phosphate, pH 6.9, as eluent with a flow rate of 0.2 ml/min. The fractions collected (0.2 ml) are analyzed by y counting and S D S - P A G E (Fig. 2). The photolabeled aggregates elute in the void volume (5.5 ml). This step is efficient, with a purification factor of 17 and a yield of 65% (Table I). From its specific activity, the protein can be considered as 50% pure. However, on S D S - P A G E it remains as an aggregate which ~ does not enter the separating gel. The next step is the dissociation of the aggregates, using the trifluoroacetic acid (TFA) treatment described by Hennessey and Scarborough. 18 Salts in the pooled fractions are diluted about 100-fold in three cycles of dilution with filtered (0.45 txm) water and centrifuged on a Centricon 30. The concentrate (about 200 /xl) is freeze-dried in 1.5 ml polypropylene Microfuge tubes with a SpeedVac concentrator (Savant, Farmingdale, NY) and resuspended in 100/xl of anhydrous trifluoroacetic acid (TFA) under a chemical hood. Anhydrous TFA (sequencing grade) is purchased ampuled under nitrogen (Pierce). The TFA is then evaporated under a stream of argon. In order to achieve maximal removal of TFA, the tube is inclined and rotated manually during this operation. After evaporation of the TFA, the dried film on the walls of the tube is resuspended in 250 txl of prefiltered 150 mM sodium phosphate, 2% (w/v) SDS, 5% (w/v) 2-mercaptoethanol, and fractionated by size-exclusion HPLC under the conditions used for isolation of the aggregates. Two radioactive peaks are eluted at 5.5 and 6.3 ml, which correspond to residual aggregates and to the purified disaggregated transporter, respectively, as assessed by SDS-PAGE. The disaggregation yield, defined as the percentage of monomeric VMAT in the total specific radioactivity, is estimated from the areas of the major peaks. A mean value of 70% + 12% (n = 14) has been calculated, with variations between 47 and 93%. The various fractions are analyzed by SDS-PAGE. Careful observation of the stained proteins shows that the monomeric transporter, which appears as a homogeneous broad band, elutes from the size-exclusion column just 17 W. Schaffner and C. Weissmann, A n a l Biochem. 56, 502 (1973). is j. p. Hennessey and G. A. Scarborough, Anal. Biochem. 176, 284 (1989).
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A *
"It
*
,It
28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 --i
--97 --66 11 - - 4 5
- --31
__22
B 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 --i
--97 --66 --45
--31
__22 --f FIG. 2. SDS-PAGE analysis of the aggregated material fractionated by size exclusion HPLC. (A) Silver-stained gel; (B) autoradiogram. The partially purified [a25I]AZIK-labeled transporter, monitored by its radioactivity (B), is quantitatively aggregated by heating at 100° in SDS/2-mercaptoethanol. The aggregate is separated from the bulk of proteins by sizeexclusion HPLC. [Reproduced from C. Sagn6, M. F. Isambert, J. P. Henry, and B. Gasnier, Biochem. J. 316, 825 (1996) with permission.]
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LABELING AND PURIFICATION OF V M A T
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after a 70-kDa doublet, which has been identified by immunoblotting as dopamine /3-monooxygenase. Only fractions devoid of dopamine /3-monooxygenase are pooled. The corresponding overall purification factor is 275, consistent with a purification of the transporter to homogeneity (Table I). However, the yield drops severely at the last step (about 10%), because of the variability of the disaggregation efficiency and of the overlap of VMAT and dopamine/3-monooxygenase peaks. Second Protocol
Purified aggregated VMAT can be useful for some purposes, such as protein sequencing a9 or antibody preparation. In this case, the protocol can be modified by adding another purification step, affinity chromatography on immobilized wheat germ agglutinin (WGA),TM after D E A E chromatography and omitting the disaggregation step, which decreases the yield of the purification. The neutralized eluate of the DEAE-MemSep cartridge is incubated with Sepharose 6MB-bound W G A (1 ml of wet resin prepared from a water suspension, Pharmacia, Piscataway, N J), with gentle mechanical stirring for 2 hr in a 10-ml closed plastic chromatographic column. The column is centrifuged at low speed, and the supernatant is discarded. The resin is washed at flow rate of 1 ml/min by 12 volumes of 0.1% Nonidet P-40 and then 25 volumes of 200 mM NaC1/0.05% SDS/10 mM Tris-HC1 (pH 6.8). The labeled protein is then eluted by incubation with 10 volumes of the last buffer containing 200 mM N-acetylglucosamine. The W G A eluate is concentrated about 50-fold by centrifugation on Centriprep 30 and Centricon 30 devices (Amicon). During this step, the buffer is exchanged for 100 mM Tris-HC1 (pH 6.8) containing 0.05% SDS. The SDS and Tris-HC1 (pH 6.8) in the concentrates are raised to 2% and 150 mM, respectively, and 2mercaptoethanol is added to a 5% final concentration. The sample (700/xl) is heated for 10 min at 100 ° in a closed vial and injected onto a size-exclusion HPLC column as before. The pooled radioactive fractions are concentrated, and their salt concentration is diluted 100fold with water by centrifugation on Centricon 30 devices. The total yield of the three-step procedure is 20%. The purity of the preparation is checked by S D S - P A G E on an aliquot previously disaggregated by treatment with anhydrous TFA, as previously described. VMAT is the major component of the preparation as shown by the fact that N-terminal sequence analysis indicated only one component with the sequence of bVMAT2. 19 C. Sagn6, M. F. Isambert, J. Vandekerckhove, J. P. Henry, and B. Gasnier, Biochemistry 36, 3345 (1997).