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Neurobiology CFIARACTIItIZATION OF A PURIFIID NICOTINIC RE(EP'i'OR FRIDM RAT BRAIN USING IDIOTYPIC ANID ANTI-IDIOTYPIC ANI'IBODIES Leo G. Abood*, John J. Langonet, Robert Bjerckefi, * * Xin Lu , and~Srabani Banerjee ((-)-6-hydroxymethylnicotine/affinity chromatography/ imnninoaffinity chromatography/nicotine/receptor purification) *Center for Brain Research and Department of Biochemistry, University of Rochester Medical Center, Rochester, NY 14642; and tDepartments of Medicine and~ Biochemistry, Baylor College of Medicine, Houston, TX 77030 Abbreviatione mAb, monoclonal antibody

c I ABSTRACT The availability of an anti-nicotine monoclonal antibody has made it possible to further establish the nature of the nicotine recognition proteins purified fr= rat brain by affinity chromatography and to provide a highly sensitive assay for determining [3H]-nicotine binding to the purified material. An enantiomeric analogue of nicotine, (-)-6-hydroxymethylnicotine was used: to prepare the affinity column. In addition, with the use of an anti-idiotypic monoclonal antibody, it was confirmed that the recognition site for nicotine resides on a protein complex comprised of two components with molecular weights of 62 and 57 kdalton. It was also demonstrated that the same two proteins could be purified by immunoaffinity chromatography with the use of an anti-idiotypic monoclonal antibody. With the use of the anti-nicotine antibody to measure [3H]-nicotine binding, the purified material was shown to have activity of 2.5 x 10 10 moles/mg protein. By utilizing a procedure whereby the purified receptor protein~was conjugated to membranes via disulfide bonds, a binding activity of 0.8 x 10 10 moles/mg was obtained. With the availability of stereospecific monoclonal antibodies to S-(-)-nicotine as well as monoclonal anti-idiotypic antibodies. derived from using the anti-nicotine antibodies as immunogens, additional procedures became available for the further characterization of the purified nicotine receptor and examining its (-)-[3H]-nicotine binding characteristics. ~ ~ ~ ~ ~ 2 ~ W

Although the nicotinic cholinergic receptor of the Torpedo electric organ and mammalian neuromuscular junction are similar in their molecular characteristics (see [1] for review), the nature of the nicotinic site in mammalian brain and its relationship to the peripheral receptor remains problematical. A number of studies have described limited cross-reactivity between antibodies against the electric organ and an oo-bungarotoxin binding site in rat brain (2-4); however, neither receptor binding studies using [3H)-nicotine and [3H)-cholinergic as ligands nor behavioral and electrophysiological studies indicate any similarity in the a-bungarotoxin and nicotine sites in rat brain. Recently, a monoclonal antibody (mAb) prepared against purifi~ed nicotinic receptors from chick brain has been used to isolate by immunoaffinity chromatography a nicotinic receptor from rat brain (4). The receptor complex, which was comprised of two proteins having molecular weights of 51 and 79 kdalton, differs somewhat from a nicotine binding complex purified by affinity chromatography using a nicotine analogue conjugated to epoxysepharaose (5). The present communication describes further studies on the characterization of the nicotinic receptor purified by affinity chromatography by making use of idiotypic monoclonal antibodies (6) to nicotine as well as their anti-idiotypes. 3

MATFRIAIS AND METHODS Preparation of Rat Brain ltieabranes. After adult male Sprague-Dawley rats were sacrificed by spinal dislocation, the whole brains were removed, washed, and homogenized with a polytron in 20 volumes of ice-cold 0.05 M NaPO4, pH 7.5. The membrane pellet obtained after centrifugation at 50,000 x g for 30 min was washed once, recentrifuged, and stored at -70°. Solubilization of Membranes with CHAPS. The solubilization of membranes was performed as described elsewhere (5) except that 10 mM CHAPS was substituted for 1% Triton X-100. Approximately 1 g of membrane protein in 200 ml of ice-cold 50 mM Tris.HCL, pH 7.5, containing 10 mM CHAPS + 10-3 M phenylmethanesulfonyl fluoride + 10-3 M EGTA was homogenized with a polytron for 60 sec (setting 5.0); and upon centrifugation of the suspension at 100,000 x g for 1 hr and concentration of the supernatant to 15 ml by flow dialysis, a final concentration of 12 mg/mL of solubilized protein was obtained. Affinity Chro®atography of Solubilized Membrane Extract. The procedure for the isolation of the nicotine binding proteins by affinity chromatography was a modification of that described previously (5). Instead of (±)-6-(2-hydroxyethyl)nicotine, (-)-6-hydroxymethylnicotine was used to prepare the affinity gel, 4

by conjugating 100 mg of the ligand to 5 g of epoxy activated Sepharose in 0.02 M Na2C03, pH 10.5. Other modification involved the use of a hydroxylapatite column (1 g in a 2 cm diameter column) prior to the affinity column (2 cm diameter). In a typical run, 50 mg of solubilized protein in 5 ml 0.05 M NaP04, pH 6.5 + 10 mM CHAPS was passed through the hydroxylapatite column,, followed by application to the affinity column. After washing the affinity column with 40 ml of 0.05 M NaP04 buffer containing 1 mM CHAPS, the nicotine binding protein was eluted with 50 ml of 10-4 M(-)-nicotine inthe same buffer. The fraction eluted from~ the affinity -column with 10-4 M (-)-[3H]-nicotine was concentrated by flow dialysis at 40 by repeated dilution with H20. If not used within 24 hr, the material was lyophilized and stored at -70° until used for further studies including NaDodSO4/polyacrylamide electrophoresis, employing the procedure of Laemmli and Favre (7). Preparation of (-)-6-hydroxymethylnicotine. The preparation of 6-hydroxymethylnicotine was based on a modification of Itokawa et al (8), involving radical hydroxymethylation of (-)-nicotine. To a mixture containing 0.5 g (-)-nicotine dissolved in 501 m1 methanol containing 1.7 g FeSO4.7H20 + 1 ml concentrated HZSO4 at 20° was added dropwise with~ stirring over a period of 15 min 0.7 g of 30% H202. After an additional 30 min, the solution was 5

neutralized with 10% NaOH in methanol and concentrated in vacuo to 10 ml, 30 mL of acetone was added and the precipitate removed by centrifugation. The acetone extract was concentrated in vacuo, and applied to an alumina (80-200 mesh) column (2 x 5 cm), which had been previously washed with 100 ml of benzene. After eluting the residual (-)-nicotine with 100 ml of benzene, 6-hydroxymethylnicotine was eluted with 100 ml chloroform: methanol•triethylamine (50:50:2). The product yield was 25% and 80% pure. Further purification (99%) was achieved by preparative cellulose thin layer chromatography (0.5 mm thickness): using cyclohexane as a solvent: Rf for nicotine was 0.9 and 0.0 for (-)-6-hydroxymethylnicotine. Purity was confirmed by MS and NMt using a standard~ provided by ,7. Seeman, Philip Morris Research~ Center. Characterization of Anti-Nicotine Monoclonal Antibody and Monocional Anti-Idiotype Antibody. Monoclonal anti-nicotine 402C10, an IgG2ak antibody is specific for naturally occurring (-)-nicotine (6) and was used as immunogen for obtaining monoclonal anti-idiotypes (9). A total of nine anti-idiotypes were selected that inhibited the binding of fluid phase anti-nicotine to immobilized nicotine-polylysine and were extensively cloned, of which one, anti-idiotype 422F11, has been extensively characterized as to ligand and receptor specificity (9). 6

Preparation of Immumoaffinity Column with Anti-Idiotype mAb, 422F11. After 2 g cyanogen bromide-activated sepharose 4B was washed with 100 ml 1 mM HC1 followed by 50 ml 0.1 M NaHC03 + 0.2 M NaCl, it was incubated for 2 hr with 2 mg of purified anti-idiotypic mAb, 422F11 overnight at 4° with gentle shaking, followed by 1 M ethanolamine for 2 hr at room temperature to block unreacted groups. The gel was then washed with 0.1 M NaPO4, pR 7.5, and packed into 1.5 x 4.0 mm column. Approximately 10 mg of CHAPS-solubilized membrane protein in a 2.0 ml volume containing 1 mM CHAPS was applied to the column; and after removing the unbound proteins with 50 m1 of 0.05 M NaP04, pH 7.5, the receptor protein was eluted with 50 ml of 0.1 M NaP04, pH 4.0. The eluant was concentrated by flow dialysi~s and either assayed immediately for [3H]-nicotine binding or dialyzed, lyophilized, and stored at -70°. Measurement of [3H]-Nicotine Binding of Conjugated Purified Receptor. A new procedure was developed for measuring binding of the purified proteins obtained by affinity chromatography which involved conjugation of the material to a rat brain membrane preparation after heat-inactivation at 90° for 15 min. To a suspension of 10 mg/ml of heat-inactivated membranes + 10 pg of the affinity gel-derived protein was added 0.04 ml of 0--mercaptoethanol to a final volume of 4 ml H20. After standing 7

at 200 for 2 hr, the contents were lyophilized to remove the 0-mercaptoethanol. The contents were then homogenized in 4 mL of 0.05 M Tris, pH 7.5 containing 5 x 10-3 M 2,2'-dithiopyridine. After standing at 20°C for 2 hr, the material was dialyzed for 16 hrs at 5° against distilled H20, lyophilized, and resuspended in 4 ml of the Tris buffer. The procedure for measuring [3H)-nicotine binding is described in detail elsewhere (4). Briefly, it consisted of incubating in an i~ce-bath for 30 min a mixture containing, 0.3 ml of the conjugated protein (representing 3 mg membrane protein + 3,og purified protein) + 1 x 10 9 M (-)-[ 3 H]-nicotine (sp. act. - 87, Ci/mmole, New England Nuclear) with or without 10-6 M unlabeled (-)-nicotine or an analogue brought to a final volume of 1.2 ml with 0.05 M Tris, pH 7.5 in a 2 ml polypropylene centrifuge tube. After sedimentation in an Eppendorf centrifuge, the tubes were washed twice with Tris (without disturbing the pellet) and the radioactivity determined in the pellet by liquid scintillation. Measurement of [3H]-Nicotine Binding to Purified Receptor by Competition with mAb 402C10. various amounts of purified nicotine receptor (0.01-1 pg) were incubated for 1 hr at 37° in Eppendorf centrifuge tubes in~ a final volume of 1.0 ml of a mixture containing 0.5 ml Tris-gel buffer (0.15 M NaCl 0.01 M Tris-HC1, pH 7.4 + 0.1% gelatin) 1 x 10 9 M (-)-[ 3 H)-nicotine at 8

vortexed, incubated overnight at 5°, and centrifuged in an Eppendorf centrifuge for 30 min at 5°. After aspirating the supernatant and carefully rinsing the tube with 2.0 ml of Tri~s-gel buffer, the bottoms of the tubes were cut off and radioactivity measured. Measurement of Effect of Anti-Nicotine mAb and Anti-Idiotype on [3H]-Nicotine Binding to Membranes. Various amounts of either mAbs 402C10 were preincubated for 30 min in an ice bath with membrane suspensions containing 2 mg protein in 1 ml 0.04 M NaP04 buffer, pH 6.5. [3HJ-nicotine binding was then determined as described above using a final concentration of 1 x 10 9 M [3H]-nicotine. Studies were also performed with longer pre-incubation periods at 37° prior to measuring [3H]-nicotine binding. Electrophoretic Transfer of Receptor Protein from Gels to Nitrocellulose. The procedure for the electrophoretic transfer of the receptor proteins from polyacrylamide gels to nitrocellulose was performed by the procedure of Towbin (10). After the receptor proteins were separated by NaDodSO4 polyacrylamide gel electrophoresis and the electrophoretic blotting to nitrocellulose performed with a Trans-Blot Cell (Bio-Rad Labs.), the nitrocellulose was immersed © with shaki~ng overnight at 37° in 0.05 K Tris-HC1 + 0.25 M NaC1 N ~ CA ~ 9 ~ ~ ~ ~

buffer, pH 7.4 containing 0.5% bovine serum albumin and 0.05 pg purified 420G11, followed by incubation with biotinylated sheep anti-mouse serum. After washing, the blots were then incubated with avidin horseradish~peroxidase, washed, exposed to a solution containing 5 x 10 3 M 3,3'-diamino-benzidine + 0.01% H202 in 10 mM Tris-HC1, pH 7.4, and washed in H20. RESULTS Fractionation of Soluble Proteins by Affinity Chromatography. The various fractions eluted from the affinity column were assayed for [3HJ-(-)-nicotine binding (Table 1). Approximately one-third of the total binding activity and almost all of the protein was present in fraction I; whereas, about two-thirds of the total binding activity was retained by the affinity column and eluted with 10-4 M(-)-nicotine (fraction II). NaDodSO4-Gel Electrophoresis of Purified Receptor Proteins. Acrylamide gel electrophoresis patterns of the receptor proteins purified by affinity (lane B, Fig,. 1) and immunoaffinity chromatography (lane C, Fig. 1Y were very similar, revealing a major component with a molecular weight of about 62 kdaltons and a minor component at 57 kdaltons. Lane D represents one-half of a sample of the receptor eluted from the affinity column, while lane E represents an immmoblot onto nitrocellulose on the other half of the purified sample. 10