the journal of biological chemistry vol. 261, no. 14, … · the journal of biological chemistry 0...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc.

Photoaffinity Labeling of the Ah Receptor*

Vol. 261, No. 14, Issue of May 15, pp. 6352-6365 1986 Printed tn ~T.s.A.

(Received for publication, November 13,1985)

Alan Poland$, Edward Glover, F. Hal Ebetino, and Andrew S. Kende From the McArdk Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706 and the Department of Chemistry, University of Rochester, Rochester, New York 14627

A series of halodibenzo-p-dioxins with the photolabile aryl azide functional group were synthesized and screened as potential photoaffinity labels for the Ah receptor, and 2-azido-3-iodo-7,8-dibromodibenzo-p- dioxin was selected for radiosynthesis with lZ6I (specific activity 2 176 Ci/mmol, equilibrium dissociation constant, KO = 0.76 nM). Following incubation of this 12‘I-labeled photoaffinity ligand with the protamine sulfate-precipitated fraction of C57BL/6J mouse liver cytosol, and irradiation with long wavelength ultra- violet light, the radiolabeled macromolecules were precipitated with acetone and analyzed by denaturing gel electrophoresis and autoradiography. Among the labeled products, two peptides with apparent molecular masses of 95,000 and 70,000 daltons had the following properties: 1) they were selectively labeled at low ligand concentrations; 2) they were labeled in approximately a 1:l ratio; 3) co-incubation with receptor agonists inhibited the photoaffinity labeling of both peptides to a similar extent, and structure activity relationship for inhibition of labeling by these agonists corresponded to that for their binding affinity to the Ah receptor; 4) upon nondenaturing chromatographic separation of photoaffinity labeled cytosol on high performance liquid chromatography size exclusion and anion exchange columns, the 95- and 70-kDa peptides coelute; 5) the migration of these peptides upon denaturing electrophoresis is the same in the presence or absence of a thiol reducing agent; and 6) proteolysis of the 95- and 70-kDa peptides produces a sim-ilar pattern of cleavage peptides. The simplest structure of the Ah receptor in mouse liver cytosol, appears to be a dimer composed of two noncovalently linked subunits of apparent molecular masses of 95 and 70 kDa, which have homologous structure and similar ligand binding sites, but other possibilities are discussed.

All of the diverse biological responses produced by 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD1) and related halogenated aromatic hydrocarbons are believed to result from the reversible, stereospecific binding of these compounds to a

* This work was supported in part by National Institute of Envi- ronmental Health Sciences Grant ES-01884 and National Cancer Institute Core Grant CA-07175. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Burroughs Wellcome Scholar in Toxicology. The abbreviations used are: TCDD, 2,3,7,8-tetrachlorodibenzo-p-

dioxin; TCDBF, 2,3,7,8-tetrachlorodibenzofuran; 1-NH2Me-TCDD, 1-aminomethyl-2,3,7,8-tetrachlor0dibenzo-p-dio~ ECm, concentration of competing ligand which reduces specific binding to one-half the initial value; KD, equilibrium dissociation binding constant; PS, protamine sulfate; MOPS, 3-(N-morpholiio)propanesulfonic acid; HPLC, high performance liquid chromatography; MS, mass spectrometry.

soluble protein, the Ah receptor, and the initiation of gene expression by the receptor-ligand complex (1). These responses include: (a) the induction of cytochrome PI-450 and other coordinately expressed “drug-metabolizing enzymes,” (b) the production of histologic lesions in a variety of tissues, the most distinctive of which involve proliferation and/or altered differentiation in epithelial tissues, and (c ) tumor promotion (2).

Agonists for the Ah receptor include: (a) chlorinated dibenzo-p-dioxin, dibenzofuran, biphenyl and azo(xy)benzene congeners, and brominated biphenyl isomers, with multiple halogen substitutions in the lateral ring positions; and (b) polycyclic aromatic hydrocarbons, i.e. 3-methylcholanthrene and benzo(a) pyrene, and some other nonhalogenated aromatic compounds, i.e. 5,6 benzoflavone (3, 4). All of the compounds are planar or can assume nearly planar configu- rations. Steroid hormones, vitamin D, vitamin A, and thyrox- ine do not compete for binding to the receptor, and at present there is no known physiologic ligand to the receptor (5).

Indirect evidence that the Ah receptor-ligand complex binds to a specific DNA sequence has been provided by Jones et al. (6) . These workers constructed a plasmid of the 5”regulatory sequence of cytochrome P1-450 and the gene for chloramphenicol acetyltransferase, transfected the DNA into cells with the Ah receptor, and showed incubation with TCDD produced expression of chloramphenicol acetyltransferase activity.

The Ah receptor has been identified and characterized in the cytosolic fraction of a variety of tissues from several mammalian species (7, 8). Following in vivo administration of [3H]TCDD the receptor-ligand is associated with the nuclear fraction and extractable by high concentration of sodium chloride (9). This and similar observations (10, 11) led to the proposal that the unoccupied Ah receptor exists in the cyto- plasm and upon ligand binding translocates to the nucleus. However, Whitlock and Galeazzi (12) have recently presented data, using a mouse hepatoma cell line, that the unoccupied receptor resides in the nucleus and its presence in the cytosol is an artifact of cell disruption in a large volume of buffer.

Identification and characterization of the Ah receptor is dependent on its reversible, high affinity binding of radiolabeled ligand (e.g. [3H]TCDD). The most frequently used source of the Ah receptor is rodent liver, an abundant tissue with relatively high receptor concentrations (50-120 fmol/mg cytosol, 5-12 pmol/g tissue). Hannah et al. (13) reported that the Ah receptor from C57BL/6J mouse liver has a Stokes radius of 75 A and an apparent M, of 250,00$; while Poellinger et al. (14) reported a Stokes radius of 61 A and an apparent M, of 110,000. Several factors have contributed to the difficulty in characterization of the protein and have frustrated attempts at purification: 1) the low receptor concentration and hence the need for extensive purification (1 to 2 pg of receptor/g of liver), 2) the hydrophobicity of the radioligands and low receptor concentration which result in appreciable

6352

Photoaffinity Labeling of Ah Receptor 6353

nonspecific binding and imprecision in many binding assays; 3) the tendency for the receptor to form aggregates, and 4) dependency on reversible ligand binding.

It is this latter point we wish to address. The unoccupied receptor gradually loses its capacity to bind ligand in solution. Dependency on reversible ligand binding precludes the use of any conditibn during purification which might result in irre- versible denaturation (e.g. chaotropic salts, many detergents, and extremes in pH). One approach to this problem is to use a radioligand which fofms a covalent linkage to the Ah receptor. In this paper, we report (a) the syntheses and scfeening of a number of photoaffinity labeled ligands for the Ah receptor, (b) the radiosynthesis of one of these photoaffiiity ligands, and (c) the use of this ligand in characterization of the Ah receptor in mouse liver.

EXPERIMENTAL PROCEDURES

Materials Acrylamide, bisacrylamide, ammonium persulfate, sodium dodecyl

sulfate, and N,N,N',N'-tetramethylethenediame were purchased from Bethesda &search Laboratories. Triethanolamine, triethanolamine hydrochloride, MOPS and salt, protamine sulfate (from Salmon, Grade X), glycine, dithiothreitol, 2-mercaptoethanol, lithium dodecyl sulfate, Coomassie Brilliant Blue R, phenylmethylsulfonyl fluoride, glutathione, and Staphylococcus aureus V-8 protease were purchased from Sigma. a-Chymotrypsin (bovine pancreas) was obtained from Calbiochem, and aprotinin from Mobay Chemical Co. Ammonium sulfate was purchased from Schwarz/Mann and Iodo- beads from Pierce Chemical Co. NOBF4, silver trifluoroacetate, Chor- amine T , thiopropionic acid, methionine methylester, 2-methylthio- ethanol, DL-thioctic acid, trans-o-dithiane 4,5-diol, ethanolamine, and dimethylaminopyridine were purchased from Aldrich.

Animals-C57BL/6J mice were purchased from the Jackson Lab- oratory and bred in our laboratory. Congenic C57BL/6J. (Ahd) mice, homozygous for the Ahd allele which determines the low affinity Ah receptor, were kindly provided by Dr. Daniel Nebert, National Insti- tute of Child Health and Human Development. These mice, C57BL/ 6N. DBA/2N(Ahd) N13F13, were maintained by a backcross-intercross mating system with C57BL/6J mice for 9 generations (C57BL/6J NsFg) and the.homozygous AhdAhd phenotype was determined by zoxazolamine paralysis tissue (15).

Syntheses l-Cyano-2,3,7,8-tetrachlorodibenzo-p-dioxin-A solution of 0.78 g

(4.4 mmol) of 3,4-dichlorocatechol and 9 ml of 1 N KOH (9 mmol) in methanol was stirred for several minutes and then evaporated to dryness. The resulting gummy solid was further dried by addition of 50 ml of benzene followed by evaporation under high vacuum (-1 mm Hg) for 6 h. The powdery dipotassium salt was then combined with 1.2 g (5 mmol) 2,3-dichlorobenzonitrile in 60 ml of dry dimethyl formamide. The reaction was placed under a nitrogen atmosphere and refluxed for 30 h with vigorous stirring. After cooling, this solution was poured over 75 ml of 2 N KOH/H20 and 75 ml of methylene chloride. The layers were separated and the aqueous fraction was reextracted with 2 X 50 ml of methylene chloride. The organic layers were then washed with 5 X 30 ml of water and 1 x 30 ml of brine. After drying over MgS04 and evaporation of solvent, the crude product was sublimed at 95 "C under a 1 mm Hg vacuum (an earlier sublimate was collected at 50-75 "C, 1 mm Hg and discarded) to yield 0.65 g (43%) of 1-cyano-2,3,7,8-tetrachlorodibenzo-p-dioxin. An analytical sample was prepared by recrystallization from acetic acid, m.p. 176-178 "C (TLC (CHCl3, silica), Rf = 0.55 (bright blue spot under 254 nm UV light), and MS m/e 282 (52), 284 (50), 286 (X), 345 (771,347 (loo), 349 (51), 351 (14)).

C13H13N02 Calculated C 45.22 H 0.87 Found C 45.01 H 0.97

1 -Aminomethyl-2,3,7,8-tetrachlorodibenzo-p-dwxin-l-Cyano- 2,3,7,8-tetrachlorodibenzo-p-dioxin (600 mg, 1.9 mmol) was taken up in 150 ml of ether in a three-neck reaction flask equipped with magnetic stirring and a gas outlet linked to a silicon oil bubbler. Over 30 min 1.2 g (22 mmol) of lithium aluminum hydride was added

portionwise at room temperature. The solution was then stirred for an additional h at room temperature. The reaction was carefully quenched by the sequential addition of 10 ml of water, 10 ml of 15% sodium hydroxide, and 10 ml of water. The mixture was filtered and the solvent was evaporated. The residue was fractionally sublimed at 140-150 "C under a 1 mm Hg vacuum (an earlier sublimate was collected at 100-120 "C, 1 mm Hg and discarded) to yield 480 mg (79%), m.p. 174-175 "C, giving: NMR 6 3.30 (2H, bs), 3.80 (2H, bs), 7.28 (lH, s), 7.38 (lH, s), 7.49 (lH, s); MS m/e 349, 351, 353 (100, Ch pattern), 314, 316 (C4 pattern); and UV X,, 309 (e = 7,600), 236 (e = 53,800). An analytical sample was prepared by fractional subli- mation.

C13H7CbNOz

Found C 44.69 H 2.19 N 3.83 Calculated C 44.48 H 2.01 N 3.99

1-(5'-Azido-2'-nitrobenzamidomethy~-2,3,7,8-tetrach~rodibenzo- p-dioxin-To a solution of 11.4 mg (0.033 mmol) of 1-NH2Me-TCDD in 0.5 ml of tetrahydrofuran (freshly distilled) was added 10 mg (0.33 mmol) of N-5-azido-2-nitrobenzoyloxysuccinimide (Pierce Chemical Co.) in 0.5 ml of tetrahydrofuran (freshly distilled). The reaction mixture was allowed to stir for 3 days in a sealed vessel a t room temperature in the dark. The resulting solution was eluted on a 2- mm silica gel prep plate with 1% methanol/methylene chloride. The major band (Rf = 0.65) was extracted with chloroform and the solvent was evaporated to yield 13.5 mg (76%) of a light yellow powder, m.p. 185-187 "C (decomposition, darkens at 163 "C) giving: MS (20 eV) m/e 539, 541, 543 (76, 100, 43), 513, 515, 517 (75, 99, 44); and UV X,. 307 (e = 11,900), 235 (e = 51,500).

1 -(4-Azido-2-hydroxybenzamidomethyl-2,3,7,8-tetrachlorodibenzo- p-dioxin-To a solution of 6.5 mg (0.019 mmol) of 1-NH2Me-TCDD in 0.15 ml of methylene chloride was added 10 mg (0.36 mmol) of N- hydroxysuccinimidyl-4-azidosalicylic acid (Pierce Chemical Co.) in 0.15 ml of methylene chloride. Triethylamine (0.01 ml) was added and the reaction mixture was allowed to stir for 2 days in a sealed vessel at room temperature in the dark. The resulting solution was eluted on a 2-mm silica gel prep plate with 1% methanol/methylene chloride. The major band (Rf = 0.45) was extracted with chloroform and the solvent was evaporated to yield 2.5 mg (26%) of a white solid, m.p. 183-186 "C giving: MS m/e 475 (7), 477 (5), 449 (loo), 451 (95), 453 (30); and W X,, 310 (E = 7,810), 273 (e = 11,500), 232 (c = 33,500).

1 -(4-Azidobenzamidomethyl)-2,3,7,8-tetrachlorodibenzo-p-dwxin- 1-NH2Me-TCDD, 8.1 mg (0.023 mmol) was taken up in 0.3 ml of methylene chloride, and to this solution was added 15 mg (0.058 mmol) of N-hydroxysuccinimidyl-4-azidobenzoate (Pierce Chemical Co.). Triethylamine (0.01 ml) was then added and this solution was allowed to stir for 3 days at room temperature in the dark. After evaporation of the methylene chloride with a nitrogen stream, the residue was stirred with 0.3 ml of tetrahydrofuran and 0.3 ml of dilute sodium bicarbonate for 4 h. The mixture was then extracted with 3 X 0.3 ml of methylene chloride and washed with 0.3 ml of aqueous saturated sodium bicarbonate. The organic solvent was dried over magnesium sulfate and evaporated to yield 4 mg of the desired amide m.p. 193.5-197 "C giving TLC Rf = 0.35 (CHCl,, Analtech uniplates, Silica Gel GF); MS (20 eV) m/e 494,496,498 (C4 pattern), 433,435, 437 (Cb pattern); and UV X,, 310 (e = 5,450), 270 (e = 19,400), 235 (e = 45,700).

1 -(6-~4-Azido-2-nitroph~nylami1w)hexamidomethyl)-2,3,7,8-tetrachlorodibenzo-p-dioxin-l-MH2Me-TCDD 6.5 mg (0.019 mmol) was taken up in 0.3 ml of methylene chloride and to this solution was added 15 mg (0.037 mmol) of N-succinimidyl 6-(4'-azido-2'-nitro- pheny1amino)hexanoate (Pierce Chemical Co.). Triethylamine (0.01 ml) was then added and this solution was allowed to stir for 3 days at room temperature in the dark. After evaporation of the methylene chloride with a nitrogen stream, the residue was stirred with 0.3 ml of tetrahydrofuran and 0.3 ml of 1 N aqueous sodium hydroxide for 4 h. The mixture was then extracted with 3 X 0.3 ml of methylene chloride and washed with 0.3 ml of aqueous saturated sodium bicarbonate. The organic solvent was dried over magnesium sulfate and evaporated to yield 7 mg (60%) of a red solid, m.p. 125-129 "C giving: NMR (CDCI3, 6) 1.45 (2H, m), 1.75 (4H, m), 2.20 (2H, t), 3.25 (2H, m), 4.61 (2H, d), 5.72 (lH, bs), 6.80-7.09 (5H, m), 7.83 (lH, d), 7.94 (lH, bs); and UV X,, 260 (e = 20,500), 239 (e = 51,700) shoulder -310.

1 -Azido-3,7,8-trichlorodibenzo-p-dioxin-l-Nitro-3,7,8-trichlorodibenzo-p-dioxin was prepared according to Chae et al. (16) and 1.85 g (5.59 mmol) was taken up in 20 ml of acetic acid and then 2 g (35.7

6354 Photoaffinity Labeling of Ah Receptor

mmol) of iron powder was added. The mixture was stirred at room temperature under nitrogen for 2 days. After pouring over 100 ml of ethyl acetate and 50 ml of water and separation, the organic extracts were washed with 30 ml of water, 2 X 30 ml of sodium carbonate, and 30 ml of brine and dried over MgSO,. Evaporation of solvent and recrystallization from toluene gave 1.2 g (71%), m.p. 217-222 "C of 1- amino-3,7,8-trichlorodibenzo-p-dioxin. An analytical sample was prepared by HPLC (85% methanol/water, 1 ml/min, Waters C-18 pBon- dapak, 11 min, m.p. 228-230 "C (lit. 228.5-230 "C giving: TLC

2.5), 6.38 (lH, d, J = 2.5), 6.96 (lH, s), 6.97 (lH, s); MS m/e 301 (M+), 303, 305 (characteristic pattern for CL); and UV Amax 305 nm ( e = 6,900), 244 nm ( e = 43,800).

TO a solution of 2 mg (0.014 mmol) of nitrosonium tetrafluoroborate in 0.25 ml of dry CH2C1, was added 4.1 mg (0.016 mmol) of 1- amino-3,7,8-trichlorodibenzo-p-dioxin in 1 ml of dry CH,C12. This solution was stirred for 2 h a t room temperature in a sealed vessel. The solvent was removed with a nitrogen stream, and the resulting diazonium salt was treated with 13 mg of sodium azide in 2 ml of acetonitrile (distilled). The reaction was allowed to stir 3-5 h at room temperature in the dark and the solvent was partially removed with nitrogen. The residue was taken up in 10 ml of ether and 5 ml of water, separated, and reextracted with 2 X 5 ml of ether. Finally, the organic extracts were washed again with water and an aqueous sodium chloride solution (4 ml) and dried over MgS04 to yield 3.1 mg (70%), m.p. 170-175 "C, of the desired product. This product was of very high purity for biological testing as demonstrated by its 400-MHz NMR spectrum in CDC13 giving (6) 6.66 (lH, d, J = 1.5), 6.70 (lH, d, J = 1.5), 6.98 (lH, s), 7.05 (lH, 5); MS 327 (M +, 20), 329 (M + 2, 201,331 (M + 4, lo), 299 (37), 301 (421,303 (21), 264 (loo), 266 (70); UV Amax 304 ( e = 4,500), 243 ( e = 34,900); and HPLC, 14.2 min (90% ethanol/water, 1 ml/min, Waters C-18 pBondapak).

1 -~zido-~-wdo-3,7,8-trichlorodibenzo-p-dwxin-To a solution of 11 mg (0.03 mmol) of l-amino-3,7,8-trichlorodibenzo-p-dioxin in 2 ml of tetrahydrofuran was added 20 mg of chloramine T (0.06 mmol) followed by 15 mg of NaI (0.6 mmol). Finally, 15 mg (0.5 mmol) of AgOCOCF3 (silver trifluoroacetate) and 10 mg (0.09 mmol) of sodium acetate were added and the reaction mixture was allowed to stir for 48 h at room temperature in the dark. HPLC analysis showed that the reaction had proceeded to 60% 1-amino-2-iodo-3,7,8-trichlorodibenzo-p-dioxin and 40% starting material. An analytical sample was prepared by reverse phase HPLC; retention time 14 min (85% methanol/water, 1 ml/min, Waters C-18 pBondapak) to yield an off-white solid, m.p. 229-231 "C giving: NMR (CDCls, 6) 3.91 (2H, bs), 6.63 (lH, s), 7.01 (lH, s), 7.12 (lH, 5); and MS m/e 427 (M +, loo), 429 (M + 2, loo), 431 (M + 4, 341,300 (20), 302 (20), 304 (8).

This amino analogue was converted to the corresponding azide by the same conditions used for the conversion of l-amino-3,7,8-trichlorodibenzo-p-dioxin. The product had a retention time of 17.9 min in HPLC (90% methanol/water, 1 ml/min Waters C-18 pBondpak) giving NMR (CDC13, 6) 6.89 (lH, s), 7.06 (lH, s), 7.14 (lH, S) MS 453 (M +, 13.1), 455 (M + 2, 14.2), 457 (M + 4, 6.0), 425 (20.2), 427 (loo), 429 (78.7); and UV L a x 254 ( e = 40,200), 310 ( e = 4,960). 2-Amino-3,7,8-trichlorodibenzo-p-dioxin-To 96.7 mg of 2,7.8-

trichlorodibenzo-p-dioxin in 5 ml of glacial acetic acid in a 10-ml round-bottom flask was added 1.0 ml of fuming nitric acid. After 10 min of stirring at room temperature, the mixture was placed in a 90 "C oil bath and held at that temperature with stirring for an additional 45 min, then the flask was allowed to cool to room temperature overnight. The reaction mixture was partitioned between 40 ml of methylene dichloride and 15 ml of H20, the organic layer was thoroughly washed with aqueous NaHC03 solution until effervescence ceased, then once with water and dried over anhydrous MgSO4. Evaporation of solvent gave 97 mg of an insoluble yellow solid, m.p. 275-278 "C, shown by mass spectrometry (M' = 331) and proton NMR to be the 3-nitro compound.

Reduction of this nitro compound was carried out by stirring it in a glacial acetic acid solution at 75 "C overnight with arr equal weight of iron powder. The reaction mixture was poured into ice water, and the precipitated amine was dried then sublimed at 130-135 "C and -0.2 mm to give a 70-75% yield of a light yellow solid, m.p. 280- 283 "C decomposition, Mt = 301. The amine was acetylated (acetic anhydride, 30 min, 90 "C) to give the colorless N-acetylamino derivative, m.p. 305-308 "C, which was sent for analysis.

(CHCl3) Rf = 0.7, NMR (CDC13, 6) 3.87 (2H, bs), 6.29 (lH, d, J =

CMH&LNO~

Found C 48.64 H 2.46 Calculated C 48.98 H 2.33

2-Azido-3,7,8-trichlorodibenzo-p-dwxin-2-Amino-3,7,8-trichlorodibenzo-p-dioxin (11 mg, 0.036 mmol) was dissolved in 1.8 ml of methylene chloride and this solution was added to a slurry of 5.1 mg (0.043 mmol) of nitrosonium tetrafluoroborate. The reaction mixture was stirred for 1 h in a sealed vessel a t room temperature and then the solvent was evaporated with a stream of nitrogen. A slurry of 36 mg (0.55 mmol) of sodium azide in 3 ml of acetonitrile (distilled from CaH,) was then added to the tetrafluoroborate salt and this mixture was stirred vigorously overight. The solvent was then evaporated with a stream of nitrogen and the residue was partitioned between 5 ml of ether and 3 ml of HzO. After separation and reextraction with 3 ml of ether, the organic phase was washed with water and brine and dried over MgSO,. The solvent was evaporated with a nitrogen stream to yield a light tan solid, m.p. 189-193 "C (decomposition) giving: TLC Silica Gel GF (CHCl,) Rf = 0.75. MS 327,329,331 (C13 pattern); and UV Amax 314 (t = 7,300), 239 ( e = 29,900). 2-Azido-3-iodo-7,8-dibronodibenzo-p-dioxin-4,5-Dibromocate-

chol (1.5 g) was dissolved in 10 ml of methanol, containing 660 mg of KOH and concentrated under vacuum. Ten ml of benzene was added and rotovaporated 3 times. 3,4-Dichloronitrobenzene (1.1 g) in 10 ml of freshly distilled dimethyl formamide was added and the mixture was refluxed overnight. The mixture was diluted to 200 ml with water and washed 2 times with ethyl acetate. The organic phase was washed with an aqueous solution of sodium hydroxide, water, aqueous hydro- chloric acid, and sodium chloride and then dried over MgS04. Follow- ing crystallization from acetic acid, 602 mg of 2-nitro-7,8-dibromodibenzo-p-dioxin was obtained, 27% yield, m.p. 215-216 "C.

C~H5N04Br2 Calculated: C 37.2 H 1.29 Found C 37.26 H 1.49

The product (602 mg) was dissolved in 12 ml of acetic acid. Iron powder (440 mg) was added and the mixture heated at 55 "C for 4 h, during which it turned from an orange to tan color. A saturated solution of sodium bicarbonate was added until the sample was weakly basic, and the mixture was twice extracted with ethyl acetate. The combined organic extracts were washed with water and aqueous sodium chloride and then dried over MgS04. The product, 400 mg with a m.p. of 186-188 "C (decomposition), was purified by thin layer chromatography using a hexane:ethyl acetate (3:l) solvent. Two hundred mg of 2-amino-7,8-dibromo-p-dioxin were obtained, m.p. 196-197 "C. Anal. Found C, 40.63; H, 2.12; Br, 45.72. This compound was converted to the iodinated derivative as described for the micro- scale radiosynthesis described below.

Radwsynthesis of 2-Azid0-3-~~~I]wdo-7,8-dibromodibenzo-p-dwxin The lZ5I photoaffinity ligand was formed in a 3-step synthesis from

2-amino-7,8-dibromodibenzo-p-dioxin as seen in Fig. 1. To 5 mCi of carrier-free NalZ5I (New England Nuclear, NE2-033L, 2350 mCi/ml in 6 mM NaOH, 115 pl of water) in a septum-sealed vial was added 2.5 nmol of 2-amino-7,8-dibromodibenzo-p-dioxin in 25 pl of methanol, 1.13 pmol of sulfuric acid in 10 pl of methanol/water (9:1), and 25 nmol of chloramine T in 5 pl of methanol (17). The iodination reaction was complete in 30 min and terminated by the addition of 500 pg of sodium metabisulfite and 5 pmol of sodium hydroxide in 35 pl of water. Two hundred fifty microliters of methylene dichloride were added and the organic phase was aspirated by syringe (gas syringe, pressure-locked plunger tip, P010032, Pierce Chemical Co.) and transferred to a 1-ml sealed conical vial (Reacti-vial, 13221, Pierce Chemical Co.) containing 10 mg of MgSO4,75 pl of methylene chloride and a microstirring bar. After stirring the MgS04 organic mixture for 45 min to remove water, the organic phase was transferred to a 1-ml sealed conical vial containing 1 mg of NOBF4 (Aldrich) in 25 pl of CHzClz and a microstirring bar. With stirring, the nitrosation reaction was complete in 45 min and the solution was transferred to a 3-ml septum-sealed conical vial containing 150 pl of CH3CN, 25 mg NaN3 and a microstirring bar. The reaction was complete after 45 min, and sodium metabisulfite (2 mg in 100 pl of water) and 200 pl

B r J p J , O r ;;lrJ=i;+{ 0 7 NOBF4: N a b ) yJ1n; Br

~ ~ 3 0 . 7 6 X 10-9~ sp. act. = 2176 Cilmmol

FIG. 1. Radiosynthesis of 2-azido-3-['261]iodo-7,S-dibromodibenzo-p-dioxin.


of methylene dichloride were added to the vial and vigorously mixed. The organic phase (300-500 pl) was transferred to another vial, the solvent was evaporated by gentle warming and a stream of NP, and the solute was redissolved in 100 p1 of methanol. The radiosynthesis was performed in a separate room with an excellent laboratory hood, with the use of sealed vials and airtight syringes to minimize escape of lP512 and monitoring with a y scintillation counter.

The product was purified by HPLC on a pBondapak C-18 reverse phase column (with a C-18 precolumn) using an isocratic elution system of methanol/water (9010) and a flow rate of 1 ml/min. The '251-labeledproduct cochromatographs with unlabeled 2-azido-3-iodo- 7,8-dibromodibenzo-p-dioxin and has a retention time of 16.3 min. The '%I photoaffinity label was stored at 1 X 10' cpm/ml in methanol and protected from light. The overall yield was usually -20%, based on incorporation of lP51 into the purified product.

Buffer The buffer MEN contained 25 mM MOPS, 1 mM EDTA, 0.02%

NaN8, pH 7.5 (at 0 "C).

Preparation of Hepatic Cytosol, the Ammonium Sulfate, and the Protamine Sulfate Fraction of Cytosol

C57BL/6J mice were killed by cervical dislocation, and their livers were removed and homogenized in 3 or 9 volumes of MEN buffer + 10% glycerol and centrifuged at 10,000 X g for 20 min at 4 "C. The supernatant fraction was carefully removed by aspiration to avoid the surface lipid layer and centrifuged at 100,000 X g for 1 h at 4 "C. The supernatant fraction was removed by aspiration to avoid the surface lipids and either stored a t -70 "C or further processed. 40-55% Ammonium Sulfate Fraction of Cytosol-A saturated so-

lution of ammonium sulfate in MEN buffer was slowly added to hepatic cytosol (10 mg of protein/ml) in MEN buffer + 10% glycerol at 0 "C to achieve a 40% salt concentration. After stirring for 30 min, the solution was centrifuged at 10,000 X g for 20 min and the supernatant was decanted. To this supernatant fraction at 0 "C, a saturated solution of ammonium sulfate was slowly added to achieve a final salt concentration of 55% and the solution was stirred for 30 min at 0 "C. The 40-55% ammonium sulfate precipitate was collected by centrifugation and stored at -70 "C until use. This precipitate contained 25% of the cytosol protein and a 2-fold enrichment in the Ah receptor.

Protamine Sulfate Precipitate of Cytosol-To hepatic cytosol (5 mg of protein/ml) in MEN buffer + 10% glycerol was added a solution of protamine sulfate so the final concentration was 0.2 mg/ml and, after standing for 15 min at 0 "C, the precipitate was collected by centrifugation at 5,000 X g for 10 min, and the pellet was stored at -70 "C until use. On the day of use the protamine sulfate-precipitated pellet (15 mg of protein, equivalent to 1 g wet weight of liver) was dissolved in 250 pl of MEN buffer + 2 M NaCl, diluted with 3 volumes of MEN to lower the NaCl concentration to 0.5 M, passed over a small column of CM-Sepharose (Pharmacia) to remove protamine sulfate, and then diluted to the appropriate concentration of protein. Protamine sulfate precipitate contains about 15% of the total cytosol protein and most of the nucleic acid and produces a 4- to &fold enrichment in the Ah receptor.

Determination of the Equilibrium Dissociation Binding Constants, KD Values, of the Photoaffinity Ligands

One ml of hepatic cytosol (2 mg of protein/ml) in MEN buffer + 10 mM dithiothreitol + 10% glycerol was incubated with: ( a ) 1 X lo-' M t3H]TCDD (-30 Ci/mmol), (b) the same concentration of radioligand plus a 200-fold molar excess of unlabeled TCDBF, or (c) the radioligand plus varying concentrations of the unlabeled photoaffinity ligands. After a 30-min incubation at 20 "C, 0.5 mi of a suspension of charcoal/dextran (3%/0.3%) was added, and after 5 min the mixture was centrifuged to remove the charcoal. The radioactivity in the supernatant was quantified by liquid scintillation spectrometry and corrected for efficiency (-40%).

Specific binding was calculated as total binding minus nonspecific binding (binding in the presence of an excess of TCDBF). The EC50, the concentration of competing ligand which reduced specific binding to one-half the initial value, was estimated from a plot of specific binding versus log of ligand concentration. The KD of the competing

ligand was estimated from the relationship (18), KO = EC5o/l + -, A KA

where A = concentration of [3H]TCDD (1 X lo-' M) and KA = equilibrium dissociation constant for TCDD (= 0.3 X lo-' M).

Photolysis

The hepatic cellular fraction containing the Ah receptor was incubated with the photoaffinity ligand to achieve equilibrium binding and then irradiated. Unless otherwise noted, this consisted of incubation of the PS fraction of liver cytosol in MEN buffer and the lP51- labeled photoaffinity ligand for 30 min at 20 "C, followed by 5 min at 0 O. Charcoal/dextran (final concentration 1%/0.1%) was added for 5 min at 0 "C to adsorb unbound ligand and then removed by centrifugation. The incubation mixture was immediately irradiated at 0 "C with Westinghouse FS-20 sunlamps (20 watts, X > 300 nm) using either of two nearly identical conditions: (a) 40 watts at a distance of 8 cm for 2 min or (b) 80 watts a t a distance of 4 cm for 20 s. Under the former conditions light intensity, measured by an International Light Radiometer at 355 nm was 2.8 X watts/cm2/s.

Following irradiation, 4 to 9 volumes of cold acetone was added immediately to precipitate the proteins, and the mixture was stored overnight a t -20 "C. The protein pellet was collected by centrifugation and washed with cold acetone/water (9:l) to remove any remaining noncovalently bound photoproducts and salt. The washed pellet was dissolved in electrophoresis sample buffer (19) (which contained 2% lithium dodecyl sulfate and 20 mM dithiothreitol) a t a concentration of 1 mg of protein/ml and placed in a boiling water bath for 2 min.

Gel Electrophoresis and Autoradiography

The photoaffinity labeled protein samples were subjected to denaturing electrophoresis on discontinuous polyacrylamide slab gels by the method of Laemmli (19) (4% stacking gel, 7.5% separating gel, and acrylamide/bisacrylamide (300.8)) 1.5 mm thick, with 15-mA current/gel. The gels were fixed and stained with 25% isopropyl alcohol, 10% acetic acid, 0.025% Coomassie Brilliant Blue R, de- stained with 10% isopropyl alcohol, 10% acetic acid, and dried. For autoradiography, the dried gel was exposed to a sheet of XAR-5 film (Eastman Kodak Co.) backed with an intensifying screen (Cronex, Dupont) and held at -70 "C until film development (20).

HPLC

The instrumentation consisted of the following components from Waters: injection port (MUGK), two-solvent delivery system (models 6000 or 510), automated gradient controller (680), and W-absorbance detector (M440). In addition, we used an ISCO V, variable wavelength absorbance monitor, a Kipp and Zonen chart recorder, a Hewlett- Packard integrator (3390A), and an LKB Helirack fraction collector. All solvents were filtered through a 0.2 pm filter and deaerated by vacuum and sonication prior to use.

The photoaffinity ligands were chromatographed on a reverse phase column (pBondapak C-18,0.46 X 25 cm, Waters) with a similar precolumn and developed by an isocratic elution system (methanol/ water (9O:lO or 85%)) a t a flow rate of 1 ml/min (21,22).

The '251-photoaffinity labeled proteins from cytosol were subjected to ion exchange chromatography and size exclusion chromatography. Ion exchange chromatography was performed on a Mono& column (0.5 X 5 cm) with prefilter (Pharmacia) using 20 mM triethanolamine buffer, pH 7.5 (at 0 "C), with a 0-500 mM NaCl gradient and a solvent flow rate of 1 ml/min. To maintain a cold temperature (essential to prevent dissociation of the reversible ligand-receptor complex), the solvents, exteriorized injection loop, and the column were submerged in ice baths.

Size exclusion chromatography was performed on a TSK G-4000 SW column (0.75 X 60 cm) with TSK SW precolumn (0.75 X 10 cm) (Toyo Soda Co.) conducted at -5 "C in the cold room. The buffer was 25 mM MOPS, 0.02% NaN3,2OO mM NaCl, pH 7.0. The sample injected was 400 pl, and the flow rate was 0.4 ml/min.

Quuntitatwn of Radioactivity

The [3H]TCDD in binding assays or chromatographic fractions was quantified by liquid scintillation spectrometry. The 'z51-photoaffinity ligand or covalently labeled macromolecules (in the acetone precipitates or gel electrophoresis bands) were quantified by y scintillation spectrometry.


TABLE I Photoaffinity ligands: structure and properties

The equilibrium dissociutwn constants, KD values, for the photoaffinity ligands were determined by a competitive binding assay in the dark as described under “Experimental Procedures.” The UV absorption spectrum for each compound dissolved in p-dioxane was measured on a Cary 118C split-beam recording spectrophotometer. e , the molar extinction cwfficient at the maximum absorbancy peak (232-254 nm), was determined. Photolysis was carried out by radiation of the ligands in p-dioxane with two 20-watt FS sunlamps at a distance of 8 cm. Photolysis was determined by the reduction in the UV absorbancy maxima or the reduction in peak area on HPLC reverse phase chromatography. From a plot of concentration uersus irradiation time, the photolysis half-life was calculated. HPLC retention time on pBondapak C-18 reverse phase column using an isocratic elution system of methanol/ water: *, methanol/water (9010) **, methanol/water (85:15) (see ‘‘Experimental Procedures” for details).

Absorption HPLC

time K d maxima photol~sis retention Tv,

1 cEJ$J CI CI

nM n n S nin

X = 243,304 0.44 t = 34,900 38 14.2*

4

cl@J;&l CI CI 2.1 X e = = 254,310 40,200 30 17.9*

X = 239,314 0.49 e = 29,900 22.0**

0.76 X = 242,316 t = 46,000 15 16.6*

5 X = 235,307 13.0 e = 51,500

6

7

n

0 U

I I

8.1

113.0

11**

X = 235,270 shoulder 310

e = 45,700 200 16.3**

X = 232,273 shoulder 310

e = 33,500 24 23**

shoulder.310 cn nnn nn

RESULTS

As potential photoaffinity ligands for the Ah receptor, we synthesized two series of halodibenzo-p-dioxins containing photolabile azide functional groups (Table I): (a) coupling 1- aminomethyl-2,3,7,8-tetrachlorodibenzo-p-dioxin to commer- cially available azidophenyl derivatives, or (b) synthesizing 1- azido- or 2-azido-halodibenzo-p-dioxins from their corresponding amino precursors. 1-NH2Me-TCDD has an equilibrium dissociation constant, KO, for the Ah receptor of 2 nM, and the introduction of bulky aryl groups reduces the receptor affinity (reversible binding measured in the dark) 4-fold or more, KD values 2 8 nM (compounds 5-8). In contrast, compounds 1-4, with the azido group directly attached

to the dibenzo-p-dioxin ring, had much greater receptor affin- ities, KO values = 0.44-2.1 nM similar to that of TCDD (KO = 0.3 nM).

Also seen in Table I are the UV absorption maxima, rate of photolysis, and chromatographic retention time (HPLC reverse phase C-18 column) for these analogues. All of the halodibenzo-p-dioxin congeners had major, absorption maxima at 232 to 254 nm and smaller absorption peaks or broad shoulders a t 304 to 314 nm. Compounds 6-8 had additional absorption maxima at 260 to 273 nm. The photolysis rates of these analogues were more rapid with irradiation at 254 nm (Sylvania germicidal lamp, 15 watts) than with longer wavelength UV light (X > 300 nm); however, the latter was quite


A

1.0

0.8

0.6 0)

c f :: Q n

0.4

0.2

0.0

240 260 200 300 320 340nm

3

I I I I I

240 260 280 300 320 340nrn

FIG. 2. UV spectrum and effect of photolysis. A, l-azido-3,7,8- trichlorodibenzo-p-dioxin. B, 2-azido-3-iodo-7,8-dibromodibenzo-p- dioxin. The compounds were dissolved in p-dioxane (10 pg/ml) and the UV absorbance spectrum was determined on a Cary 118C split- beam recording spectrophotometer at time 0 and after irradiation for varying intervals (FS sunlamps, 40 watts at 8 cm distance).

adequate and was used in all subsequent studies to minimize direct UV damage to protein. The UV absorption spectra and changes with photolysis for two analogues are seen in Fig. 2.

The capacity of these analogues to covalently bind to the Ah receptor upon UV irradiation was assessed by an indirect

assay, receptor inactivation, i.e. the loss of reversible ligand binding. This avoided the need to radiolabel each compound. This assay is based on one used by Katzenellenbogen et al. (34) for photoaffinity labeled estrogen inactivation of the estrogen receptor. Hepatic cytosol was incubated with the photoaffinity ligands at concentrations equivalent to 3, 10, and 30 KO, and one sample was irradiated, while an identical sample was held in the dark. Following procedures to remove the ligand or noncovalently bound photoproducts, the specific binding of [3H]TCDD was determined (specific binding = total binding ([3H]TCDD) minus nonspecific binding (3HTCDD plus a 200-fold molar excess of unlabeled TCDBF)). The difference between the specific binding in the nonirradiated.and irradiated samples is a measure of receptor inactivation. Reduction in reversible specific binding may be caused by: (a) true photoaffinity labeling, (b) pseudoaffinity labeling (35), (c) failure to completely remove the photoaffinity ligand or noncovalently labeled photoproducts, and (d) direct irradiation damage to the receptor.

In Table I1 are seen data from two typical experiments. The specific binding of C3H]TCDD to cytosol that was treated with the extraction/adsorption procedure is equated with 100% binding. Irradiation of cytosol in the absence of ligand produced no reduction in the specific binding of [3H]TCDD. Following incubation of high affinity ligands with cytosol in the dark and their subsequent removal by the extraction/ adsorption procedure, the specific binding of [3H]TCDD was approximately that of control cytosol (first three compounds in Table 11). Irradiation of these samples produced a concentration-related reduction in the specific binding of [3H]TCDD. Incubation of lower affinity ligands with cytosol in the dark produced a concentration-dependent reduction in specific binding, suggesting the incompleteness of removal of ligand at high concentrations (last example, Table 11). Irradiation of these samples produced a further reduction in specific binding of [3H]TCDD. This indirect assay, despite its limitations, permitted the identification of the most promising photoaffinity ligands for radiolabeling, 2-Azido-3-iodo-7,8-dibromodibenzo-p-dioxin was selected

for radiosynthesis, based on its high affinity reversible binding (Kd = 0.76 nM), capacity for receptor inactivation, and ease of synthesis. The three-step radiosynthesis of this ligandusing carrier-free NalZ5I and subsequent purification by HPLC had an overall yield of 20% and the compound had a specific activity of 2176 Ci/mmol (see “Experimental Procedures”). 2-Azido-3-~251]wdo-7,8-dibromodibenzo-p-dioxin Photoaf-

finity Labeling-The PS fraction of cytosol was incubated with varying concentrations of the 1251-photoaffinity ligand in the presence or absence of excess TCDBF, the mixture was irradiated, and the products were analyzed as described under “Experimental Procedures.” As seen in Fig. 3, several peptide bands are labeled. Two peptides with apparent molecular masses of 95,000 and 70,000 daltons are of particular interest: (a) they are selectively labeled at low ligand concentrations (0.05 KO = 0.038 nM) and (b) labeling is inhibited by co- incubation with an excess of a receptor agonist. In addition, there are two or three other peptides (molecular masses between 70 and 95 kDa) which are labeled to a lesser extent and whose labeling is blocked by co-incubation with TCDBF. Inbred strains of mice with the Ahd allele have a much lower affinity Ah receptor (5). Photoaffinity labeling of the PS fraction of liver cytosol of congenic C57BL/6J (Ahd) mice produces no labeled peptides that are specifically blocked by co-incubation with TCDBF (last two lanes in Fig. 3).

The apparent M, of these two peptides was determined by subjecting the 1251-photoaffinity labeled PS fraction of cytosol, along with lZ5I-labeled marker proteins, to denaturing electro-


TABLE II Ah receptor inactivation

Mouse liver cytosol (5 mg of protein/ml) in 1 ml of MEN buffer + 10% glycerol and 2 mM 2-mercaptoethanol was incubated with the photoaffinity ligand (at concentrations of 0, 3, 10, and 30 x KD) for 30 min at 20 “C, and one sample was held in the dark, while a second identical sample was irradiated (FS 20 sunlamps, 40 watts at 8 cm for 10 min). Triton X-100 (final concentration 0.5%) was added and, following a 20-min incubation, 1 g of SM- 2 Biobeads (Bio-Rad) was added for 20 min. The resin and adsorbed detergent was removed by filtration through a sintered glass funnel. A suspension of charcoal/dextran (final concentration l/0.1%) was added to the filtrate and, following a 20-min incubation, the charcoal was removed by centrifugation. Specific binding was determined by the addition of [3H]TCDD (80,000 dpm/ml = 1.3 nM) in the absence or presence of a 200-fold molar excess of TCDBF. In control samples the specific binding was typically 70-80% of total binding.

Dark UV irradiated % Differ- ence

Specific % Control Specific % Control d& - lJIJ binding binding biidiig binding irradiated

Control (untreated) Control (extraction/adsorption)

Control (untreated) 11,176 Control (extraction/adsorption) 10,533

CPm 16,070 10,985

10,577 9,954

10,192

12,561 114 5,846 46 68 12,584 115 8,730 69 46 12,169 111 3,807 30 81

11,517 11,027

9,928

10,418 99 8,846 78 21 10,346 96 8,450 74 22

7,555 72 4,978 44 25

10,379 99 7,972 70 29 9,077 86 7,318 64 22 7,022 67 5,895 52 15

phoresis on a 5-20% polyacrylamide gradient, long slab gel (Hoefer Scientific Instruments). In Fig. 4a, Coomassie Blue- stained gel is seen in lanes 14, and the autoradiograph in lanes 5-8. The apparent M, of the two photoaffinity labeled bands, blocked by an excess unlabeled agonist (lane 7 versus 8) determined by a plot of the migration distance verws log M, of the protein standards (Fig. 4b), was determined to be 95,000 and 70,000.

Pseudoaffinity Labeling: Photoaffinity Labeling Stoichiome- try and Specificity-Prior to further characterization of these labeled peptides, it is useful to address three aspects of photoaffinity labeling: (a) pseudoaffinity labeling (35), (b) specificity of labeling, and (c) stoichiometry. We will concentrate on the 95-kDa peptide for simplicity and assume that it is part of the Ah receptor, as will be documented below.

The 1251-photoaffinity ligand was incubated with the PS fraction of cytosol and irradiated for varying periods of time, and the proteins were immediately precipitated with acetone and analyzed as in previous experiments. As seen in Fig. 5, upon irradiation, the time course OE (a) total covalent binding

100

cPm 16,016 12,577 100

96 8,869 71 25 91 7,185 57 40 93 6,923 55 38

100

109 105

94

10,011 11,363

9,264 6,918 6,572

100

81 61 58

28 44 36

-

(cpm in acetone precipitate), (b) covalent binding to the 95- kDa peptide, and (c) photolysis of the radioligand are similar, with half-lives of approximately 15 s. However, this overall stoichiometry is misleading.

Pseudoaffinity Labeling-If the same incubation mixture is irradiated and the addition of acetone delayed, the total covalent binding and the labeling of the 95-kDa peptide increase over the interval following irradiation until the addition of acetone (Fig. 6A). The specificity of labeling, 95-kDa labeling/ total covalent binding x 100, decreases over this interval from 5.1 to 3.2%. This data suggests that (a) irradiation of photoaffinity ligand generates a long-lived reactive intermediate, (b) this long-lived electrophile has less specificity of labeling than the initial photoproducts, (c) the addition of acetone stops labeling by this long-lived species by separating the precipitated protein from the acetone-soluble photoproducts.

A more direct demonstration of this photolysis-generated long-lived intermediate is seen in Fig. 6B. The 1251-photoaffinity ligand was irradiated in aqueous buffer and at subsequent times an aliquot of the solution was added to the PS fraction


B 6 (A hb) B 6 (Ahd 1 0.5KD 0.15KD 0.05KD 1.5KD

"

FIG. 3. Photoaffinity labeling of the PS fraction of liver cytosol: analysis of the ''%labeled proteins by denaturing electrophoresis. The PS fraction of mouse liver cytosol (300 pg of protein/ml) was incubated with varying concentrations of '"I-photoaffinity ligand (0.05,0.15,0.5, and 1.5 KD; 1.5 KD = 1.14 nM) in the absence or presence of an excess of TCDBF, the mixture was irradiated, and the acetone-precipitated proteins were analyzed by denaturing electrophoresis and autoradiography as described under "Ex- perimental Procedures." B6(Ahb), normal C57BL/6 mice; the Ahb allele determines for the high affinity receptor. B6(Ahd), congenic C57BL/6 mice, homozygous for Ahd allele, which determines a much lower affinity receptor. Alternating lanes are incubation of protein labeled with just 1251-photoaffinity ligand and with photoaffinity ligand plus an excess of TCDBF.

Q

205 - 170-

116-

97 -

68 - 55 - 45-

36- 29-

20 -

10' -

c- I W

5 x 104 - ? -

i 0 5 t

t

of cytosol. Labeling by this photogenerated reactive species decreases modestly over the first 2 h and then remains nearly constant for at least 48 h. The addition of 2-mercaptoethanol to the irradiated solution rapidly reduces this species (dotted lines in Fig. 6B).

Arylazides, upon irradiation, form short-lived singlet and triplet nitrenes, and the singlet aryl nitrenes are thought to rearrange to relatively long-lived electrophilic intermediates, arylaziridines and cycloazaheptatetrenes (24-26). The effects of long-lived intermediates produced by the irradiation of 2- azido-3-['251]iodo-7,8-dibromodibenzo-p-dioxin can be mini- mized by rapid addition of acetone or a nucleophilic scavenger. It is important to store the photoaffinity ligand secure from any UV light to avoid the generation of this very persistent species.

Specificity of Photoaffinity Labeling-The specificity of photoaffinity labeling (ie. receptor labeling/total protein labeling) can be affected by a number of variables: (a) ligand affinity for the receptor and radiospecific activity (which are fixed in our experiments), (b ) ligand concentration, ( c ) receptor concentration, (d ) receptor purity, (e) removal of nonspecifically bound ligand prior to photolysis, and ( f l the use of scavengers. In general, most procedures which increase specificity decrease the absolute amount of labeling. The effects of some of these variables are seen in Table 111. As the ratio of ligand to receptor is decreased, the absolute labeling of the receptor decreases, but the specificity of labeling increases. Removal of nonspecifically bound 2-azido-3-[ '251]iodo-7,8-dibromodibenzo-p-dioxin by adsorption with charcoal/dextran is less effective than adsorption of [3H]TCDD and significant only at low protein concentration: at 0.27 mg of protein/ml, absorption reduces the ligand concentration by 77% (8.31 to 1.95 X lo5 cpm) while, at 8.56 mg of protein/ml, adsorption removes only 9% of the ligand (2.60 to 2.41 X lo4 cpm).

U I I I

.2 .4 .6 .e MOBILITY

FIG. 4. Determination of the apparent molecular mass of photoaffinity-labeled peptides. The PS fraction of liver cytosol (320 pg of protein/ml) was incubated with '251-labeled photoaffinity ligand (0.76 nM) in the absence or presence of an excess of unlabeled TCDBF, and the samples were irradiated and precipitated by acetone. The photoaffinity-labeled samples along with '251-labeled marker proteins (prepared by the Iodobead method (23)) were subjected to denaturing electrophoresis on a 5-20% polyacrylamide gradient, long slab gel. a, the first four lanes show the Coomassie Blue-stained gel and the last four lanes are the autoradiograph of the same gel. The protein standards are in lanes 1 ,4 ,5 , and 8. Comparison of lanes 6 and 7 reveals the photoaffinity-labeled peptides whose labeling is blocked by co-incubation with TCDBF. Protein standards (myosin, 205 kDa; m - macroglobulin, 170 kDa; 8-galactosidase, 116 kDa; phosphorylase b, 97.4 kDa; glutamate dehydrogenase, 55.4 kDa; bovine serum albumin, 68 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 29 kDa; and soybean trypsin inhibitor, 20 kDa) were obtained from Sigma, Bio-Rad, and Boehringer-Mannheim. B, A plot of the log molecular mass versus peptide mobility.

I .I 0


Partial purification and enrichment of the receptor relative to other proteins enhances the specificity of labeling (as seen in Fig. 7, e.g. cytosol = 1, ammonium sulfate fractionation 2- 3 times, PS fractionation 4-7 times).

For many photoaffinity ligands, the addition of scavengers (amine or thiol compounds, or macromolecular nucleophiles) is found to reduce pseudoaffinity labeling and increase the selectivity of labeling. Thiol compounds, especially dithiols react chemically with arylazides to produce the corresponding amines (27). We tested effects of a number of scavengers added immediately prior to irradiation: 2-mercaptoethanol (100 mM), glutathione (100 mM), thiopropionic acid (30 mM), methionine (10 mM), methionine methylester (10 mM), 2- (methy1thio)ethanol (10 mM), DL-thioctic acid (100 mM),

b r + I I

Time of Irradiation (min) FIG. 5. Time course of UV irradiation on photoaffinity la-

beling and ligand degradation. The PS fraction of liver cytosol was incubated with the ‘251-photoaffinity ligand (as described under “Experimental Procedures”) and irradiated for varying periods of time, followed immediately by the addition of acetone. The covalent labeling of total protein (cpm/2OO pg of protein) was determined in the acetone pellet, and the label in the 95-kDa band was determined following denaturing electrophoretic separation. The 1251-photoaffinity ligand remaining after irradiation was determined by thin layer chromatography of the acetone layer and by scanning densitometry of the TLC autoradiograph.

trans-o-dithiane 4,5-diol (100 mM), ethanolamine (60 mM), pyridine (10 mM), imidazole (10 mM), dimethylaminopyridine (10 mM), and Tris (10 mM). These compounds had no effect or produced only modest increases in selectivity of labeling with a reduction in absolute labeling.

Stoichiometry-Irradiation of an equilibrated incubation of 1251-photoaffinity ligand, at a half-saturating concentration (1 X KO = 0.76 nM) and the PS fraction of cytosol (Ah receptor concentration -0.15 nM) produced covalent labeling of the 95-kDa peptide that was equivalent to only about 0.5% of the sites estimated to be occupied by the reversibly bound ligand (data not shown). This low fractional receptor labeling might be due to (a) a rapid dissociation rate of the ligand-receptor complex, and hence much less than half the receptor sites are occupied at the instant of photolysis; or (b) lack of a good nucleophilic site in the receptor that is immediately adjacent to the ligand binding site, and hence the majority of the photogenerated products do not react with the receptor. Based on the assumption that the covalently labeled 95-kDa peptide is a constant fraction of the reversibly bound complex of lZ5I- photoaffinity ligand and this peptide, we estimated the half- life of the reversibly bound complex to be 4 h at 0 “C (Fig. 8). Hence, the low fractional labeling is not due to rapid dissock- tion of the reversibly bound ligand.

Relationship of the 95- and 70-kDa Peptides to the Ah Receptor-We now consider the relationship of the two peptides, 95 and 70 kDa, that are specifically labeled by low concentrations of the 1251-photoaffinity ligand, and whose labeling is blocked by competing unlabeled TCDBF. We first addressed the possibility that the 70-kDa peptide is a proteolytic degradation product formed from the 95-kDa peptide in two experiments. The PS fraction of cytosol was prepared fresh from mouse liver in the presence or absence of protease inhibitors (phenylmethylsulfonyl fluoride, lo-* M, and aprotinin, 100 units/ml) and photoaffinity labeled. The labeling of the 95- and 70-kDa bands remained unchanged (data not shown). In a second experiment, we photoaffinity labeled liver cytosol and the PS fraction of cytosol (immediately after irradiation, 2-mercaptoethanol was added to quench the long- lived photogenerated species), and then stored the incubation mixtures at 20 “C for 0 to 4 days to permit proteolysis before acetone precipitation and electrophoresis. As seen in Fig. 9, labeling of the 95- and 70-kDa peptides remained unchanged with storage, and no new labeled bands appeared.

FIG. 6. Pseudoaffinity labeling. A, the PS fraction of cytosol was incubated with 1251-photoaffinity ligand (0.76 nM) and irradiated as described under “Ex- perimental Procedures.” At varying time periods postirradiation, cold acetone was added. Total covalent binding (nonex- tractable radioactivity in the acetone pellet) and labeling of the 95-kDa peptide were determined. B, the lmI-photoaffinity ligand (1.5 nM) in MEN buffer was irradiated, and at varying time intervals after irradiation a 250-pl aliquot of the irradiated ligand solution was added to 250 pl of a solution of PS fraction of cytosol (330 pg of protein) in MEN buffer + 10% glycerol. The mixture was allowed to stand overnight before the addition of acetone. Total covalent binding and labeling of the 95- kDa band were determined.

A

c I x I

h 30 60 90 I20

Time of Irradiation ( m i d

Total Prolein

n Y


TABLE 111 Specificity of photoaffinity labeling: effect of protein and ligand concentration and charcoal adsorption

The PS fraction of cytosol at varying concentrations of protein was incubated with the 12sII-labeled photoaffinity ligand, the unbound ligand was adsorbed with charcoal/dextran, the samples were irradiated, and the total covalently bound radioactivity (acetone-precipitated pellet) and the radioactivity in the 95-kDa peptide were determined (see "Experimental Procedures").

Lipand/protein 95-kDa Specificity covalently of labeling Protein Ligand

After charcoal After irradiation concentration Initial covalently bound 95 kDa/total

adsorption bound radioactivity covalent bound

m.e/m[ nM cpmI200 M cpm 76 0.27 0.23 8.31 X 105 1.95 X lo5 5.38 X 10' 1220 2.3 0.54 0.23 4.16 X loK 1.68 X 10' 3.52 X 10' 1168 3.3 1.07 0.23 2.08 X lo5 1.14 X lo5 2.08 X 10' 512 3.4 2.14 0.23 1.04 X 10' 6.60 X 10' 1.06 X 104 484 4.6 4.28 0.23 5.20 X 10' 4.26 X 10' 6.1 X lo2 317 5.2 8.56 0.23 2.60 X 104 2.41 X 10' 3.6 X 10' 306 8.5

1.07 0.76 6.94 X lo5 4.52 X 10' 8.88 X 10' 2285 2.6 1.07 0.23 2.10 x 10' 1.46 X 10' 3.10 X 10' 1001 3.2 1.07 0.08 6.98 X 10' 5.15 X 10' 1.04 X 10' 402 3.8 1.07 0.02 2.08 X 104 1.74 X 10' 3.7 x lo2 186 5.2

Cytosol AS PS

FIG. 7. '251-Photoaffinity labeling of liver cytosol and en- riched fractions of cytosol. Equivalent amounts of cytosol, the 40- 555 ammonium sulfate (AS) fraction of cytosol, and the PS fraction of cytosol, with protein concentrations of 1.0, 0.24, and 0.125 mg/ml, were incubated with the "'I-photoaffinity ligand (0.76 nM) in the absence or presence of TCDBF (15.2 nM). Following irradiation and acetone precipitation, 150 pg of protein from each sample were analyzed by denaturing gel electrophoresis and autoradiography. Lanes I , 3, and 5 are samples incubated with only the photoaffinity ligand; lanes 2 ,4 , and 6 are samples incubated with the photoaffinity ligand + TCDBF. The radiolabel in the 95-kDa band was 919 cpm in cytosol, 2857 cpm in AS, and 4771 cpm in PS.

We examined the effects of co-incubation with various receptor agonists on photoaffinity labeling of the 95-kDa and 70-kDa peptides (Table IV). These peptides are labeled in approximately a 1:l ratio, and co-incubation with varying concentrations of TCDD inhibits labeling of both peptides to a similar extent. Co-incubation with other receptor agonists inhibits labeling of the 95- and 70-kDa peptides to a similar extent and the structure-activity relationship for this inhibition of labeling corresponds to that established for binding to the Ah receptor (4, 5). The 95- and 70-kDa peptides appear to be present in a 1:l molar ratio and their binding sites have equivalent affinity and ligand specificity.

When photoaffinity labeled PS fraction of cytosol was

?z '"""c T offraie=250min. 1/2

300 t I I I I I I 0 1 2 3 4 5

Time (hours) FIG. 8. Dissociation rate of the '251-photoaff'inity ligand

from the 95-kDa peptide. The PS fraction of cytosol (1.52 mg of protein/ml) was incubated with the "'1-photoaffinity ligand (1.14 nM) as described under "Experimental Procedures," diluted with 9 volumes of a suspension of charcoal/dextran (l%/O.l%), and held at 0 "C for varying times before rapid centrifugation and irradiation. Following irradiation, an aliquot of the solution was precipitated with acetone, and the radiolabel associated with the 95-kDa peptide was determined.

subjected to denaturing gel electrophoresis in the presence or absence of 20 mM dithiothreitol, the mobility of the 95- and 70-kDa peptide bands was unchanged, suggesting these peptides are not covalently linked by sulfhydryl bonds (data not shown).

Nondenaturing Chromatography-The 40-55% ammonium sulfate fraction of mouse liver cytosol was incubated with ["]TCDD in the absence or presence of an excess of unlabeled TCDBF and subjected to HPLC size exclusion chromatography a t 5 "C under nondenaturing conditions (28) (see "Experimental Procedures"). As seen in Fig. 10, the specifically bound ["HITCDD eluted as a fairly sharp peak with a maximum a t fraction 46. The ammonium sulfate fraction of cytosol was photoaffinity labeled and subjected to the same chromatographic separation, and the fractions were analyzed by denaturing electrophoresis. The 95- and 70-kDa peptides were found to coelute, and the peak fraction for elution


Storage Tlme at 2OoC After Irradiation

Days+ 0 I 2 3 4

97K-

68 K- - FIG. 9. Effect of storage at 20 "C on the photoaffinity la-

beled 95- and 70-kDa peptides. The PS fraction of cytosol was incubated with the ""I-photoaffinity ligand and irradiated in the usual manner (see "Experimental Procedures"), and 2-mercaptoethanol was immediately added to a final concentration of 140 mM. The sample was stored for 0 to 4 days a t 20 "C before addition of acetone to permit proteolysis, and the labeled proteins were analyzed by denaturing electrophoresis.

1400-

1200-

1000-

C 0 ._ t 800- ?

& 600-

L \

0

400-

200 -

25 30 35 40 45 5 0 5 5 60

Fract ion Number FIG. 10. HPLC size exclusion chromatography. The 40-50%

ammonium sulfate fraction of liver cytosol (3.4 mg of protein/ml) in 25 mM MOPS, 0.2% NaN3, 200 mM NaCI) was incubated with ['HI TCDD (1.5 X IO5 dpm/ml) in the presence or absence of a 200-fold excess of TCDBF for 30 min a t 20 "C, followed by the addition of charcoal/dextran (final concentration l%/O.l%) for 5 min and its removal by centrifugation. Four hundred pl of sample (1.36 mg of protein) were subjected to chromatography on a TSK G-4000 SW column a t 4 "C (see "Experimental Procedures"). 0, ["HITCDD; 0, ["]TCDD + TCDRF.

TABLE IV Inhibition of photoaffinity labeling of the 95- and 70-kDa peptides by

Ah receptor agonists The PS fraction of cytosol (150 pg protein/ml) was incubated with

'"I-photoaffinity ligand (0.76 nM) in the absence or presence of competing ligands, and the solution was irradiated and subjected to denaturing electrophoresis to determine the covalent labeling of 95- and 70-kDa peptide bands (see "Experimental Procedures").

Ligand Covalent binding

95 kDa 70 kDa con-

tration

Control (n = 2) TCDD TCDD TCDD TCDD TCDD TCDD TCDD

Control TCDBF TCDD 2,3,7-Trichlorodibenzo-p-dioxin 2,7-Dichlorodibenzo-p-dioxin 1,6-Dichlorodibenzo-p-dioxin

Control (n = 6) TCDBF (n = 3) 2,8-Dichlorodibenzofuran 2,3,6,7-Tetrachlorobiphenylene 3,3',4,4'-Tetrachlorobiphenyl 3,3',4,4'-Tetrachlorobiphenyl 2,2',3,3'-Tetrachlorobiphenyl 1,2,5,6-Dibenz(a)anthracene 1,2,3,4-Dibenz(a)anthracene Benzo(a)pyrene Benzo(a)pyrene 3-Methylcholanthrene 3-Methylcholanthrene 5,6-Benzoflavone Fluorene Pyrene Anthracene Phenanthracene

R M

0.19 0.38 0.76 1.5 3.0 6.1

15.2

15.2 15.2 15.2 15.2 15.2

15.2 15.2 15.2 15.2 60.4 15.2 15.2 15.2 15.2 45.6 15.2 45.6 15.2 15.2 15.2 15.2 15.2

1421

671 464 356 227 206 176

1186 205 174 453

1477 1225

5953 1044 6057 1263 3460 1926 5779 933

1025 2858 1092 904 634

1327 5291 5864 5170 5523

824 1448 822 738 460 370 274 261 174

1249 206 157 448

1448 1283

5680 877

5023 951

3324 1763 5192 685 902

2718 1947 942 722

1223 5204 5691 5223 5699

corresponded to the peak for ["]TCDD reversible ligand. When the photoaffinity labeled sample was analyzed by size exclusion chromatography in the presence of 6 M guanidine hydrochloride, the peak of the 95-kDa peptide eluted 3 or 4 fractions before that of the 70-kDa peptide (data not shown).

In Fig. 1lA is seen the chromatographic separation of the PS fraction of cytosol incubated with ["HJTCDD in the absence or presence of an excess of unlabeled TCDBF, on an anion exchange column (Mono Q ) using a sodium chloride gradient (see "Experimental Procedures"). The peak of reversibly bound ligand eluted a t approximately 340 mM NaCl. When the photoaffinity labeled PS fraction of cytosol was subjected to the same separation (Fig. 11R), the 95- and 70- kDa peptides coeluted in the same peak fractions and this corresponded to the peak of reversibly bound ligand.

Proteolysis of the 95- and 70-kDa Peptides-The photoaffinity labeled 95- and 70-kDa peptide bands were cut from the gels, the peptides were eluted and subjected to proteolysis by chymotrypsin and Staphylococcus aureus V-8 protease, and the peptide fragments were analyzed by denaturing electrophoresis (29). As seen in Fig. 12, the pattern of proteolytic fragments from both peptides was very similar, suggesting considerable homology.


s 0 CD N

0 0

0

A 1

I 0.002 Absorbonce

b

-1600

-1400

-1200

30 31 32 33

Fraction Number Fraction FIG. 11. HPLC anion exchange chromatography of [%]TCDD bound to PS fraction of cytosol. A, the

PS fraction of cytosol (620 pg of protein/ml) in 20 mM triethanolamine buffer (TEA) pH 7.5 (0 "C) + 25 mM NaCl was incubated with [3H]TCDD (1 X lo6 dpm/ml) in the absence or presence of 200-fold excess TCDBF for 30 min at 20 "C, and then for 5 min at 0 'C. Charcoal/dextran was added to a final concentration of 1%/0.1%, incubated for 5 min at 0 "C, and removed by centrifugation. One ml of the supernatant (565 pg of protein) was subjected to HPLC anion exchange chromatography (Mono Q 5/5 column) at 0 "C, flow rate 1.0 ml/min, for 6 min with 20 mM triethanolamine buffer, pH 7.5, then for 28.5 min with 0-500 mM NaCl gradient in triethanolamine buffer. One- ml fractions were collected and the radioactivity was quantified by liquid scintillation spectrometry. 0, [3H]TCDD. 0, [3H]TCDD f 200 X TCDBF. B, the PS fraction of cytosol was labeled with the '251-photoaffinity ligand, the sample was subjected to the same chromatographic separation, and the fractions were acetone-precipitated and analyzed by denaturing gel electrophoresis and autoradiography.

DISCUSSION

In this paper we have characterized a series of halodibenzo- p-dioxin congeners bearing the arylazide functional group as potential photoaffinity labels for the Ah receptor. 2-Azido-3- iodo-7,8-dibromodibenzo-p-dioxin (KD = 0.76 nM) was radio- synthesized using carrier-free 1251 and used to characterize the Ah receptor in mouse liver. The photoaffinity labeled products in cytosol were analyzed by denaturing gel electrophoresis and autoradiography. We observed 1) two peptides of apparent M , of 95,000 and 70,000 that were selectively labeled at low ligand concentrations ( i e . 0.05 KO); 2) that these two peptides were labeled in an approximately 1:l ratio; 3) that co-incubation with receptor agonists inhibited the labeling of both peptides to approximately the same extent, and the structure activity relationship among these congeners for inhibition of photoaffinity labeling corresponds to that for the receptor binding; 4) that in nondenaturing chromatographic separation of photoaffinity labeled cytosol, on HPLC size exclusion and anion exchange columns, the 95- and 70- kDa peptides comigrate; 5) that these two peptides are not covalently linked by disulfide bonds; and 6) that the 95- and 70-kDa peptides, upon proteolytic digestion, give very similar patterns of proteolytic fragments.

Based on the above data, the simplest structure for the Ah receptor in mouse liver is a heterodimer, composed of two

noncovalently linked subunits of molecular masses of 95,000 and 70,000 daltons. These subunits have homologous struc- tures (as indicated by their similar proteolytic fragmentation patterns in Fig. 12, A and B ) and each has a similar ligand binding site. The homologous structure of these peptides suggests the possibility that the 70-kDa peptide may be a proteolytic product the 95-kDa subunit. While we have been unable to demonstrate this conversion on cytosol, we cannot eliminate this degradation occurring in the whole cell or homogenate. Thus, the possibility exists that the intact receptor is a homodimer of two identical 95-kDa subunits.

Two experimental observations should be noted in light of this model. First, in addition to the major peptides, we observed selective photoaffinity labeling, but to a lesser extent of two or three other peptides which had apparent M , between 70,000 and 95,000, and which comigrated with the 70- and 96- kDa peptides on nondenaturing size exclusion and anion exchange chromatography. These peptides may represent proteolytic degradation products of the 95-kDa peptide. Second, we observed that in various preparations of cytosol the ratio of photoaffinity labeling of the 95- to 70-kDa peptide varied from 1:l to 1:0.8. The deviation in the stoichiometry of labeling may represent: (a) greater lability of the 70-kDa peptide or greater difficulty in extracting this subunit from cells; or (b) if the receptor is really a homodimer, variable proteolytic conversion of the 95-kDa to the 70-kDa peptides.


B V-8 Protease (yg)

0.4 2.0 I O

A Control Chymotryps 9 5 K 7 0 K 0.1 I .O

3 r"

e

FIG. 12. Proteolysis of the 95- and 70-kDa peptide bands. The PS fraction of cuytosol was photoaffinity labeled with '*'I-ligand and subject to denaturing electrophoresis, along with dansylated bovine serum albumin (68 kDa) and phosphorylase b (97.5 kDa) which served as fluorescent markers to locate the 95- and 70-kDa bands. These bands were cut from the gel (each band had 3000-4000 cpm/3OO pg of protein loaded per lane of a 3 mm thick gel), chopped into small pieces with a razor blade, and twice extracted with 5 ml of 0.1% sodium dodecyl sulfate in 50 mM NH.HC03 buffer, pH 8.0. The combined extracts were filtered through a GF/C glass filter, dialyzed overnight against water a t 4 "C, and lyophilized. The lyophilisates were dissolved along with y-globulin (50 pg/ml) in water. Stock solutions of the proteolytic enzymes, a-chymotrypsin, and Staphylococcus aureus V-8 protease were prepared 1 mg/ml enzyme, 50 pg/ml y-globulin in 10 mM Tris, pH 6.8. Ten pl of the appropriate dilution of the enzyme solution was mixed with 20 pl of the solution of 95- or 70-kDa peptide (-2000 cpm) and the mixture was incubated for 30 min a t 37 "C. The reaction was terminated by the addition of 170 pl of electrophoresis sample buffer (containing 2% lithium dodecyl sulfate and 20 mM dithiothreitol) and boiled for 10 min. The entire sample (200 pl) was subjected to denaturing electrophoresis on a 15% acrylamide slab gel and the fragments were visualized by autoradiography. The 95- and 70-kDa fragments were run in alternate lanes. A, chymotrypsin digestion. B, V-8 protease digestion.

Halogenated aromatic hydrocarbons act on the Ah receptor to evoke two distinct pleiotropic responses: 1) the induction of cytochrome P1-450 (and/or other coordinately expressed drug-metabolizing enzymes); and 2) a variety of tissue-specific morphologic changes, most notably cellular proliferation and/ or differentiation in some epithelial tissues (1). The former response is often viewed as an adaptive response to a foreign chemical, to enhance the metabolism of that compound to polar metabolites and hasten its elimination from the body; the "purpose" of the latter response is not so easily rational- ized. The Ah receptor has no known endogenous ligand,' and the physiologic role of this receptor, if any, is unknown. The Ah receptor bears many physicochemical similarities to the steroid hormone receptors (31,32). Many experiments suggest a two-state model for these receptors (31): 1) a "nontransformed-nonactivated" state in which in the absence of ligand the receptor has a low affinity for the nucleus and has a sedimentation coefficient of 8 S; and 2) a "transformed-acti- vated" state in which the receptor has a higher affinity for

* Kurl and Villee (30) reported that lumichrome, a metabolite of riboflavin a t very high concentrations, produced modest inhibition of [3H]TCDD binding to the Ah receptor from rat liver cytosol.

the nucleus and polyanions such as DNA, and usually has a sedimentation coefficient of 4-5 S in a high salt gradient. The Ah receptor has a sedimentation coefficient of 8-9 S in a low salt gradient, and 4 S in a high salt gradient (7, 9). The nontransformed-nonactivated form of most steroid receptors is stabilized by molybdate (33), but molybdate has no apparent affect on the stability of the Ah receptor.3

Photoaffinity labeling, the covalent attachment of radioligand to the Ah receptor, permits more direct characterization of this protein (gel electrophoresis) rather than hydrodynamic properties of the reversible ligand-receptor complex. The photoaffinity ligand should prove useful in the further characterization and purification of the Ah receptor.

Acknowkdgmnts-We wish to thank Dr. Joyce Knutson for mea- suring the 'ZSI-photoaffinity degradation rate in the experiment in Fig. 5, and Dr. William Lamph for his advice in the proteolysis experiment in Fig. 12.

REFERENCES 1. Poland, A., and Knutson, J. (1982) Annu. Rev. Pharmacol. Toxi-

CO~. 22,517-554

A. Poland, E. Glover, F. H. Ebetino, and A. S. Kende, unpublished results.


2. Poland, A., Palen, D., and Glover, E. (1982) Nature 300, 271- 274

3. Goldstein, J. A. (1980) in Halogenated Biphenyls, Terphenyls, Naphthalenes, Dibenzodwxins and Related Products (Kim- brough, R., ed) pp. 151-190, Elsevier, Amsterdam

4. Poland, A., Greenlee, W. F., and Kende, A. S. (1979) Ann. N. Y. Acad. Sci. 320,214-230

5. Poland, A., Glover, E., and Kende, A. S. (1976) J. Bwl. Chem. 251,4936-4945

6. Jones, P. B. C., Galeazzi, D. R., Fisher, J. M., and Whitlock, J. P. (1985) Science 227,1499-1502

7. Mason, M. E., and Okey, A. B. (1982) Eur. J. Biochem. 123,

8. Gasiewicz, T., and Rucci, G. (1984) Mol. Pharmacol. 26,90-98 9. Okey, A. B., Bondy, G. P., Mason, M., Kahl, G. F., Eisen, H. J.,

Guenthner, T. M., and Nebert, D. W. (1979) J. Bwl. Chem.

10. Okey, A. B., Bondy, G. P., Mason, M. E., Nebert, D. W., Forster- Gibson, C. J., Muncan, J., and Dufresne, M. J. (1980) J. Bwl. Chem. 255,11415-11422

11. Greenlee, W. F., and Poland, A. (1979) J. Biol. Chem. 254,9814- 9821

12. Whitlock, J. P., Jr., and Galeazzi, D. R. (1984) J. Biol. Chem.

13. Hannah, R., Nebert, P. W., and Eisen, H. J. (1981) J. Biol. Chem.

14. Poellinger L., Lund, J., Gillner, M., Hansson, L.-A., and Gustaf-

15. Poland, A., and Glover, E. (1980) Mol. Pharmacol. 17,86-94 16. Chae, K., Cho, L. K., and McKinney, J. D. (1977) J. Agric. Food

17. Denny, J. B., and Blobel, G. (1984) Proc. Natl. Acad. Sei. U. S.

209-215

254,11636-11648

259,980-985

256,4584-4590

son, J.-A. (1983) J. Bwl. Chem. 258, 13535-13542

Chem. 25, 1207-1209

A. 81,5286-5290

18. Cheng, Y.-C., and Prusoff, W. H. (1973) Biochem. Pharmucol.

19. Laemmli, U. K.. (1970) Nature 227,680-685 20. Laskey, R. A., Mills, A. D. (1977) FEBS Lett. 82,314-316 21. Pfeiffer, C. D., Nestrick, T. J., and Kocher, C. W. (1978) A d .

22. Gasciewicz, T., and Neal, R. A. (1979) Toricol. Appl. Pharmacol.

23. Markwell, M. A. K. (1982) Anal. Biochem. 125,427-432 24. Nielsen, P., and Buchard, 0. (1982) Photochem. Photobiol. 35,

25. Iddon, B., Meth-Cohn, O., Scriven, E. F. V., Suschitzky, H., and Gallager, P. T. (1979) Angew Chem. Int. Ed. Engl. 18,900-917

26. Bayley, H., and Staros, J. V. (1984) in Azides and Nitrenes: Reactivity and Utility (Scriven, E. F. V., ed) pp. 433-490, Academic Press, New York

27. Staros, J. V., Bayley, H., Standring, D. N., and Knowles, J. R. (1978) Biochem. Bwphys. Res. Commun. 80,568-572

28. Gasiewicz, T. A., and Rucci, G. (1984) Bwchim. Bwphys. Acta 798,3745

29. Cleveland, D. W., Fischer, S. G., Kirschner, M. W., and Laemmli, U. K. (1977) J. Biol. Chem. 252, 1102-1106

30. Kurl, R. N., and Villee, C. A. (1985) Pharmacology 30,241-244 31. Baulieu, E. E. (1984) Biochem. Pharmacal. 33,905-912 32. Eriksson, H., and Gustafsson, J.-A. (e&) (1983) Steroid Hormone

Receptors: Structure and Function, pp. 95-99, Elsevier, Am-

33. Dahmer, M. K., Housley, P. R., Pratt, W. B. (1984) Annu. Reo. sterdam

Physiol. 46, 67-81 34. Katzenellenbogen, J. A., Johnson, H. J., Jr., Carlson, K. E., and

Myers, H. N. (1974) Biochemistry 13,2986-2994 35. Ruoho, A., Kiefer, H., Roeder, P. E., and Singer (1973) Proc.

Natl. Acad. Sci. U. S. A. 70, 2567-8571

22,3099-3108

Chem. 50,800-804

51,329-339

317-323

the journal of biological chemistry vol. 261, no. 14, … · the journal of biological chemistry 0...

Documents