The identity and nuclear uptake of a cytosolic binding protein for 3-methylcholanthrene

The ability of 3-methylcholanthrene to interact noncovalently with rat liver cytosolic proteins was studied using Sephadex G200 chromatography. A specific 3-methylcholanthrene binding fraction from Sephadex G200 chromatography, termed peak B, when incubated with rat liver nuclei was able to translocate 3-methylcholanthrene into the nucleus. This translocation occurred faster and was quantitatively greater than the binding of 3-methylcholanthrene in buffer to nuclei. In addition, the nuclear uptake of peak B was increased by prewarming, suggesting that a heat-sensitive activation step may occur prior to the translocation process. However, no evidence was found on sucrose gradients for any conformational change in the protein fraction studied here. The translocation to the nucleus was temperature and time dependent. An examination of the characteristics of this 3-methylcholanthrene binding protein using Sephacryl S200 column chromatography showed a small number of high-affinity, saturable, binding sites to be present. These had an apparent dissociation constant, K_d, of 2.8 nm and a binding capacity of 770 fmol/mg of cytosolic protein. The selectivity of this protein was examined by competition studies and, in general, polycyclic hydrocarbons competed for the binding site, except for anthracene and phenanthrene. Of the inducers studied, 5,6-benzoflavone was a strong competitor. No competition was found with 12-O-tetradecanoyl phorbol-13-acetate, 2,6-ditertbutyl-p-cresol, β-retinyl acetate, or a number of steroids, except for 17β-estradiol which exhibited moderate binding. Peak B had a sedimentation coefficient of 4.2 S when analyzed on a linear sucrose gradient. Chromatography of peak B on a calibrated Sephacryl S200 column gave a molecular weight corresponding to 44,600 ± 4000.     

Purification and photoaffinity labelling of lipid methyltransferase from rat liver.

An enzyme that catalyses the three-step methylation of phosphatidylethanolamine to phosphatidylcholine as well as the methylation of fatty acids and that uses S-adenosylmethionine as the methyl donor has been purified about 200-fold from rat liver. Irradiation of the purified enzyme with a short-wavelength u.v. light in the presence of [methyl-3H]8-azido-S-adenosylmethionine followed by electrophoresis results in the incorporation of radioactivity into a single protein band of about 25 kDa. It is concluded that a single catalytic subunit catalyses the conversion of phosphatidylethanolamine into phosphatidylcholine and fatty acid methylation.     

An assay for the detection of specific binding of 3-methylcholanthrene to rat liver cytosolic proteins using DEAE-cellulose

A method for the detection of the specific binding of 3-methylcholanthrene to rat liver cytosolic proteins is described. The separation of the protein-bound 3-methylcholanthrene from the free 3-methylcholanthrene was achieved using a batch DEAE-cellulose technique. Extraction of the DEAE-cellulose with 0.3 M KCl allowed the selective release and measurement of the amount of protein-bound 3-methylcholanthrene. The assay was optimized for the following parameters: time of incubation with DEAE-cellulose, time required for salt extraction, protein concentration, the concentration of KCl required to elute the specific binding proteins, the amount of DEAE-cellulose required to bind the specific binding proteins, and ligand specificity. The sedimentation properties of those 3-methylcholanthrene-binding proteins which were extracted with salt from DEAE-cellulose were examined on 5 to 20% sucrose gradients; the major binding species sedimented as a broad peak at 4.5 S.     

Differences in the binding of 3-methylcholanthrene and phenobarbital to rat liver cytosolic and nuclear protein fractions

The inducers of cytochrome P-450c and P-450b, 3-methylcholanthrene and phenobarbital, respectively, have been studied in their interaction with subcellular fractions from rat liver. 3-Methylcholanthrene bound to both nuclear and cytoplasmic components as demonstrated by DNA-cellulose chromatography. The binding of 3-methylcholanthrene to cytosolic proteins, on DNA-cellulose, was approximately 27 fmol/mg of applied protein, whereas the binding to nuclear proteins was 250–570 fmol/mg applied protein. Phenobarbital did not bind to proteins of rat serum, rat liver cytosol, or rat liver nuclei which could bind to DNA-cellulose. Further examination of the potential interaction of phenobarbital to rat liver cytosolic proteins was carried out using either DEAE A-50 Sephadex chromatography, charcoal dextran analysis, or sucrose density gradients. No binding of phenobarbital to rat liver cytosolic proteins was observed under these experimental conditions. In contrast, the binding of 3-methylcholanthrene to cytosolic proteins showed four peaks of radioactivity after DEAE A-50 Sephadex chromatography, two peaks by sucrose density gradient analysis, and specific binding (0.13 pmol/mg protein) was observed using the charcoal dextran technique. One of the peaks on sucrose gradients was labile in the presence of salt. The uptake and intranuclear distribution of 3-methylcholanthrene and phenobarbital were markedly different after incubation with whole nuclei: 64% of the available 3-methylcholanthrene but only 3% of the available phenobarbital radioactivity became associated with nuclei. Of this radioactivity, the highest specific activity of the 3-methylcholanthrene radioactivity was associated with the 2 m KCl-resistant nuclear pellet and the highest specific activity of the phenobarbital radioactivity was associated with the nuclear fraction soluble in the absence of salt. These results are interpreted in regard to the induction of cytochrome P-450c.     

Purification and characterization of NADPH-dependent cytosolic 3,5,3'-triiodo-L-thyronine binding protein in rat kidney

The NADPH-dependent cytosolic 3,5,3'-triiodo-L-thyronine(T3)-binding protein (CTBP) has been purified over 30,000-fold from rat kidney by using charcoal extraction, Mono Q-Sepharose, Blue Sepharose CL-6B, and Sephacryl S-200 column chromatography. Purified CTBP had a sedimentation coefficient of 4.7 S, Stokes radius of 32.5A, and calculated molecular weight of 58,000. The apparently homogeneous protein consisted of a single polypeptide chain with Mr of 58,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Scatchard analysis of T3 binding showed that NADPH increases maximal binding capacity without changes in the affinity constant (Ka = 2.43 X 10(9) M-1). Double reciprocal analysis of NADPH and binding capacity gave maximal binding capacity of 16,400 pmol/mg of CTBP, Mr = 58,000. The order of affinity of iodothyronine analogues to purified CTBP was as follows: L-T3 = D-T3 greater than triiodothyroacetic acid greater than L-thyroxine. [125I]T3 bound to purified CTBP spontaneously dissociated from CTBP at 20 degrees C (t 1/2 = 22 min) in the absence of NADPH, whereas the dissociation was not observed in the presence of NADPH. The optimal pH for T3 binding was 7.2-7.5 Na+, K+, Ca2+, and Mg2+ (0-200 mM) did not influence T3 binding to CTBP. The purified CTBP did not bind to DNA and was not adsorbed to concanavalin A-Sepharose.     

Demonstration of a rat liver microsomal binding protein specific for beta-glucuronidase.

A binding protein with apparent specificity for beta-glucuronidase has been partially purified from a Triton X-100 extract of rat liver microsomes by affinity chromatography on glucuronidase-Sepharose 2B. It appears that once removed from the membrane, this binding protein self-aggregates to form large macromolecular complexes. With the use of polyacrylamide gel electrophoretic and sucrose density gradient ultracentrifugation assays to monitor the conversion of glucuronidase tetramer to a very high molecular weight complex, it was shown that the binding activity is heatlabile and protease-sensitive. However, binding activity is not influenced by salts, carbohydrates, other proteins or glycoproteins, or by extensive periodate oxidation of beta-glucuronidase, nor does binding occur with any other protein tested. The binding protein does not discriminate against any form of beta-glucuronidase from any rat organ tested. However, the binding protein does show organ localization, being present in the liver and kidney but not the spleen. The possible relationship of this binding protein to egasyn, a membrane protein which stabilizes beta-glucuronidase in mouse liver endoplasmic reticulum, is discussed.     

Yeast phenylalanyl-tRNA synthetase. Affinity and photoaffinity labelling of the stereospecific binding sites.

The localization of the binding sites of the different ligands on the constitutive subunits of yeast phenylalanyl-tRNA synthetase was undertaken using a large variety of affinity and photoaffinity labelling techniques. The RNAPhe was cross-linked to the enzyme by non-specific ultraviolet irradiation at 248 nm, specific irradiation in the wye base absorption band (315 nm), irradiation at 335 nm, in the absorption band of 4-thiouridine (S4U) residues introduced in the tRNA molecule, or by Schiff's base formation between periodate-oxidized tRNAPhe (tRNAPheox) and the protein. ATP was specifically incorporated in its binding site upon photosensitized irradiation. The amino acid could be linked to the enzyme upon ultraviolet irradiation, either in the free state, engaged in the adenylate or bound to the tRNA. The tRNA, the ATP molecule and the amino acid linked to the tRNA were found to interact exclusively with the beta subunit (Mr 63000). The phenylalanine residue, either free or joined to the adenylate, could be cross-linked with equal efficiency to eigher type of subunit, suggesting that the amino acid binding site is located in a contact area between the two subunits. The Schiff's base formation between tRNAPheox and the enzyme shows the existence of a lysyl group close to the binding site for the 3'-terminal adenosine of tRNA. This result was confirmed by the study of the inhibition of yeast phenylalanyl-tRNA synthetase with pyridoxal phosphate and the 2',3'-dialdehyde derivative of ATP, oATP.     

Purification of a benzo[a]pyrene binding protein by affinity chromatography and photoaffinity labeling

Binding proteins for the polycyclic aromatic hydrocarbon carcinogen benzo[a]pyrene (B[a]P) have been purified from C57B1/6J mouse liver. Following affinity chromatography on aminopyrene-Sepharose, a single polypeptide of 29,000 daltons was isolated. The photolabile compound 1-azidopyrene was developed as a photoaffinity labeling agent to identify the protein during its purification. 1-Azidopyrene was found to be a competitive inhibitor of [3H]B[a]P binding. Affinity labeling studies with [3H]-1-azidopyrene in unfractionated cytosol, and in purified preparations, yielded a single covalently labeled protein of 29,000 daltons. The formation of this labeled species was blocked by preincubation with excess unlabeled B[a]P. A native molecular weight of 30,000 was estimated by gel filtration chromatography of [3H]B[a]P- and [3H]-1-azidopyrene-labeled cytosol proteins. An equilibrium dissociation constant of 2.69 +/- 0.66 nM and a maximum number of binding sites of 2.07 +/- 0.10 nmol of [3H]B[a]P bound/mg of protein were estimated for the pure protein. Two-dimensional gel electrophoresis further resolved the purified 29,000-dalton protein into three major isoelectric variants, each of which was specifically labeled by [3H]-1-azidopyrene.     

Bacterial expression and photoaffinity labeling of a pheromone binding protein.

The first high-level production of a binding-active odorant binding protein is described. The expression cassette polymerase chain reaction was used to generate a DNA fragment encoding the pheromone binding protein (PBP) of the male moth Antheraea polyphemus. Transformation of Escherichia coli cells with a vector containing this construct generated clones which, when induced with isopropyl beta-D-thiogalactopyranoside, produced the 14-kDa PBP in both the soluble fraction and in inclusion bodies. Purification of the soluble recombinant PBP by preparative isoelectric focusing and gel filtration gave > 95% homogeneous protein, which was immunoreactive with an anti-PBP antiserum and exhibited specific, pheromone-displaceable covalent modification by the photoaffinity label [3H]6E,11Z-hexadecadienyl diazoacetate. Recombinant PBP was indistinguishable from the insect-derived PBP, as determined by both native and denaturing gel electrophoresis, immunoreactivity, and photoaffinity labeling properties. Moreover, the insoluble inclusion body protein could be solubilized, refolded, and purified by the same procedures to give a recombinant PBP indistinguishable from the soluble PBP. Proton NMR spectra of the soluble and refolded protein provide further evidence that they possess the same folded structure.     

Beta-endorphin: photoaffinity labelling of a non-opioid binding site in a human neuroblastoma

A photoaffinity reagent 2-nitro-4-azidophenylsulfenyl chloride (2,4-NAPS-Cl) and synthetic analogs of human beta-endorphin (beta h-EP) were employed to demonstrate the presence of receptor sites specific for beta h-EP but of non-opioid character in a human neuroblastoma cell line (IMR-32). The radioactive photoaffinity probe was carried out using [125I-Tyr1,2,4-NAPS-Trp27]-beta h-EP and IMR-32 cell membranes. After solubilization with sodium dodecyl sulfate (SDS) and SDS polyacrylamide gel electrophoresis, a single labelled protein band was identified with a molecular weight of 72,000. Labelling was blocked by beta h-EP or beta h-EP-(6-31) but remained in the presence of beta h-EP-(1-27). The specificity of this band is thus identical to that of the non-opioid site previously characterized. Various nonionic or zwitterionic detergents did not extract the labelled non-opioid site.     

A specific binding protein for 1,25-dihydroxyvitamin D3 in rat intestinal cytosol.

In the presence of 0.3 M potassium chloride and 0.5 mM dithiothreitol, rat intestinal cytosol contains two binding proteins for 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃)¹ having sedimentation coefficients of 3.2S and 5–6S. The 3.2S protein is specific for 1,25-(OH)₂D₃ as determined by competition analysis, whereas the 5–6S protein binds 25-hydroxyvitamin D₃ (25-OH-D₃) exclusively.     

Purification and characterization of rat brain cytosolic 3,5,3'-triiodo-L-thyronine-binding protein. Evidence for binding activity dependent on NADPH, NADP and thioredoxin.

A rat brain cytosolic 3,5,3'-triiodo-L-thyronine-(T3)-binding protein (CTBP) was purified using, successively, carboxymethyl-Sephadex, DEAE-Spherodex, T3-Sepharose-4B affinity chromatography and Sephacryl S-200. The molecular mass determined by SDS/PAGE wa 58 kDa. The binding characteristics determined by Scatchard analysis revealed a single class of binding sites with a Ka of 1.56 nM-1 and a maximal binding capacity of 7500 nmol T3/g protein. The relative binding affinities of iodothyronine analogues were D-T3 > L-T3 > L-T4 > 3,3'-5-triiodothyroacetic acid > reverse T3. The optimum pH for binding was 7.5. Purified brain CTBP was reversibly inactivated by charcoal. NADPH, NADP and thioredoxin restored binding activity to a level higher than that of the control; this effect was concentration dependent. Maximal activation was observed at 25 nM NADPH. NADP was effective only in the presence of 1 mM dithiothreitol; maximal activity was obtained at 10 nM NADP. At concentrations higher than 50 nM NADP, the binding gradually decreased. Thioredoxin in the presence of 1 mM dithiothreitol activated CTBP; maximal binding was obtained with 4 microM thioredoxin. In the presence of NADPH, NADP or thioredoxin the maximal binding capacity increased 2-4 times and the Ka was 2.6 nM-1. These results show that the activity of purified cytosolic brain T3-binding protein may be modulated by NADPH, NADP or thioredoxin.     

Spacer-modified saccharides for the regioselective photoaffinity labelling of the binding site of an immunoglobulin.

The spacer-modified trisaccharides that mimic (1----6)-linked beta-D-galactotetraose (Gal4), namely, O-beta-D-galactopyranosyl-(1----6)-S-beta-D-galactopyranosyl-(1----11)-8 -azi- 6,7,8,9,10-pentadeoxy-11-thio-D-galacto-undecose (12) and O-beta-D-galactopyranosyl-(1----6)-O-beta-D-galactopyranosyl- (1----13)-8-azi-6,7,8,9,10,11,12-heptadeoxy-D-galacto-tri decose (20) were synthesised by coupling disaccharide derivatives with 8-azi-6,7,8,9,10-pentadeoxy-1,2:3,4-di-O-isopropylidene-11-O -tosyl-alpha-D-galacto-undecopyranose (10) and 8-azi-6,7,8,9,10,11,12-heptadeoxy-1,2:3,4-di-O-isopropyli den e-alpha-D-galacto- tridecopyranose (17), respectively. Compounds 12 and 20 had affinities for the combining sites of the antibodies IgA X24 and IgA J 539 similar to those of O-beta-D-galactopyranosyl-(1----6)-O-beta-D- galactopyranosyl-(1----11)-8-azi-6,7,8,9,10-pentadeoxy-D-gal acto-undecose (7) and the native ligand Gal4. Tritium-labelled 7 chemically modified the heavy and light chains of IgA J 539, whereas 8-azi-6,7,8,9,10-pentadeoxy-D-(11-3H)galacto-undecose (5a) reacted only with the heavy chain.     

ATP binding to bovine heart cytochrome c oxidase. A photoaffinity labelling study.

ATP influences the kinetic properties of cytochrome c oxidase. A photoactivatable radioactive ATP analogue was used to localize the nucleotide-binding site on the bovine heart enzyme. Subunits IV and VIII were specifically labelled, suggesting that these two nuclear-coded polypeptides may play a regulatory role on the oxidase functions.     

Specific photoaffinity labelling of a thylakoid membrane protein with an azido-cytokinin agonist

A cytokinin-binding peptide (CBP) of 46 kDa (Thy46) has been identified in thylakoid membranes of pea chloroplasts, by photoaffinity labelling with tritiated 1-(2-azido-6-chloropyrid-4-yl)-3-phenylurea ([³H]azidoCPPU), a urea-type cytokinin agonist. The labelled peptide is also detected in Nicotiana plumbaginifolia, Nicotiana tabacum and spinach thylakoid membranes, but is absent in thylakoid membranes of Chlamydomonas reinhardtii. A pharmacological study of the interaction of this peptide with different cytokinin agonist molecules has been achieved. Urea derivatives are the most efficient competitors of photolabelling, and this efficiency is in good agreement with the cytokinin activity of these compounds. A quantitative analysis of the displacement of the photoaffinity labelling of the peptide by increasing concentrations of CPPU indicates an apparent dissociation constant of 1 M for this ligand. Purine-type cytokinins are weaker competitors than urea-type molecules, but the efficiency of the competition is also correlated to their respective cytokinin activity. A partial purification of Thy46 by a protocol involving ion exchange chromatography and 2D-gel electrophoresis is described.     

Purification of cytosolic cAMP-independent protein kinases from rat ventral prostate

Two cAMP-independent protein kinases were purified from rat ventral-prostate and liver cytosol, and were designated PK-C1 and PK-C2 to distinguish them from the nuclear protein kinases described in the preceding paper. The yield of the prostate enzymes was about 5% each, and about 10% each for the liver enzymes. The average fold purification of the prostatic enzymes was 1892 and 3176 for protein kinase C1 and C2, respectively. Their average respective specific activity towards casein was 40,111 and 67,340 nmol 32P incorporated/hr per mg of enzyme protein. protein kinase C1 comprised one polypeptide of Mr 39,000 which underwent phosphorylation in the presence of Mg2+ + ATP. Protein kinase C2 comprised three polypeptides of Mr 41,000; 38,000; 26,000. Of these only the Mr 26,000 polypeptide was autophosphorylated. The Mg2+ requirement for protein kinase C1 and C2 was between 1 and 4 mM depending on the nature of the protein substrate. Both enzymes were stimulated by 100-200 mM NaCl. Km for ATP for C1 and C2 kinases was 0.01 mM; GTP could be used only by protein kinase C2 but with a markedly lower affinity. The enzymes were active towards casein, phosvitin, dephosphophosvitin, and spermine-binding protein in vitro, but demonstrated little activity towards histones. Despite several similarities in these general properties of cytosolic protein kinases C1 and C2 with those of nuclear protein kinases N1 and N2, a number of differences are also noted.     

Characterization by photoaffinity labeling of a steroid binding protein in rat liver plasma membrane

Summary The mechanism of steroid uptake by the cell remains controversial. [³H]R5020 was utilized to characterize by photoaffinity labeling the steroid binding site in plasma membrane. This binding was saturable, reversible and had one type of binding site (K _d = 33 ± 4 nm, B _max = 32 ± 2 pmol/mg). [³H]R5020 could be prevented from binding by a variety of steroids (cortisol, progesterone, deoxycorticosterone, and levonorgestrel); estradiol did not have affinity for this binding site. The kinetics of R5020 photoactivation was time dependent and saturable. SDS-PAGE showed a specific band which corresponded to a 53-kDa peptide. The sucrose density gradient analysis has revealed the existence of a protein with a sedimentation coefficient of 3.6 ± 0.2 S. This polypeptide shows different characteristics than cytosolic steroid receptor or serum steroid binding proteins. This binding protein could correspond to the steroid binding site previously found in the plasma membrane.This work was supported by grants PB85-0461 from the Comisión Asesora de Investigatión Científica y Técnica and PGV-8612 from the Departamento de Educatión, Universidades e Investigation del Gobierno Vasco. We thank Roussel-Uclaf (France) for the nonradioactive RU-steroids kindly provided.     

In vitro binding of labeled 20-methylcholanthrene to a specific cytosol-protein

In vitro binding kinetics and specificity of [20-³H]methylcholanthrene ([³H]MC) interaction with mouse epidermal cytosol in the presence or absence of microsomal metabolizing systems were investigated. After an incubation period of 30 roin at 22°, the samples were dialyzed, subjected to gel filtration, ultrafiltration, and analyzed by 7 % basic polyacrylamide gel electrophoresis. A major portion of the binding appeared in a single peak on the gel which had the same mobility as bovine or mouse serum albumin. In vitro competition by other hydrocarbons or by promoters for binding of MC to this cytosol receptor protein showed an impressive correlation to carcinogenic and promoting activities. Essentially 100% of the MC bound to cytosol without a microsomal metabolizing system was non-covalent. However, binding Of MC to cytosol in the presence of microsomes plus reduced NADPH was party covalent which has previously been reported by Grover and Sims and Meunier and Chaveau. When bovine (BSA) or mouse serum albumin (MSA) was incubated with radioactive MC in the presence of competing non-radioactive carcinogens or promoters, little or no inhibition of binding was found. The finding that both a powerful tumor promoter and a strong carcinogen are competitors for the specific MC binding to the cytosol receptor protein indicates that it may represent a critical interaction for the promoting stage of chemical carcinogenesis in mouse skin.