Purification and protein sequence analysis of rat liver prolactin receptor

Prolactin receptors were purified from rat liver membranes by single-step immunoaffinity chromatography using a specific monoclonal antibody to the rat liver prolactin receptor. Scatchard analysis of 125I-human growth hormone binding to the purified receptor revealed two classes of specific binding sites with Ka = 18.5 x 10(9) and 1.2 x 10(9) M-1. Considering that both classes of binding sites are responsible for high affinity prolactin binding, the partially purified receptor preparation had a binding activity of 1.69 nmol/mg protein, representing 1000-fold purification over microsomal receptors with a recovery of 52%. From three separate purifications, 6 mg of partially purified prolactin receptor were obtained with a purity of approximately 4 to 6.5%. Thus, the use of monoclonal antibody for affinity chromatography resulted in a large improvement of prolactin receptor purification compared to previous hormone affinity chromatography (300-fold purification, 15% recovery). The purified receptor was run on preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis, and a homogeneous preparation of prolactin receptor was obtained by electroelution from gel slices corresponding to Mr 38,000-43,000. Immunoblot analysis using a radiolabeled monoclonal antibody revealed two separate but closely located bands of Mr 42,000 and 40,000 in microsomal, partially purified, and electroeluted preparations. The homogeneous receptor protein was extensively digested with L-1-tosylamido-2-phenylethyl chloromethyl ketone trypsin, and 10 internal amino acid sequences of the rat liver prolactin receptor were determined by gas-phase sequence analysis. Oligonucleotide probes were prepared against two of these internal sequences, and a prolactin receptor cDNA was isolated from a rat liver library using one of these probes (Boutin, J. M., Jolicoeur, C., Okamura, H., Gagnon, J., Edery, M., Shirota, M., Banville, D., Dusanter-Fourt, I., Djiane, J., and Kelly, P. A. (1988) Cell 53, 69-77). The amino acid sequence deduced from the cDNA reveals three potential sites of N-linked glycosylation, two of which were confirmed during protein sequencing. The prolactin receptor was characterized by affinity labeling with 125I-human growth hormone. Cross-linking of microsomes revealed a single band for the hormone-receptor complex with Mr 62,000. On the other hand, cross-linking of Triton X-100-solubilized or partially purified receptor with labeled hormone resulted in the appearance of two bands with Mr 62,000 and 102,000, suggesting the existence of a subunit structure of the prolactin receptor, or alternatively, the existence of two types of prolactin receptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Solubilization and purification of a membrane-associated 3,3'',5-tri-iodo-L-thyronine-binding protein from rat erythrocytes.

3,3',5-Tri-iodo-L-thyronine (L-T3) binding sites from rat erythrocyte membranes were solubilized in an active form by using the zwitterionic detergent CHAPS or the anionic detergent lauroylsarcosine. The binding protein was successively purified by Sephadex G-200 and affinity chromatography. The purified material retained its binding activity and exhibited high affinity and specificity compared with those displayed in the original membrane. Yield was about 10% of the starting activity. The specific binding activity was enriched by approx. 100-fold, which represents a purity of only 0.1%. Analysis of the purified preparation on SDS/PAGE showed two major protein bands (Mr 64,000 and Mr 50,000), but these could not represent the binding protein since the purity obtained was low. However, affinity-labelling experiments with N-bromoacetyl-L-[125I]T3 in intact membranes showed that two proteins (also with Mr values of 64,000 and 50,000) bound the hormone specifically, suggesting a co-migration of hormone receptors and contaminants on gel electrophoresis.     

The rat liver insulin receptor

Using insulin affinity chromatography, we have isolated highly purified insulin receptor from rat liver. When evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, the rat liver receptor contained the Mr 125,000 alpha-subunit, the Mr 90,000 beta-subunit, and varying proportions of the Mr 45,000 beta'-subunit. The specific insulin binding of the purified receptor was 25-30 micrograms of 125I-insulin/mg of protein, and the receptor underwent insulin-dependent autophosphorylation. Rat liver and human placental receptors differ from each other in several functional aspects: (1) the adsorption-desorption behavior from four insulin affinity columns indicated that the rat liver receptor binds less firmly to immobilized ligands; (2) the 125I-insulin binding affinity of the rat liver receptor is lower than that of the placental receptor; (3) partial reduction of the rat liver receptor with dithiothreitol increases its insulin binding affinity whereas the binding affinity of the placental receptor is unchanged; (4) at optimal insulin concentration, rat liver receptor autophosphorylation is stimulated 25-50-fold whereas the placental receptor is stimulated only 4-6-fold. Conversion of the beta-subunit to beta' by proteolysis is a major problem that occurs during exposure of the receptor to the pH 5.0 buffer used to elute the insulin affinity column. The rat receptor is particularly subject to destruction. Frequently, we have obtained receptor preparations that did not contain intact beta-subunit. These preparations failed to undergo autophosphorylation, but their insulin binding capacity and binding isotherms were identical with those of receptor containing beta-subunit. Proteolytic destruction and the accompanying loss of insulin-dependent autophosphorylation can be substantially reduced by proteolysis inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization and affinity purification of the Y2 receptor for neuropeptide Y and peptide YY from rabbit kidney.

Neuropeptide Y (NPY) is an important neuropeptide in both central and peripheral neurones whereas peptide YY (PYY) is a gut hormone present in endocrine cells in the lower bowel. Both peptides interact with multiple binding sites that have been further classified into Y1 and Y2 receptors. We have solubilized native Y2 receptors both from basolateral membranes of proximal convoluted tubules from rabbit kidney and from rat hippocampal membranes. Solubilization of functional Y2 receptors was obtained with both 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and digitonin and resulted in each case in a single class of high affinity binding sites. The soluble receptor retained the binding specificity for different peptides and long C-terminal fragments of NPY exhibited by membrane preparations. Gel filtration of solubilized receptors resulted in a single peak of specific PYY binding activity corresponding to Mr = 350,000 whereas affinity labeling revealed a major band of Mr = 60,000. Since this binding activity was inhibited by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) the Y2 receptor is probably solubilized as a receptor complex containing a G-protein along with the ligand binding protein. Y2 receptor binding sites from kidney tubular membranes were purified to homogeneity by a three-step procedure employing Mono S cation-exchange adsorption, affinity chromatography on wheat germ lectin-agarose beads, and affinity chromatography on NPY-Affi-Gel. Electrophoresis and silver staining of the final receptor preparation revealed a single protein with Mr = 60,000 whereas gel filtration showed a single peak at approximately Mr = 60,000. The purified protein can be affinity labeled with [125I-Tyr36]PYY, indicating that the Mr = 60,000 protein contains the ligand binding site of the Y2 receptor, and this binding is not affected by GTP gamma S. Scatchard transformation of binding data for the purified Y2 receptors was compatible with a single class of binding sites with Kd = 76 pM. The purified Y2 receptors retain their binding properties with regard to affinity and specificity for different members of the pancreatic polypeptide-fold peptide family. The specific activity of purified Y2 receptors was calculated to approximately 14.7 nmol of ligand binding/mg of receptor protein, which is consistent with the theoretical value (16.6 nmol/mg) for a pure Mr = 60,000 protein binding one PYY molecule. Purification to homogeneity thus reveals the Y2 receptor as an Mr = 60,000 glycoprotein.     

Mammalian alpha 1-adrenergic receptor. Purification and characterization of the native receptor ligand binding subunit

alpha 1-Adrenergic receptors from a cultured smooth muscle cell line (DDT1 MF-2) have been solubilized with digitonin and purified to apparent homogeneity by sequential chromatography on a biospecific affinity support (Sepharose-A55453 (4-amino-6,7-dimethoxy-2-[4-[5-(4-amino-3-phenyl) pentanoyl]-1-piperazinyl]-quinazoline), an alpha 1 receptor-selective antagonist), a wheat germ agglutinin-agarose gel, and a high performance steric exclusion liquid chromatography column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of iodinated purified receptor preparations reveals a peptide with an apparent Mr = 80,000 that co-migrates with the peptide labeled by the specific alpha 1-adrenergic receptor photoaffinity probe 4-amino-6,7-dimethoxy-2-[4-[5-(4-azido-3-[125I]iodophenyl)pentanoyl] -1-piperazinyl] quinazoline. The specific activity (approximately 13,600 pmol of ligand binding/mg of protein) of purified receptor preparations is consistent with that expected for a pure peptide of Mr = 80,000 containing a single ligand binding site. Overall yields approximate 14% of initial crude particulate binding. The purified receptor preparations bind agonist and antagonist ligands with appropriate alpha 1-adrenergic specificity, stereoselectivity, and affinity. Peptide maps of the pure alpha 1-adrenergic receptor and the pure human platelet alpha 2-adrenergic receptor (Regan, J.W., Nakata, H., DeMarinis, R.M., Caron, M.G., and Lefkowitz, R.J. (1986) J. Biol. Chem. 261, 3894-3900) using several different proteases suggest that these two receptors show little if any structural homology.     

Purification and characterization of the rat pancreatic cholecystokinin receptor

Cholecystokinin (CCK) is a peptide hormone that has a variety of physiologically important functions in the gastrointestinal tract, in which distinct high affinity receptors have been identified. We describe here the purification of the digitonin-solubilized rat pancreatic receptor as an initial step in the determination of its primary structure. Solubilization of total pancreatic membranes using 1% digitonin resulted in a single class of binding sites with a specific content of 4 pmol/mg as measured in a soluble binding assay using the nonpeptidyl CCK antagonist [3H]3S[-]-N-[2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4- benzodiazepine-3-yl]-1H-indole-2-carboxamide [( 3H]364,718). The solubilized receptor was purified using the following chromatographic steps: 1) cation exchange; 2) Ulex europaeus agglutinin-I-agarose; and 3) Sephacryl S-300. The final preparation of the purified receptor had a specific content of 8,055 pmol/mg, which represented a 9,051-fold purification from intact membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified receptor preparation under reducing conditions resulted in a predominant polypeptide with an Mr = 85,000-95,000 and minor polypeptides of Mr = 57,000 and 26,000 as determined by radiolabeling and silver staining. Solubilized pancreatic membranes were affinity labeled with the peptidyl CCK agonist 125I-D-Tyr-Gly-[(Nle28,31,6-NO2-Phe33)CCK-26-33] and chromatographed under conditions similar to those described for untreated membranes. Elution of radioactive peaks from each chromatographic column was coincident with [3H]364,718 binding activity and resulted in a labeled polypeptide having the same electrophoretic mobility as receptor derived from freshly labeled membranes and purified from untreated membranes. High performance liquid-gel exclusion chromatography of the crude digitonin-solubilized membrane preparation revealed an estimated molecular size for the [3H]364,718-binding activity of 370,000, which was consistent with the size determined by nondenaturing gel electrophoresis of the purified receptor complexed with the labeled nonpeptidyl antagonist. Binding of [3H]364,718 to the purified receptor preparation was comparable to that observed with the crude solubilized pancreatic membrane preparation; and both the homologous ligand 364,718 (Ki = 0.5 nm) and CCK-8 (Ki = 1.4 microM) competed for binding to both preparations in a similar manner.     

Purification and characterization of prolactin receptors from rat ovary

Prolactin receptors were purified to homogeneity by two affinity chromatography steps using concanavalin A-Sepharose and human growth hormone (hGH)-Sepharose. The purified receptors showed specificity and high affinity for lactogenic hormones and a binding capacity of 20 nmol/mg of protein. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis revealed that purified receptors were composed of two major protein bands of Mr = 41,000 and 88,000, which were identified as radioactive bands by binding of 125I-hGH to blotted renatured receptors and by autoradiogram of free and 125I-radiolabeled purified receptors. Autoradiographic analysis of SDS-polyacrylamide gel electrophoresis of cross-linked 125I-hGH-receptor or hGH-125I-iodinated receptor complexes showed two radioactive bands of Mr = 63,000 and 106,000. Analysis of the free receptors by high performance liquid chromatography using Superose 12 revealed two peaks of binding activity for 125I-hGH eluting in the positions of Mr approximately 150,000 and 250,000. After cross-linking with 125I-hGH, SDS-polyacrylamide gel electrophoresis analysis revealed that both peak fractions contained two binding species with Mr = 63,000 and 106,000. Chromatography of 125I-hGH-receptor complexes showed two radioactive fractions with approximate Mr approximately 180,000 and 300,000. The treatment of 125I-iodinated receptors with SDS and reductant resulted in the dissociation of the higher Mr form into the lower Mr form upon gel filtration. Chromatofocusing of free receptors showed three isoforms with pI 4.0, 5.0, and 5.3. These results indicate that detergent-solubilized prolactin receptors appear to be aggregated forms of holoreceptor containing two binding species of Mr = 41,000 +/- 2,000 and 88,000 +/- 3,000.     

Characterization of ovarian gonadotropin receptor. Monomer and associated form of the receptor

We have purified luteinizing hormone/human choriogonadotropin (hCG) receptor from rat ovary by sequential affinity column on wheat germ lectin-Sepharose and hCG-Sepharose chromatography. The purified receptor, previously identified as a single protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Kusuda, S., and Dufau, M.L. (1986) J. Biol. Chem. 261, 16161-16168), was further characterized by radioiodination with 1,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenylglycouril, and column chromatography on wheat germ lectin-Sepharose. Autoradiography of SDS-PAGE analysis under reducing conditions showed a single radiolabeled band of Mr = 80,000. The radioiodinated receptors treated with peptide:N-glycosidase F migrated at Mr = 54,000. Treatment with neuraminidase alone caused only a minor reduction in molecular weight, and subsequent treatment with endo-alpha-N-acetyl-D-galactosaminidase had little further effect on the receptor. When the radioiodinated receptor was analyzed by fast protein liquid chromatography, a single broad peak was eluted with Mr of approximately 350,000. The higher Mr of radioiodinated receptors than that of native receptors (Mr = 190,000 dimeric form) could be due to the aggregation of labeled molecules. These complexes dissociated into the monomeric form in the presence of SDS. To determine whether the monomers can bind hormone, the purified unlabeled receptors resolved with SDS were electroblotted to nitrocellulose membranes and incubated with 125I-hCG. Autoradiograms of the blots showed a band of monomer (Mr = 78,000) as well as one of dimer (Mr approximately 150,000). These studies have demonstrated that the luteinizing hormone/hCG receptors are predominantly N-linked glycosylated and suggest that the native receptor is a dimer of identical hormone binding subunits associated by noncovalent interactions. Although the individual subunits can bind hormone, it is conceivable that the dimeric form is necessary for signal transduction.     

Purification of vasoactive intestinal peptide receptor from porcine liver by a newly designed one-step affinity chromatography

Vasoactive intestinal peptide (VIP) receptors were solubilized from porcine liver membrane using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. The solubilized VIP receptor has been purified approximately 50,000-fold to apparent homogeneity by a one-step affinity chromatography using a newly designed VIP-polyacrylamide resin. The purified receptor bound 125I-VIP with a Kd of 22.3 +/- 0.7 nM and retained its peptide specificity toward VIP-related peptides. The specific activity of the purified receptor (16,400 pmol/mg of protein) was very close to the theoretical value (18,900 pmol/mg of protein) calculated assuming one binding site/protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified receptor revealed a single band with an Mr of 53,000 after either silver staining or radioiodination. Affinity labeling of the purified receptor with 125I-VIP using dithiobis(succinimidyl propionate) gave a single radioactive band, the labeling of which was completely inhibited by an excess of unlabeled VIP. In conclusion, an Mr 53,000 protein containing the VIP-binding site was purified to homogeneity by a one-step affinity chromatography using immobilized VIP.     

A1 adenosine receptor of rat testis membranes. Purification and partial characterization

Purification of an A1 adenosine receptor of rat testes was performed using a newly developed affinity chromatography system (Nakata, H. (1989) Mol. Pharmacol. 35, 780-786). The A1 adenosine receptor was solubilized with digitonin from rat testicular membranes and then purified more than 25,000-fold by sequential use of affinity chromatography on xanthine amine congener-immobilized agarose, hydroxylapatite chromatography, re-affinity chromatography on xanthine amine congener-agarose, and finally gel permeation chromatography on TSK-3000SW. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the final preparation showed a single broad band of Mr 41,000 by autoradiography after radioiodination. This Mr 41,000 peptide was also specifically labeled with an A1 adenosine receptor affinity labeling reagent. A high affinity A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-[3H]dipropylxanthine, bound saturably to the purified receptor with a KD of approximately 1.4 nM. The purified receptor also showed essentially the same specificity for adenosine agonists and antagonists as the unpurified receptor preparations, although the affinities of the purified adenosine receptor for agonists were significantly low compared to those of unpurified receptor preparations indicating that the purified A1 adenosine receptor exists as a low agonist-high antagonist affinity state. Deglycosylation of the purified testis adenosine A1 receptors with endoglycosidase F produced an increase in the mobility of the receptor protein to an apparent Mr 30,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, similar to that of deglycosylated A1 adenosine receptors of rat brain membranes. Peptide maps of the purified testis and brain A1 adenosine receptors using trypsin and V8 protease suggest that these receptors show some structural homologies.     

Solubilization and purification of rat pituitary gonadotropin-releasing hormone receptor

Gonadotropin-releasing hormone (GnRH) receptors were solubilized from rat pituitary membrane preparations in an active form by using the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid). The solubilized receptor exhibits high affinity, saturability, and specificity. The soluble supernatant retained 100% of the original binding activity when stored at 4 or -20 degrees C in the presence of 10% glycerol. The receptors were resolved into two components on the basis of chromatography on wheat germ agglutinin-agarose. Homogeneous receptor preparation was obtained by two cycles of affinity chromatography on immobilized avidin column coupled to [biotinyl-D-Lys6]GnRH. The overall recovery of the purified receptor was 4-10% of the initial activity in the CHAPS extract, and the calculated purification -fold was approximately 10,000 to 15,000. Analysis of iodinated purified GnRH receptors by autoradiography indicated the presence of two bands, Mr = 59,000 and 57,000. This was confirmed by photoaffinity labeling of the partially purified receptors and suggests that both components can specifically bind the hormone.     

The fat cell beta-adrenergic receptor. Purification and characterization of a mammalian beta 1-adrenergic receptor

The beta 1-adrenergic receptor of rat fat cells was effectively solubilized with digitonin and purified by affinity chromatography and steric exclusion high pressure liquid chromatography (HPLC). The purification strategy described permits an approximately 24,000-fold purification of the beta 1-adrenergic receptor of fat cells with an overall recovery of approximately 70%. Purified receptor preparations demonstrate a specific activity for (-) [3H]dihydroalprenolol binding of 12 nmol/mg of protein. The purified receptor was shown to migrate in steric exclusion HPLC as a Mr = 67,000 protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioiodinated purified receptor revealed a single, major peptide of Mr = 67,000. The binding of (-) [3H]dihydroalprenolol to purified receptor preparations displayed stereoselectivity and affinities for antagonists similar in nature to the membrane-bound and digitonin-solubilized beta 1-adrenergic receptor. In addition to the Mr = 67,000 component, a Mr = 140,000 form of the receptor was identified in HPLC runs of freshly prepared, affinity chromatographed receptor preparations that had not been frozen. This larger form of the receptor yielded binding activity of Mr = 67,000 on sequential HPLC runs and was shown to contain the Mr = 67,000 peptide. The beta 1-receptor from this mammalian source, composed of a single Mr = 67,000 peptide, is clearly quite distinct from the purified avian beta 1-, amphibian beta 2-, and mammalian beta 2-adrenergic receptors described by others.     

The mammalian beta 2-adrenergic receptor: purification and characterization

The beta 2-adrenergic receptors from hamster, guinea pig, and rat lungs have been solubilized with digitonin and purified by sequential Sepharose-alprenolol affinity and high-performance steric-exclusion liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of iodinated purified receptor preparations reveal a peptide with an apparent Mr of 64 000 in all three systems that coincides with the peptide labeled by the specific beta-adrenergic photoaffinity probe (p-azido-m-[125I]iodobenzyl)carazolol. A single polypeptide was observed in all three systems, suggesting that lower molecular weight peptides identified previously by affinity labeling or purification in mammalian systems may represent proteolyzed forms of the receptor. Purification of the beta-adrenergic receptor has also been assessed by silver staining, iodinated lectin binding, and measurement of the specific activity (approximately 15 000 pmol of [3H]dihydroalprenolol bound/mg of protein). Overall yields approximate 10% of the initial crude particulate binding, with 1-3 pmol of purified receptor obtained/g of tissue. The purified receptor preparations bind agonist and antagonist ligands with the expected beta 2-adrenergic specificity and stereoselectivity. Peptide mapping and lectin binding studies of the hamster, guinea pig, and rat lung beta 2-adrenergic receptors reveal significant similarities suggestive of evolutionary homology.     

Interactions of the nuclear thyroid hormone receptor with core histones

These studies concern the interactions of the rat liver thyroid hormone nuclear receptor with histones and factors influencing the receptor's assay and stability. Heating certain crude receptor preparations at 50 degrees C produces a selective loss of triiodothyronine (T3) but not thyroxine (T4) binding activity, whereas, with more purified preparations, such heating decreases both T3 and T4 binding. The selective T3-binding loss in crude preparations was found to be due to the simultaneous denaturation of the receptor's high-affinity hormone-binding activity for both T3 and T4 and generation of new low-affinity T4-binding sites. The fraction in which T4 binding can be activated could be separated from the receptors by Sephadex G-100 chromatography. Core histones stimulated both T3- and T4-binding activity of 6-fold-purified receptor preparations, and data from several different experimental approaches suggest that this stimulation is due to the capability of the core histones to prevent the receptor from binding to or being denatured by Sephadex G-25 assay columns. The core histones were also found to stabilize 500-fold-purified but not 6-fold-purified or crude receptor preparations. A number of other acidic or basic proteins had little or none of these stimulatory effects, whereas a few proteins (such as the insulin B chain and histone H1) did have activity, although it was less than that of the core histones. There were no significant differences between the purified core histone subfractions (H2A, H2B, H3, and H4). That core histones can interact with the thyroid hormone receptors was demonstrated more directly by the finding that the receptors bind to histone-Sepharose but not Sepharose or insulin- or ovalbumin-Sepharose columns and that this binding was blocked by core histones at concentrations suggestive of an affinity for the receptor-core histone interaction of around 3 microM at 0.15 M salt concentration. The results demonstrate the utility of the histones in the assay and stabilization of purified thyroid hormone receptors, but they fail to support our previous hypothesis of a receptor subunit where T3- but not T4-binding activity is regulated selectively by histones. However, the results indicate that histones may interact with the receptors with some degree of specificity, and they raise the possibility that the histones participate in the nuclear localization of the receptors.     

Identification of a high affinity leukotriene C4-binding protein in rat liver cytosol as glutathione S-transferase

A soluble high affinity binding unit for leukotriene (LT) C4 in the high speed supernatant of rat liver homogenate was characterized at 4 degrees C as having a single type of saturable affinity site with a dissociation constant of 0.77 +/- 0.27 nM (mean +/- S.E., n = 5). The binding activity was identified as the liver cytosolic subunit 1 (Ya) of glutathione S-transferase, commonly known as ligandin, by co-purification with the catalytic activity during DEAE-cellulose column chromatography and 11,12,14,15-tetrahydro-LTC4 (LTC2)-affinity gel column chromatography; resolution into two major bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Mr 23,000 and 25,000, of which only the smaller protein was labeled with [3H]LTC4 coupled via a photoaffinity cross-linking reagent; and immunodiffusion analysis with rabbit antiserum to glutathione S-transferase which showed a line of identity between the purified LTC4-binding protein and rat liver glutathione S-transferase. The affinity-purified binding protein bound 800 pmol of [3H] LTC4/mg of protein and possessed 12 mumol/min/mg of glutathione transferase activity as assayed with 1-chloro-2,4-dinitrobenzene as substrate. The enzyme activity of the cytosolic LTC4-binding protein was inhibited by submicromolar quantities of unlabeled LTC4, and the binding activity for [3H]LTC4 was blocked by the ligandin substrates, hematin and bilirubin. The high affinity interaction between LTC4 and glutathione S-transferase suggests that glutathione S-transferase may have a role in LTC4 disposition and that previous studies of LTC4 binding to putative receptors in nonresponsive tissues may require redefinition of the binding unit.     

Unesterified long-chain fatty acids inhibit thyroid hormone binding to the nuclear receptor. Solubilized receptor and the receptor in cultured cells

Unesterified long-chain fatty acids strongly inhibited thyroid hormone (T3) binding to nuclear receptors extracted from rat liver, kidney, spleen, brain, testis and heart. Oleic acid was the most potent inhibitor, attaining 50% inhibition at 2.8 microM. Oleic acid similarly inhibited the partially purified receptor and enhanced dissociation of the preformed T3-receptor complex. The fatty acid acted in a soluble form and in a competitive manner for the T3-binding sites, thereby reducing the affinity of the receptor for T3. The affinity of the receptor for oleic acid (Ki) was 1.0 microM. In HTC rat hepatoma cells in culture, fatty acids added to the medium reached the nucleus and inhibited nuclear T3 binding; oleic acid being the most potent. T3 binding of the cells was reversibly restored in fresh medium free of added fatty acids. Oleic acid did not affect all the T3-binding sites in the HTC cells: one form (80%) was inhibited and the other was not and these two forms were commonly present in all rat tissues examined. Thus, fatty acids inhibited the solubilized nuclear receptor as well as a class of nuclear T3-binding sites in cells in culture.     

Purification and characterization of the D2-dopamine receptor from bovine anterior pituitary

The D2-dopamine receptor from bovine anterior pituitary has been purified approximately 33,000-fold to apparent homogeneity by sequential use of affinity chromatography on immobilized carboxymethyleneoximinospiperone-Sepharose, Datura stramonium lectin-agarose, and hydroxylapatite chromatography. The purification yields a single polypeptide band of Mr approximately 120,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed by labeling with radioiodinated Bolton-Hunter reagent, Coomassie Blue, or silver staining. The purified D2 receptor preparations display a specific activity of approximately 5.3 nmol of [3H]spiperone bound per mg of protein. In detergent solutions, the purified receptor has a KD for [3H]spiperone of 5-8 nM; however, after reinsertion of the purified protein into phospholipid vesicles, a KD of approximately 160 pM is obtained, similar to that found for the receptor in crude membrane preparations. Several lines of evidence document that this polypeptide contains the ligand binding site as well as the functional activity of the D2 receptor. The Mr approximately 120,000 peptide can be covalently labeled by the affinity probe, 125I-bromoacetyl-N-(p-aminophenethyl)spiperone, with the pharmacological specificity expected of a D2-dopamine receptor. Agonist and antagonist ligands compete for [3H]spiperone binding to purified receptors in phospholipid vesicles with a rank order of potency and selectivity typical of a D2-dopamine receptor. Moreover, when reinserted into phospholipid vesicles with purified brain Gi/Go, the purified D2 receptors mediate the agonist stimulation of 35S-labeled guanosine 5'-O-(thiotriphosphate) binding to brain G-proteins with a typical D2-dopaminergic order of potency. These data suggest that we have purified an intact functional D2-dopamine receptor.     

N-methyl-D-aspartate/phencyclidine receptor complex of rat forebrain: purification and biochemical characterization

The N-methyl-D-aspartate (NMDA)/phencyclidine (PCP) receptor from rat forebrain was solubilized with sodium cholate and purified by affinity chromatography on amino-PCP-agarose. A 3700-fold purification was achieved. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol revealed four major bands of Mr 67,000, 57,000, 46,000, and 33,000. [3H]Azido-PCP was irreversibly incorporated into each of these bands after UV irradiation. The dissociation constant (Kd) of [1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) binding to the purified NMDA/PCP receptor was 120 nM. The maximum specific binding (Bmax) for [3H]TCP binding was 3.3 nmol/mg of protein. The pharmacological profile of the purified receptor complex was similar to that of the membranal and soluble receptors. The binding of [3H]TCP to the purified receptor was modulated by the NMDA receptor ligands glutamate, glycine, and NMDA.     

Purification of the neurotensin receptor from mouse brain by affinity chromatography

The neurotensin receptor was purified from newborn mouse brain by affinity chromatography. Active neurotensin binding sites were solubilized from brain homogenates using the nondenaturing detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) in the presence of cholesteryl hemisuccinate. Chromatography of the soluble extract on SP-Sephadex C-25 and hydroxylapatite eliminated 50% of proteins without loss of neurotensin binding activity. This prepurified material was loaded into an affinity column prepared by coupling neurotensin (2-13) to glutaraldehyde-activated Ultrogel AcA22. Nonspecifically adsorbed proteins were eliminated by extensive washing, and the receptor was eluted with a buffer containing 1 M NaCl, 0.1% CHAPS, and 0.02% cholesteryl hemisuccinate. After desalting, the purified receptor bound 125I-labeled neurotensin with a dissociation constant of 0.26 nM and retained its specificity towards a series of neurotensin analogues. The desalted NaCl eluate appeared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a major band of molecular weight 100,000 which was identified as the receptor by affinity labeling with 125I-labeled neurotensin in the presence of disuccinimidyl suberate. The purity of the mouse brain receptor eluted from the affinity column was estimated to be 78%. Electroelution of the 100-kDa protein band gave an homogenous preparation of receptor. Very similar results were obtained with CHAPS-solubilized neurotensin receptors from rat and rabbit brain.     

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.