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PubMed1982

Purification and characterization of androgen receptor from steer seminal vesicle.

The androgen receptor has been purified from steer seminal vesicle cytosol by a combination of differential DNA-Sepharose 4B chromatography and testosterone 17 beta-hemisuccinyl-3,3'-diaminodipropylamine-Sepharose 4B affinity chromatography. The procedure produced about 3 micrograms of receptor protein from 35 g of steer seminal vesicle, with a yield of 48%. The receptor protein, as a complex with unlabeled testosterone, was purified approximately 540000-fold. A single band, migrating at 60000 daltons, was observed following electrophoresis on a polyacrylamide gel containing sodium dodecyl sulfate (NaDoDSO4). This was confirmed by affinity labeling of the partially purified receptor with both 17-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one and 17 beta-hydroxy-[1,2,4,5,6,7,16,17-3H8)-5 alpha-androstan-3-one 17-(2-bromoacetate), which showed a peak of radioactivity migrating at 60000 daltons by NaDoDSO4 gel electrophoresis. The receptor had an estimated Stokes radius of 35 A and a sedimentation coefficient of 3.8 S in the presence of 0.3 M NaCl. The calculated molecular weight and frictional ratio for the androgen binding activity were 57000 and 1.42, respectively. Chromatofocusing of the purified receptor protein revealed an isoelectric point of 6.6. [3H]Methyltrienolone, bound to the purified receptor, was displaced with methyl-trienolone greater than testosterone greater than 5 alpha-dihydrotestosterone much greater than 3 beta-hydroxy-delta 5-androsten-17-one much greater than 5 alpha-androstane-3 alpha,17 beta-diol. The physicochemical properties of the purified receptor were similar to those of the receptor in crude cytosol. These results demonstrate that the androgen receptor from steer seminal vesicle was substantially purified without significant modification of its physicochemical or steroid binding properties.

Affinity Labels↗
PubMed1995

Sex hormone-binding globulin/androgen-binding protein: steroid-binding and dimerization domains.

Plasma sex hormone-binding globulin (SHBG) and testicular androgen-binding protein (ABP) are homodimeric glycoproteins that share the same primary structure, and differ only with respect to the types of oligosaccharides associated with them. The biological significance of these differences is not understood, but enzymatically deglycosylated SHBG and a non-glycosylated SHBG mutant both bind steroids normally. Various affinity-labelling experiments, and studies of recombinant SHBG mutants have indicated that a region encompassing and including Met-139 in human SHBG represents an important component of its steroid-binding site. Analyses of chimeric proteins comprising various portions of human SHBG and rat ABP have also indicated that residues important for the much higher affinity of human SHBG for steroid ligands are probably located within the N-terminal portion of these molecules. Recent studies of SHBG mutants have confirmed this, and a deletion mutant containing only the first 205 N-terminal residues of human SHBG has been produced which dimerizes and binds steroids appropriately. The introduction of amino-acid substitutions between Lys-134 and Phe-148 of SHBG has also indicated that residues including and immediately N-terminal of Met-139 may influence steroid-binding specificity, while those immediately C-terminal of Met-139 represent at least a part of the dimerization domain. These studies have also demonstrated that dimerization is induced by the presence of steroid ligand in the binding site, and that divalent cations play an important role in this process. Together, these data have led us to conclude that SHBG is a modular protein, which comprises an N-terminal steroid-binding and dimerization domain, and a C-terminal domain containing a highly-conserved consensus sequence for glycosylation that may be required for other biological activities, such as cell-surface recognition.

Affinity Labels↗
PubMed1991

Specific binding of antigenic peptides to cell-associated MHC class I molecules.

T lymphocytes recognize antigen in the form of peptides that associate with specific alleles of class I or class II major histocompatibility (MHC) molecules. By contrast with the clear MHC allele-specific binding of peptides to purified class II molecules purified solubilized class I molecules either bind relatively poorly or show degenerate specificity. Using photo-affinity labelling, we demonstrate here the specific interaction of peptides with cell-associated MHC class I molecules and show that this involves metabolically active processes.

Affinity Labels↗
PubMed1992

An oxidized analog of alpha-human atrial natriuretic polypeptide is a selective agonist for the atrial-natriuretic-polypeptide clearance receptor which lacks a guanylate cyclase.

The differences in biological functions between alpha-human atrial natriuretic polypeptide (alpha-hANP) and its oxidized analog, MetSO-alpha-hANP, have been investigated. Analysis of the ANP receptor subtypes by affinity labeling has shown that a bovine pulmonary aortic endothelial cell line (CPAE cells) primarily expresses ANP-R1 (R, receptor) coupled to particulate guanylate cyclase, while Hela cells from human cervical carcinoma predominantly express ANP-R2, which lacks a guanylate cyclase. alpha-hANP could bind to both ANP receptor subtypes with high affinity, while MetSO-alpha-hANP showed more selective binding to ANP-R2 than to ANP-R1. The activity of MetSO-alpha-hANP for stimulation of guanylate cyclase coupled to ANP-R1 was about 520-fold less than that of alpha-hANP (median effective dose = 2.5 nM for alpha-hANP, 1.3 microM for MetSO-alpha-hANP), indicating that MetSO-alpha-hANP was a partial agonist for this receptor. While this oxidized analog could inhibit the cAMP production through ANP-R2, with 0.15 times the activity of alpha-hANP (median concentration = 0.31 nM for alpha-hANP, 2.0 nM for MetSO-alpha-hANP). In in vivo studies, the diuretic activity of MetSO-alpha-hANP was 25-100-fold less than that of alpha-hANP. In addition, MetSO-alpha-hANP could potentiate the diuretic activity of alpha-hANP that was also caused by C-ANF4-23, a specific agonist for ANP-R2. These results demonstrate that MetSO-alpha-hANP can act as an agonist more selective for ANP-R2 than for ANP-R1, both in vivo and in vitro. The relationship between receptor selectivities and the conformation of alpha-hANP or MetSO-alpha-hANP was also discussed.

Affinity Labels↗
PubMed1977

Photochemical attachment of cyclic AMP binding protein(s) to the nuclear genome.

The nature of the cyclic AMP-receptor-nucleus interactions was examined by a novel combination of two photoreactions. A photosensitive derivative of cyclic AMP, N6-butyryl cyclic AMP, was covalently attached to its cytoplasmic receptod by photo-affinity labelling and this receptor complex was photo-crosslinked by the DNA in the rat liver nuclei. The photolytic reactions seemed to be specific since stable links were formed only when substantial noncovalent binding occured.

Affinity Labels↗
PubMed1990

Receptors for insulin and IGF-I in bovine lens epithelium.

The presence of insulin receptors has not yet been established in lens epithelial cells. Our goal was to understand the involvement of insulin in lens pathology, especially diabetic cataractogenesis. As a first step in this process, we evaluated the presence of insulin receptors in bovine lens epithelium. Insulin bound to insulin receptor with a high affinity (KD 7.0 nM), although this binding of insulin to its receptor could be replaced by IGF-I (KD 41 nM). Similarly IGF-I binding to IGF-I receptor (KD 0.38 nM) could be inhibited by insulin (KD 53.7 nM). SDS-PAGE and subsequent autoradiography of affinity labeled insulin receptor show the presence of a 130 kDa band, indicating that the lens epithelium insulin receptor may be similar to insulin receptors isolated from other tissues.

Affinity Labels↗
PubMedDate not supplied

The effect of photogenerated site-directed free radicals on surface dihydropyridine binding sites identified with photoaffinity probe (-)-[3H]-azidopine on cultured monkey renal cells.

With the use of the photoactivable dihydropyridine (DHP)-type calcium (Ca2+ channel antagonist (-)-[3H]-azidopine, a specific photoaffinity probe for Ca2+ channels, we tested the hypothesis of the existence of a separate subsite in the DHP receptor region on native polarized, stimulated depolarized and UV irradiated green monkey renal (GMR) cells preincubated with selected DHPs. Our results demonstrate that specific binding of (-)-[3H]-azidopine on GMR cells is of high affinity, stereoselective and dependent mainly on the inactivation of the membrane bound Ca2+ channel. Preincubation of the GMR cells with the DHP Ca2+ channel agonist BAY-K-8644 significantly reduced specific photolabeling. The site-directed free radicals generated after UV irradiation in DHP-preincubated renal cells inactivated Ca2+ channels and did not significantly affect the specific photoincorporation of (-)-[3H]-azidopine. (+)-Niguldipine, a DHP with the voluminous substituent on the DHP ring, significantly reduced the photolabeling. Low affinity labeling was partially prevented in (+)-nimodipine and (+)-niguldipine preincubated photoirradiated cells. The results strongly support the existence of central and peripheral subsites of the DHP region on GMR cells, with the former incorporating on photoactivation the intrinsically photoactive DHPs and with the latter labeled with a side chain bearing nitrene-generating photoreactive group, the photoaffinity probe, (-)-[3H]-azidopine.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1990

Integrin (alpha v beta 3)-ligand interaction. Identification of a heterodimeric RGD binding site on the vitronectin receptor.

The vitronectin receptor mediates cell adhesion to the extracellular matrix proteins vitronectin, fibrinogen, von Willebrand factor, and thrombospondin in an RGD-dependent manner. We previously demonstrated the direct interaction between the vitronectin receptor and an RGD-containing peptide by photoaffinity labeling the receptor with 125I-sulfosuccinimidyl-2-(p-azido-salicylamido)-1,3'-dithioprop ion ate (SASD)-GRGDSPK (Smith, J. W., and Cheresh, D. A. (1988) J. Biol. Chem. 263, 18726-18731). In that report, we identified amino acid residues 61-203 of the beta-subunit as proximal to the ligand binding site. Here we demonstrate that 125I-SASD-GRGDSPK affinity labels the alpha-subunit of the receptor at least two distinct sites within the region encompassing residues 139-349. Both of these regions are within the putative divalent cation binding region of the alpha-subunit. Collectively, our results suggest that discrete amino-terminal domains of both subunits of the receptor contribute to the structure of the ligand binding domain and furthermore that the ligand and divalent cation binding domains are spatially and functionally linked.

Affinity Labels↗
PubMed1988

Preparation of 6-125I-labeled amiloride derivatives.

Amiloride and certain of its derivatives are effective inhibitors of Na/H antiporters and of epithelial Na channels. We describe a simple method for the preparation of a variety of pharmacologically active 6-iodoamiloride derivatives that are labeled with 125I at high specific radioactivity. 6-Dechloroamiloride derivatives (bearing a hydrogen atom instead of the chlorine at the 6 position of the amiloride molecule) are reacted with 125ICl, prepared by the oxidation of the iodide in Na125I preparations. The 125I-labeled derivatives are separated from free 125I by anion exchange chromatography, or purified by thin layer chromatography. Both 6-dechloroamiloride and 5-(N-alkyl)-6-dechloroamiloride derivatives can be labeled by this method, with yields varying between 10 and 70%, depending on the ICl concentration and the structure of the 5-N-alkyl group. Efficient radiolabeling at high specific radioactivity also depends on the use of freshly prepared batches of 125I. Using carrier-free 125I, [125I]6-iodoamiloride and [125I]6-iodo-5-(N-tert-butyl)amiloride were prepared with yields of 27 and 22%, respectively. Potential applications of the 125I-labeled amiloride derivatives include ligand binding and affinity labeling experiments.

Amiloride↗
PubMed1992

Synthesis and properties of N-bromoacetyl-L-thyroxine.

A one-step bromoacetylation of L-thyroxine (T4) produces N-bromoacetyl-L-thyroxine (BrAcT4) in good yield. The reaction product is best purified by high-speed countercurrent chromatography. While HPLC is satisfactory only for purification of microgram and submicrogram quantities, amounts ranging from about 1 ng to 1 g of BrAcT4 can be processed by high-speed countercurrent chromatography (HSCCC), a method which we have previously used for the purification of N-bromoacetyl-3,3',5-triiodo-L-thyronine (BrAcT3). Operating conditions for the one-step synthesis of BrAcT4 and BrAcT3 differ due to differences in solubility and reactivity of the two hormones. BrAcT4 purified by HSCCC and shown to be pure by analytical HPLC has been characterized by alpha max and epsilon max in the near and far uv in several solvents, mass spectrum, 1H NMR spectrum, TLC in three solvent systems, retention time in reverse-phase HPLC (C18) in relation to the retention times of two internal standards, 3,3',5-triiodo-L-thyronine and T4, and melting point. Corresponding data for BrAcT3, not previously reported, have also been determined. The described procedure can provide not only substantial amounts of highly purified BrAcT4 for competition studies, but also 125I-labeled BrAcT4 of high specific activity for affinity labeling. Since solutions of BrAcT4 and of BrAcT3 undergo partial decomposition on evaporation to dryness, suitable procedures for the preparation of these hormones in solid form and for storage in solutions have been devised.

Chemical Phenomena↗
PubMed2000

Western blotting for detection of glucocorticoid receptors in the brain and pituitary gland from adrenal intact pigs.

The objectives of the experiments presented here were to develop a method for measuring total glucocorticoid receptors (GRs), in adrenal intact pigs, and to describe the distribution of this receptor in the pituitary gland and selected brain regions. Six male pigs, 14 days of age, were euthanized and the pituitary gland, hypothalamus, prefrontal cortex, and hippocampus were prepared for western blotting using a traditional soluble cytosolic preparation or a whole cell extract preparation. Samples were electrophoresed and immunoblotted using the anti-human GR antibodies #57 and #51. The #57 antibody labeled a doublet at 95 and 87 kDa whereas the #51 GR antibody labeled a single band at 87 kDa. Affinity labeling of GR with [(3)H] dexamethasone mesylate revealed a single band at 87 kDa. Using the soluble cytosolic preparation GR concentrations were highest in the pituitary gland followed by the hypothalamus (P<0. 0001). The amount of GR in the hippocampus and prefrontal cortex was equivalent but lesser (P<0.0001) than that found in the pituitary gland or hypothalamus. With the whole cell extract tissue preparation, GR concentrations were highest (P<0.05) in the prefrontal cortex and hippocampus with lower (P<0.05) levels detected in the hypothalamus. Taken together, these results indicated that the 87-kDa band was the functional GR and that regional distribution of GRs is specific to the type of tissue preparation used. In addition, we have described a procedure (from whole cell extracts) for measuring total GRs in neural and nonneural tissues from adrenal intact pigs.

Adrenal Glands↗
PubMed1999

Molecular and biochemical characterization of lecithin retinol acyltransferase.

The enzyme responsible for conversion of all-trans-retinol into retinyl esters, the lecithin retinol acyltransferase (LRAT) has been characterized at the molecular level. The cDNA coding for this protein was cloned and its amino acid sequence deduced. LRAT is composed of a polypeptide of 230 amino acid residues with a calculated mass of 25.3 kDa. Tissue distribution analysis by Northern blot showed expression of a 5.0-kilobase transcript in the human retinal pigment epithelium as well as in other tissues that are known for their high LRAT activity and vitamin A processing. Affinity labeling experiments using specific compounds with high affinity for LRAT and monospecific polyclonal antibodies raised in rabbits against two peptide sequences for LRAT confirmed the molecular mass of LRAT as a 25-kDa protein. High performance liquid chromatography analysis of the reaction product formed by HEK-293 cells transfected with LRAT cDNA confirmed the ability of the transfected cells to convert [3H]all-trans-retinol into authentic [3H]all-trans-retinyl palmitate as chemically determined.

Acyltransferases↗
PubMed1980

Hormone binding alters the conformation of the insulin receptor.

Fat cells or fat cell membranes were briefly subjected to mild proteolysis under conditions where insulin receptors were either free or bound to (125)I-labeled insulin. When receptors were then affinity-labeled to visualize the effects of this treatment, it was observed that receptors that had been occupied by ligand during proteolysis exhibited greater rates of degradation than unoccupied receptors. These results demonstrate that insulin-receptor interaction induces a change in receptor structure that may be related to signal transmission.

Adipose Tissue↗
PubMed1989

Covalent labeling of the somatostatin receptor in rat anterior pituitary membranes.

The molecular characteristics of the somatostatin (SRIF) receptor were investigated by covalently cross-linking [125I-Tyr11]SRIF to rat anterior pituitary membranes using three heterobifunctional cross-linking agents, N-5-azido-2-nitrobenzoyloxysuccinimide, N-hydroxysuccinimidyl-4-azidobenzoate, and N-succinimidyl-6-(4'-azido-2'-nitrophenylamino) hexanoate, and the homobifunctional agent disuccinimidyl suberate. Sodium dodecyl sulfate-gel electrophoresis followed by autoradiography revealed two SRIF-binding proteins with apparent mol wt (Mr) of 69,000 and 66,000 that were selectively labeled by the four cross-linking agents. When cross-linking was performed with N-5-azido-2-nitrobenzoyloxysuccinimide, both proteins migrated as a broad band centered at 68,000; however, with N-hydroxysuccinimidyl-4-azidobenzoate, the band was resolved into 69,000 and 66,000 Mr components. N-Succinimidyl-6-(4'-azido-2'-nitrophenylamino) hexanoate covalently labeled the 69,000 Mr protein and a minor species with a Mr of 45,000-47,000. Cross-linking with disuccinimidyl suberate labeled only the 66,000 Mr band. Labeling of both bands was specific, since affinity labeling with each of the four agents was abolished when 1 microM cyclic SRIF was included in the binding reaction. Binding of [125I-Tyr11]SRIF to membranes and labeling of the 69,000 and 66,000 Mr SRIF-binding species were similarly inhibited in a dose-dependent manner by unlabeled SRIF. Radiolabeling of both proteins was specifically displaced by 1 microM SRIF-28 and [D-Trp8,D-Cys14]SRIF, but not by oxytocin. Moreover, the extent of radiolabel incorporation into both components was dependent on the concentration of [125I-Tyr11]SRIF in the binding reaction. These results demonstrate the presence of two SRIF-binding proteins in rat anterior pituitary membranes that show characteristics of the SRIF receptor.

Animals↗
PubMed1990

Muscarinic acetylcholine receptors. Peptide sequencing identifies residues involved in antagonist binding and disulfide bond formation.

Muscarinic acetylcholine receptors (mAChR) were purified from rat brain and labeled either with the site-directed affinity label [3H]propylbenzilylcholine mustard (PrBCM) or with the sulfhydryl-specific label [3H]N-ethylmaleimide (NEM), using a protocol designed to give selective incorporation of the label into disulfide-bonded cysteines. m1 mAChRs were purified from CHO-K1 cells stably expressing the cloned receptor sequence and labeled with [3H]PrBCM. The labeled receptors were cleaved with the lysine-specific protease Lys-C and, after fractionation of the products, subcleaved with cyanogen bromide. Two major CNBr cleavage products were found with a molecular mass of approximately 3.9 and approximately 2.4 kDa, labeled either by [3H]PrBCM or [3H]NEM. The results obtained from CNBr cleavage of purified forebrain receptors were consistent with those obtained from the purified cloned m1 mAChR. Edman degradation was applied to the CNBr peptides. The results were compatible with the attachment of the [3H]PrBCM label to a conserved aspartic acid residue in transmembrane helix 3 of the mAChR (corresponding to Asp-105, m1 sequence) and of [3H]NEM to a conserved cysteine residue (corresponding to Cys-98, m1 sequence). These results support the hypothesis that the cysteine residue participates in a disulfide bond on the extracellular surface of the mAChRs and related G-protein-coupled receptors, while the aspartic acid residue is involved in binding the positively charged headgroup of muscarinic antagonists.

Amino Acid Sequence↗
PubMed1986

Purification and properties of testicular 3 beta-hydroxy-5-ene-steroid dehydrogenase and 5-ene-4-ene isomerase.

Through the treatment of rat testicular microsomes with sodium cholate, 3 beta-hydroxy-5-ene-steroid dehydrogenase and 5-ene-4-ene isomerase (abbreviated as the 3 beta-hydroxysteroid dehydrogenase and isomerase, respectively) were solubilized, and then purified by DEAE and hydroxylapatite column chromatographies. The findings were as follows: With this purification procedure, the 3 beta-hydroxysteroid dehydrogenase activity could not be separated from the isomerase. For 3-oxo-4-ene-steroid formation from 3 beta-hydroxy-5-ene-steroids, NAD+ was required as a cofactor. While the 3 beta-hydroxysteroid dehydrogenase required NAD+, the isomerase also required NAD+ or its reduced form, in contrast to the microbial enzyme. On treatment of the purified enzyme with 5'-p-fluorosulfonyl-benzoyladenosine (FSBA), both enzyme activities were markedly reduced. The enzyme, affinity labeled with [adenine-8-14C]FSBA, showed a mol. wt of 46.8 K. During 4-androstenedione production from DHA, 5-androstenedione was detected as an intermediate.

3-Hydroxysteroid Dehydrogenases↗
PubMedDate not supplied

Anti-sulfonylbenzoate antibodies as a tool for the detection of nucleotide-binding proteins for functional proteomics.

Proteins that bind ATP and GTP are important cellular components. We developed an immunological approach to selectively tag nucleotide-binding proteins based on the use of 5'-[4-(fluorosulfonyl)benzoyl]adenosine and 5'-[4-(fluorosulfonyl)benzoyl]guanosine affinity tags and an antibody against 4-(sulfonyl)benzoate. Detection follows affinity labeling, gel electrophoresis, and ester bond cleavage to expose the epitope. Trial analyses of labeled proteins from lymphoid cells identified multiple ATP-binding proteins, including chaperones, actin, kinases, an RNA splicing factor, a membrane ATPase, and ATP synthase.

Adenosine Triphosphate↗
PubMed1992

Analysis of the human insulin receptor.

The insulin derivative 4-azidosalicyloyl-[B1-biocytin-B2-lysine]insulin was used to photo-affinity-label the highly purified insulin receptor from human placenta. As shown by SDS-polyacrylamide gel electrophoresis, the 5 monoiodo isomers, with iodine in positions B1, B16, B26, A14 or A19, gave different labelling patterns. After complete tryptic digestion of the covalent receptor complex with 125I-Asa-[BctB1,LysB2]insulin, a stable fragment of 18 kDa was isolated, which was further purified by HPLC. This tryptic fragment of the intact receptor corresponds, according to HPLC, Tricin-SDS-PAGE and 2D-electrophoresis, to the similarly labelled sequenced domain of the receptor ectodomain (Fabry, M. et al. (1992) J. Biol. Chem. 267, 8950-8956). We thus conclude that insulin is bound to identical contact sites of native receptor and truncated ectodomain.

Affinity Labels↗