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PubMed1982

Retinoid affinity label for the binding site of retinol-binding protein.

Three radioactive retinoid bronoacetates were synthesized as potential retinoid affinity labels for the retinol binding site of human plasma retinol-binding protein (RBP). The compounds synthesized were beta-[9-3H]ionyl bromoacetate (IBA), beta-[11-3H]ionylideneethyl bromoacetate (IEBA), and [15-3H]retinyl bromoacetate (RBA). When excess ligand was incubated with RBP for 5 h at 37 degrees C, IBA and IEBA formed nearly 1:1 molar complexes with RBP, whereas RBA bound only approximately one-third as well. Subsequent addition of retinol to the retinoid-RBP complex resulted in complete displacement of IBA and RBA from the protein, whereas a large proportion (37%) of the [3H]IEBA remained bound to the retinol binding site of RBP. For maximization of covalent bonding of IEBA to RBP, IEBA was incubated with RBP for varying lengths of time, followed, in each instance, by addition of retinol to displace noncovalently bound IEBA. The amount of IEBA remaining bound to RBP increased with increasing incubation time, reaching a maximum of about 0.66 mol/mol of RBP at 18 h. Moreover, at each time point, the binding of retinol to RBP was inhibited to an extent that was equivalent to the amount of [3H]IEBA that was not displaced from RBP by retinol. Only a very small proportion of the bound [3H]IEBA that was not displaced with retinol could be extracted from the protein with chloroform-methanol. Taken together, these several lines of evidence strongly suggest that the IEBA was bound in the retinol binding site of RBP and was attached to the protein in a covalent manner. Thus, IEBA appears to be an effective affinity label for the retinol binding site of RBP.

Affinity Labels↗
PubMed1984

Affinity labeled somatomedin-C-binding proteins in rat sera.

We have developed an affinity labeling technique that uses disuccinimidyl suberate to covalently cross-link [125I]somatomedin-C (Sm-C) to specific binding proteins in rat serum. Normal rat serum contains four major classes of intensely labeled [125I]Sm-C-binding protein complexes which are sensitive to competition with unlabeled Sm-C with relative molecular masses of 95, 49, 36-33, and 26-23 K. In addition, less intensely labeled complexes are observed migrating between 175 and 115 K. Of the Sm-C binding complexes observed in normal serum, hypophysectomized (hypox) serum contains only an intensely labeled 36-33-K complex and a faint 49-K complex. Chronic administration of ovine (100 micrograms, ip, daily) to hypox rats induces the 95-K complex and possibly complexes between 175-115 K. With increasing duration of treatment, these complexes as well as the 49-K complex appear to increase in intensity. Binding proteins in both hypox and normal sera do not appear to distinguish between Sms, since both unlabeled Sm-C and multiplication-stimulating activity were equally potent in competing with [125I]Sm-C for binding. This affinity labeling technique appears to be a useful investigative tool to study the physiology and structure of Sm-binding proteins.

Animals↗
PubMed1985

Covalent labeling of the beta-adrenergic ligand-binding site with para-(bromoacetamidyl)benzylcarazolol. A highly potent beta-adrenergic affinity label.

para-(Bromoacetamidyl)benzylcarazolol (pBABC) was synthesized and found to be an extremely potent affinity label for beta-adrenergic receptors. Its interaction with mammalian (rabbit and hamster lung) and nonmammalian (turkey and frog erythrocyte) beta-adrenergic receptors was similar, displaying EC50 values of 400-900 pM for inhibiting 125I-cyanopindolol binding to these receptors. pBABC reduced the number of beta-adrenergic receptors in frog erythrocyte membranes, without any change in the affinity of the remaining sites for [125I]iodocyanopindolol. pBABC has been radioiodinated. As assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this affinity probe specifically labeled the beta-adrenergic peptide of a purified preparation of hamster lung, with high efficiency (approximately 40%) and with a pharmacological specificity characteristic of an interaction at the beta 2-adrenergic receptor ligand-binding site. Comparison of the proteolyzed products derived from purified receptor labeled with [125I]pBABC and with the photoaffinity agent [125I]p-azidobenzylcarazolol suggested that covalent labeling of the beta-adrenergic receptor by these probes occurs at similar domains of the beta-adrenergic receptor. Because of the much higher level of incorporation of this affinity probe as opposed to photosensitive probes, pBABC should prove to be a useful tool for structural studies of purified beta-adrenergic receptors.

Affinity Labels↗
PubMed1997

Identification of the subdomain in the nuclear receptor for the hormonal form of vitamin D3, 1 alpha,25-dihydroxyvitamin D3, vitamin D receptor, that is covalently modified by an affinity labeling reagent.

Multiple physiological actions of the hormonal form of vitamin D3, 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3), are mediated by a genomic pathway which is initiated by the highly specific recognition and binding by its cognate receptor (vitamin D receptor, VDR) in the target cells. Thus, knowledge of the three-dimensional geometries of the ligand, i.e., 1,25(OH)2D3, and the 1,25(OH)2D3-binding domain of VDR is crucial for a better understanding of diverse physiological roles of this hormone. Recently our laboratory has developed 1 alpha,25-dihydroxyvitamin D3-3 beta-bromoacetate (1,25(OH)2 D3-3-BE) as an affinity labeling reagent for covalently modifying the hormone binding domain of native VDRs from calf thymus and rat osteosarcoma cells and baculovirus-expressed recombinant human VDR (hVDR). In the present report, we report affinity labeling of the hormone binding domain of hVDR, expressed in Escherichia coli as a glutathione S-transferase fusion partner, site-specific cleavage of the affinity-labeled VDR with 3-bromo-3-methyl-2-(2-nitrophenylmercapto)- 3H-indole, and identification of the C-terminal subdomain of human VDR containing the putative hormone binding site.

Affinity Labels↗
PubMed1975

Catalytic competence, a new criterion for affinity labeling. Demonstration of the reversible enzymatic interconversion of estrone and estradiol-17 beta covalently bound to human placental estradiol-17 beta dehydrogenase.

Human placental estradiol-17beta dehydrogenase is rapidly inactivated upon treatment with 3-bromoacetoxyestrone. Pseudo-first order kinetic data are obtained and inactivation is accompanied by incorporation of 1 mol of 3-acetoxyestrone/mol of subunit (Mr =34,000). Treatment of the inactivated enzyme with (4S)-[4-2H]DPNH results in the formation of covalently bound [17alpha-2H]estradiol-17beta, which can be released by hydrolysis and identified by gas chromatography-mass sepctrometry. When (4R)-[4-2H]DPNH was used, deuterium was not transferred. Thus, the normal stereochemistry of hydridetransfer is preserved for both partners. After treatment with p-mercuribenzoate, affinity-labeled estradiol-17beta dehyrogenase is no longer able to caralyze reduction its covalently bound estrone; in the presence of DPNH and native enzyme, however, reduction occurs, demonstrating that affinity-labeled enzyme can itself serve as subtrate for native estradiol-17beta dehydrogenase. The reversible enzymatic interconversion of covalently bound estrone was demonstrated using a transhydrogenase assay. The ability of an enzyme to catalyze its normal reaction with a covalently bound substrate is termed catalytic competence, and is considered to be a new criterion for affinity labeling.

Affinity Labels↗
PubMed1988

Enzyme-gold affinity labelling of cellulose.

The enzyme-linked colloidal gold affinity labelling technique was tested as a method to localize cellulose on thin sections of plant cell walls and slime mold spores. Commercially available cellulase from cultures of Trichoderma reesei, the main components being cellobiohydrolase I and II (CBH I, CBH II) and endoglucanase (EG), was linked to colloidal gold by using standard techniques and applied as a dilute, buffered suspension to thin sections. After brief exposure, e.g., 15-30 minutes, cellulose exposed on the surface of sections was labelled with the enzyme-gold complex. Poststaining did not appear to have a deleterious effect on the labelled sections. The specificity of labelling was demonstrated by its complete inhibition when carboxymethylcellulose was incorporated in the labelling mixture, by lack of labelling of 1,4-beta-mannans or 1,3-beta-xylans in noncellulosic walls of marine algae, by lack of labelling of 1,4-beta-glucans in chitin, by much lower labelling density when done at 4 degrees C, and by lack of labelling when sections were predigested with cellulase. Labelling with the crude commercial cellulase was compared to labelling with purified CBH I-, CBH II-, and EG-linked colloidal gold, and the labelling pattern was similar. This method was found useful on conventionally fixed material and required no special preparation other than the use of inert (Ni or Au) grids and 0.5% gelatin to reduce nonspecific binding of the gold complex. Labelling was similar in the several embedding resins tested: LR White, Lowicryl K4M, Epon 812, and Spurr's.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1991

Benextramine irreversibly inhibits [125I]neuropeptide Y affinity labeling of the Y2 binding protein in bovine hippocampus.

Affinity labeling of iodinated neuropeptide Y (NPY) to bovine hippocampal binding proteins revealed that benextramine inhibited specific NPY labeling of the 50 kDa NPY binding protein (Y2 binding protein) in a dose-dependent manner (IC50 = 33 microM). Hippocampal membranes, which were pretreated with benextramine and washed, exhibited decreased [125I]NPY labeling of binding proteins in a similar dose-dependent manner. These findings demonstrate that benextramine irreversibly blocks specific NPY binding to the 50 kDa NPY Y2 binding protein.

Affinity Labels↗
PubMed1984

Digitalis receptors affinity labelling and relation with positive inotropic and cardiotoxic effects.

Affinity labelling of the digitalis receptor has indicated that it is situated on the N-terminal part of the alpha-subunit of the (Na+,K+)ATPase. Biochemical and pharmacological properties of the (Na+,K+)ATPase studied on intact chick embryonic hearts and under heart cell culture conditions have indicated the existence of two families of ouabain binding sites i.e.: a low affinity binding sites with a dissociation constant (Kd) of 2-6 microM for the ouabain-receptor complex and a high affinity binding site with a Kd of 26-48 nM. High and low affinity sites also are present at all embryonic stages studied. Inhibition of 86Rb+ uptake in cultured cardiac cells and increase in intracellular Na+ concentration, due to (Na+,K+)ATPase blockade, occur in an ouabain concentration range corresponding to the saturation of the low affinity ouabain site. Ouabain stimulated 45Ca2+ uptake increases in parallel with the increase in the intracellular Na+ concentration. It is suppressed in Na+ free medium or when Na+ is replaced by Li+ suggesting that the increase is due to the indirect activation of the Na+/Ca2+ exchange system in the plasma membrane. Dose-response curves for the inotropic effects of ouabain on papillary muscle and on ventricular cells in culture indicate the development of the cardiotonic properties is parallel to the saturation of the low affinity binding site for ouabain. Therefore, inhibition of the cardiac (Na+,K+)ATPase corresponding to low affinity ouabain binding sites seems to be responsible for both the cardiotonic and cardiotoxic effects of the drug.

Affinity Labels↗
PubMed1991

Lys631 residue in the active site of the bacteriophage T7 RNA polymerase. Affinity labeling and site-directed mutagenesis.

A highly selective affinity labeling of T7 RNA polymerase with the o-formylphenyl ester of GMP and [alpha-32P]UTP was carried out. The site of the labeling was located using limited cleavages with hydroxylamine, bromine, N-chlorosuccinimide and cyanogene bromide and was identified as the Lys631 residue. Site-directed mutagenesis using synthetic oligonucleotides was used to substitute Lys631 by a Gly, Leu or Arg residue. Kinetic studies of the purified mutant enzymes showed alterations of their polymerizing activity. For the Lys----Gly mutant enzyme, anomalous template binding was observed.

Affinity Labels↗
PubMed1990

Highly selective affinity labeling of the primer-binding site of E. coli DNA polymerase I.

Highly selective affinity labeling of the primer site of E. coli DNA polymerase I was performed with the 5'-reactive derivatives of oligothymidylate in the presence of poly(dA) template. Subtilysine cleavage proved that the site of affinity modification belonged to the 'Klenow' part of DNA polymerase I. If taken separately, Klenow fragment was not labeled by these oligonucleotide derivatives. The site of affinity labeling were tested in the structure of DNA polymerase I by hydroxylamine cleavage. At least two sites of labeling were revealed. The main one was localized between Gly-833 and His-928.

Affinity Labels↗
PubMed1993

Use of trinitrobenzensulfonate for affinity labeling of lysine residues at phosphate binding sites of some enzymes.

Trinitrobenzensulfonate, a reagent for lysine residues, inactivates lamb liver 6-phosphogluconate dehydrogenase through affinity labeling. Complete inactivation is due to the binding of only one residue of reagent per enzyme subunit. Other enzymes with a phosphate binding site are also inactivated by affinity labeling. It appears that trinitrobenzensulfonate, when used at low concentrations, first binds to a phosphate binding site, then reacts with a nearby lysine residue. This reagent presents some advantages over pyridoxal phosphate, which has similar characteristics.

Affinity Labels↗
PubMed1984

Identification of human leukemic glucocorticoid receptors using affinity labeling and anti-human glucocorticoid receptor antibodies.

Antisera raised against human lymphoid glucocorticoid receptors were used in combination with the glucocorticoid receptor affinity label [3H]dexamethasone 21-mesylate [( 3H]DM) to identify the glucocorticoid receptors of the human B-lymphoblastoid cell line IM-9 and the human T-cell leukemic cell line CEM-C7. Antisera were obtained following immunization of New Zealand White rabbits with [3H]triamcinolone acetonide [( 3H]TA)-glucocorticoid receptor complexes partially purified by two-stage DNA-cellulose chromatography. The presence of anti-human glucocorticoid receptor antibodies was verified by: (a) adsorption of [3H]TA-receptor-antibody complexes to Protein A; (b) a shift to higher apparent molecular weight in the elution position from Sephacryl S300 of [3H]TA-receptor complexes incubated with immune serum; and (c) the ability of immune serum to displace [3H]TA-receptor complexes on sucrose gradients. These antibodies also recognized rat liver and murine S49 cell glucocorticoid receptors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]DM-labeled IM-9 cytosol identified a major competable band with a molecular weight of approximately 90,000, three minor competable components with molecular weights of approximately 78,000, approximately 51,000, and approximately 38,500, and at least 21 other noncompetable components. Following immunoprecipitation of [3H]DM-labeled cytosol with immune serum, only the Mr 90,000 and 78,000 components were seen. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]DM-labeled CEM-C7 cytosol revealed a larger number of [3H]DM-labeled components. However, after immunoprecipitation of [3H]DM-labeled CEM-C7 cytosol, a predominant competable component with a molecular weight of 90,000 was easily identified. This component was markedly diminished when cytosols from the glucocorticoid receptor-deficient cell line ICR-27 were used. Thus, the combination of affinity labeling and anti-human glucocorticoid receptor antibodies is capable of providing direct physical identification of human lymphoid glucocorticoid receptors.

Affinity Labels↗
PubMed1987

2-[(4-Bromo-2,3-dioxobutyl)thio]- and 2-[(3-bromo-2-oxopropyl)thio]adenosine 2'5'-bisphosphate: new nucleotide analogues that act as affinity labels of nicotinamide adenine dinucleotide phosphate specific isocitrate dehydrogenase.

Two new reactive adenine nucleotide analogues have been synthesized and characterized: 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 2',5'-bisphosphate (2-BDB-TA-2',5'-DP) and 2-[(3-bromo-2-oxopropyl)thio]adenosine 2',5'-bisphosphate (2-BOP-TA-2',5'-DP). Starting with NADP+, 2'-phospho-adenosine 5'-(diphosphoribose) (PADPR) was generated enzymatically and was converted to PADPR 1-oxide by reaction with m-chloroperoxybenzoic acid. Treatment with NaOH followed by reaction with carbon disulfide yielded 2-thioadenosine 2',5'-bisphosphate (TA-2',5'-DP). Condensation of TA-2',5'-DP with 1,4-dibromobutanedione or 1,3-dibromo-2-propanone gave the final products 2-BDB-TA-2',5'-DP and 2-BOP-TA-2',5'-DP, respectively. The structure of these new reagents was determined by UV, 1H NMR, 31P NMR, and 13C NMR spectroscopy as well as by bromide and phosphorus analysis. Both of these reagents exhibit properties expected for an affinity label of the coenzyme site of NADP+-dependent isocitrate dehydrogenase. With both reagents, biphasic kinetics of inactivation are observed that can be described in terms of a fast initial phase of inactivation resulting in partially active enzyme of 6-7% residual activity, followed by a slower phase leading to total inactivation. The inactivation rate constants for both reagents exhibit a nonlinear dependence on reagent concentration, consistent with the formation of a reversible complex with the enzyme prior to irreversible modification. The enzyme incorporates both reagents to a limited extent and is protected against inactivation by NADP+ and NADPH. The reaction of these new nucleotide analogues with isocitrate dehydrogenase is compared to the much slower inactivation caused by bromoacetone, indicating the importance of the nucleotide moiety in the functioning of the affinity labels. It is likely that 2-BDB-TA-2',5'-DP and 2-BOP-TA-2',5'-DP will have general applicability as affinity labels for other NADP+ binding enzymes.

Adenosine Diphosphate↗
PubMed1972

Affinity labeling of the acetylcholine receptor in the electroplax: electrophoretic separtion in sodium dodecyl sulfate.

Electroplax, single cells dissected from electric tissue of Electrophorus, are labeled in a two-step procedure: reduction by dithiothreitol followed by alkylation by the affinity label 4-(N-maleimido)-alpha-benzyltri-[methyl-(3)H]methylammonium iodide, either alone or in combination with [2,3-(14)C]N-ethylmaleimide. Electrophoresis in sodium dodecyl sulfate on polyacrylamide gel of an extract, prepared with this detergent, of single-labeled or of double-labeled cells results in a major peak of (3)H activity, with a mobility corresponding to a polypeptide of molecular weight 42,000. In addition, in the double-labeled samples, there is a unique peak in the ratio of (3)H to (14)C that is coincident with the (3)H peak. The electrophoretic patterns of extracts of cells in which affinity alkylation of the reduced receptor has been suppressed by dithiobischoline, an affinity oxidizing agent, by cobratoxin, an irreversible ligand, or by hexamethonium, a reversible ligand, show a considerably diminished peak of (3)H activity in the region of molecular weight 42,000. This is the predominant difference between the electrophoretic patterns of extracts of unprotected and of protected cells. Furthermore, extracts of cells protected with dithiobischoline before labeling with both tritiated affinity label and [(14)C]N-ethylmaleimide do not show the peak in the (3)H to (14)C ratio seen in the absence of protection. Thus, by several diverse criteria, the peak of (3)H activity corresponding to a molecular weight of 42,000 contains affinity-labeled acetylcholine receptor or receptor subunit.

Acrylamides↗
PubMed1980

Affinity labeling and characterization of the active site histidine of glucosephosphate isomerase. Sequence homology with triosephosphate isomerase.

N-Bromoacetylethanolamine phosphate was found to act as a specific affinity label for the active center of glucosephosphate isomerase. The inactivation process followed pseudo-first order kinetics, was irreversible and exhibited rate saturation kinetics with minimal half-lives of inactivation of 4.5 and 6.3 min for the enzyme isolated from human placenta and rabbit muscle, respectively. The pH dependence of the inactivation process closely paralleled the pH dependence of the overall catalytic process with pKa values at pH 6.4 and 9.0. The stoichiometry of labeling of either enzyme, as determined with N-bromo[14C2]acetylethanolamine phosphate, was 1 eq of the affinity label/subunit of enzyme. After acid hydrolysis and amino acid analysis of the radioactive affinity-labeled human enzyme, only radioactive 3-carboxymethyl histidine was found. In the case of the rabbit enzyme, the only radioactive derivative obtained was 1-carboxymethyl histidine. Active site tryptic peptides were isolated by solvent extraction, thin layer peptide fingerprinting, and ion exchange chromatography before and after removal of the phosphate from the active site peptide. Amino acid analysis of the labeled peptides from the two species were very similar. Using high sensitivty methods for sequence analysis, the primary structure of the active site was established as Val-Leu-His-Ala-Glu-Asn-Val-Asp (Gly,Thr,Ser) Glu-Ile (Thr-Gly-His-Lys-Glx)-Tyr-Phe. Apparent sequence homology between the catalytic center of glucosephosphate isomerase and triosephosphate isomerase suggest that the two enzymes may have evolved from a common ancestral gene.

Affinity Labels↗
PubMed1983

Molecular properties of the slow inward calcium channel. Molecular weight determinations by radiation inactivation and covalent affinity labeling.

The slow inward calcium channel, identified by physiologic and pharmacologic responses and [3H]nitrendipine-specific binding, has been characterized by radiation inactivation and covalent affinity labeling. Target size analysis of guinea pig ileum longitudinal smooth muscle membranes indicates a molecular weight of 278,000 for the calcium channel. An affinity label analog of nifedipine and nitrendipine, 2,6-dimethyl-3,5-dicarbomethoxy-4-(2-isothiocyanatophenyl)-1,4-dihydropyridine, was found to inhibit the calcium channel by a covalent interaction with a protein subunit (Mr = 45,000) of the calcium channel.

Affinity Labels↗
PubMed1985

Demonstration and affinity labeling of a stereoselective binding site for a benzomorphan opiate on acetylcholine receptor-rich membranes from Torpedo electroplaque.

The interaction of an optically pure benzomorphan opiate, (-)-N-allyl-N-normetazocine [(-)-ANMC], with the nicotinic acetylcholine receptor from Torpedo electroplaque was studied by using radioligand binding and affinity labeling. The binding was complex with at least two specific components having equilibrium dissociation constants of 0.3 microM and 2 microM. The affinity of the higher affinity component was decreased by carbamoylcholine but not by alpha-bungarotoxin. The effect of carbamoylcholine was not blocked by alpha-bungarotoxin. In comparison, the affinity of [3H]phencyclidine, a well-characterized ligand for a high-affinity site for noncompetitive blockers on the acetylcholine receptor, is increased by carbamoylcholine and the increase is blocked by alpha-bungarotoxin. The binding of (-)-[3H]ANMC was inhibited by a number of other benzomorphans, with (-) isomers being 4- to 5-fold more potent than (+) isomers. Phencyclidine inhibits the binding of (-)-[3H]ANMC to its high-affinity site by a mechanism that is not competitive. UV-catalyzed affinity labeling indicated that the high-affinity-binding site for (-)-[3H]ANMC is at least partially associated with the delta subunit. Tryptic degradation of the Torpedo marmorata delta chain suggested that (-)-ANMC labeled a 16,000-dalton COOH-terminal portion of the subunit. In contrast, 5-azido-[3H]trimethisoquin, a photoaffinity label of the high-affinity site for noncompetitive blockers, labels a 47,000-dalton NH2-terminal fragment of the delta subunit. These results suggest that (-)-[3H]ANMC binds to sites completely distinct from the binding sites for acetylcholine. The high-affinity-binding site for (-)-ANMC and that for phencyclidine and 5-azidotrimethisoquin are allosterically coupled but are regulated differently and are probably physically distinct.

Affinity Labels↗