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PubMedDate not supplied

[125I]iododesethyl tamoxifen aziridine: synthesis and covalent labeling of the estrogen receptor with an iodine-labeled affinity label.

Iododesethyl tamoxifen aziridine (I-Tam-Az), an analog of the estrogen receptor-affinity label tamoxifen aziridine (Tam-Az) in which the ethyl group has been replaced by an iodine, has been prepared by two routes: (a) metallation of a bromotriarylethylene system, followed by reaction with iodine, and aziridinylation, and (b) direct iodination of a trimethylstannyl triarylethylene system that is the immediate precursor of I-Tam-Az. The latter method can be used to prepare [125I]I-Tam-Az rapidly and in good yield, both at carrier-added and no-carrier-added levels; specific activities greater than 200 Ci/mmol have been obtained. In competitive radiometric binding assays with the estrogen receptor, I-Tam-Az has an apparent affinity of ca. 20%, equivalent to that of Tam-Az. It also undergoes rapid and selective time-dependent, irreversible binding to the estrogen receptor. [125I]I-Tam-Az reacts covalently with estrogen receptor in uterine cytosol preparations; its attachment is rapid and efficient, but somewhat less selective than that of Tam-Az. Estrogen receptor in intact MCF-7 human breast cancer cells can also be labeled with [125I]I-Tam-Az, and autoradiographic analysis of salt extracts of labeled nuclear estrogen receptor on SDS-polyacrylamide slab gels shows highly selective labeling of a 65K protein. [125I]I-Tam-Az is an efficient, selective affinity label for the estrogen receptor, available at high specific activity, and should be useful in studies on estrogen receptor structure, dynamics, and chromatin interactions.

Affinity Labels↗
PubMed1983

Structural similarities in the plasma membrane 3,3',5-triiodo-L-thyronine receptors from human, rat and mouse cultured cells. Analysis by affinity labeling.

Affinity labeling of Swiss 3T3-4 mouse fibroblasts with 0.3 nM N-bromoacetyl-3,[125I]3',5-triiodo-L-thyronine (BrAc[125I]T3) at 4 C for 0.5 h showed three labeled protein bands with apparent molecular masses of 55, 53 and 33 kilodaltons (kDal) in a ratio of 85:8:7. Only the labeling of 55-kDal protein was selectively reduced to approximately 50% by 15 microM unlabeled T3. Affinity labeling of the purified plasma membranes under identical conditions yielded similar results. One-dimensional peptide mapping by Staphylococcus aureus V8 or elastase digestion of the 55-kDal proteins from cells or plasma membranes gave identical peptide fragments. Thus the 55-kDal protein is a membrane-associated protein. Furthermore, affinity labeling of human epithelioid carcinoma A431 cells and purified plasma membranes gave similar results as those of Swiss 3T3 cells. Peptide mapping of elastase or S. auerus digestion of 55-kDal proteins from A431, Swiss 3T3 and GH3 rat pituitary tumor cells gave identical patterns. These results indicate that plasma membrane T3 receptors from three species have structural similarity in the hormone binding domains. Thus, the plasma membrane T3 receptor probably are highly conserved.

Affinity Labels↗
PubMed1986

Location of long chain fatty acid-binding sites of bovine serum albumin by affinity labeling.

Affinity labeling with palmitic acid was used to identify long chain fatty acid-binding sites of bovine serum albumin. [1-14C]Palmitic acid was activated by esterification with N-ethyl-5-phenyl-isoxazolium-3'-sulfonate (Woodward's Reagent K). The product was purified by chromatography and shown to compete with unesterified fatty acids for binding sites on bovine serum albumin. Activated [14C]palmitic acid coupled covalently to albumin producing [14C]palmitoyl-albumins containing from 0.12 to a maximum of 6.9 mol of attached label per mol of albumin. The presence of the covalently attached affinity label depressed binding of other long chain fatty acids to albumin. Albumin carrying 1 eq. of [14C]palmitate was cleaved using cyanogen bromide, pepsin, and trypsin. Radioactive peptides were isolated by high pressure liquid chromatography. Three peptides accounted for greater than 90% of the label. Residues labeled with [14C]palmitate were identified as Lys-116, Lys-349 and Lys-473, and the relative distribution of label was 10, 45, and 45% respectively, consistent with the presence of two strong binding sites in the COOH-terminal half of albumin and a somewhat weaker site in the NH2-terminal half.

Affinity Labels↗
PubMed1989

Taipoxin-binding protein on synaptic membranes: identification by affinity labeling.

Affinity labeling techniques were used to identify the neuronal membrane molecules involved in the binding of taipoxin, a neurotoxic protein with phospholipase A2 activity. After [125I]taipoxin had bound to synaptosomes from guinea pig brain, treatment with disuccinimidyl suberate resulted in the formation of a predominant radioactive conjugate of 60,000 Da. Notexin and some other PLA2s are weakly inhibitory to this conjugation, while beta-bungarotoxin and some others are not inhibitory. The 60K conjugate was not detected when plasma membranes from several nonneuronal tissues were used. We concluded that a 45,000 Da protein specifically present in neuronal membranes is (a subunit of) the major molecule responsible for taipoxin binding.

Affinity Labels↗
PubMed1985

Phenylalanyl-tRNA synthetase from E. coli MRE-600: analysis of the active site distribution on the enzyme subunits by affinity labelling.

Affinity labelling has been employed to localize the substrate-binding sites on the enzyme subunits of phenylalanyl-tRNA synthetase (L-phenylalanine:tRNAPhe-ligase, EC 6.1.1.20) of Escherichia coli MRE-600 (alpha 2 beta 2-type). N-Chlorambucilylphenylalanyl-tRNA, N-bromoacetylphenylalanyl-tRNA, tRNAPhe containing an azido group at the eighth position of the molecule (S4U), tRNAPhe containing azido groups at different points of the molecule, p-azidoanilidate of phenylalanine, adenosine 5'-trimethaphosphate and N-bromoacetyl-L-phenylalaninyladenylate were used in experiments. It has been shown that tRNA-binding sites are formed on heavy beta-subunits of the enzyme. Phenylalanyl-tRNA is also localized on beta-subunits, while the aminoacyl moiety of aminoacyl-tRNA is localized near the contact region of subunits. The phenylalanine-binding site is located on light alpha-subunits of the enzyme. Adenosine 5'-trimethaphosphate and the analogue of phenylalanyladenylate modify both types of enzyme subunits. In our opinion, the catalytic center of tRNA aminoacylation is formed in the contact region of subunits, and the aminoacyl moiety is transferred into tRNA (from the alpha- into beta-subunit in the region of their contact).

Adenosine Triphosphate↗
PubMed1985

Reductive alkylation with oxidized nucleotides. Use in affinity labeling or affinity chromatography.

A study has been made of the products of a reaction of oxidized ribonucleotides with a primary amine. As a model reaction, periodate-oxidized adenosine was combined with glycine in the presence of NaCNBH3. The purified major product of this reaction, adenine 9,2'-(4'-carboxymethyl-6'-hydroxymethylmorpholine), was characterized by 13C and 1H NMR spectroscopy, ultraviolet spectroscopy, and thin layer chromatography. When used to generate affinity columns, oxidized adenosine or oxidized ATP formed stable products with immobilized diaminohexane when treated with NaCNBH3. Failure to treat with NaCNBH3 yielded an unstable affinity matrix. These results are used in the interpretation of differing results when oxidized nucleotides have been used as affinity labels for different proteins.

Adenosine↗
PubMed2002

Naphthalene dicarboxaldehyde as an electrophilic fluorogenic moiety for affinity labeling: application to opioid receptor affinity labels with greatly improved fluorogenic properties.

To develop ligands with fluorogenic properties amenable for following the kinetics of cross-linking to receptors, a naphthalene dicarboxaldehyde moiety has been attached to an opiate pharmacophore 2 and evaluated in mu-opioid receptors. The fluorescence of the benzo[f]isoindole formed upon cross-linking of mu-opioid receptors by 2 permitted the time-course of covalent bonding to be followed. This demonstrated proof-of-concept suggests the usefulness of naphthalene dicarboxaldehyde-containing affinity labels as kinetic probes.

Affinity Labels↗
PubMed1990

Cysteinyl peptides of rabbit muscle pyruvate kinase labeled by the affinity label 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-triphosphate.

The affinity label 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-triphosphate (8-BDB-TA-5'-TP) reacts covalently with rabbit muscle pyruvate kinase, incorporating 2 mol of reagent/mol of enzyme subunit upon complete inactivation. Protection against inactivation is provided by phosphoenolpyruvate, K+, and Mn2+ and only 1 mol of reagent/mol of subunit is incorporated [DeCamp, D.L., Lim, S., & Colman, R.F. (1988) Biochemistry 27, 7651-7658]. We have now identified the resultant modified residues. After reaction with 8-BDB-TA-5'-TP at pH 7.0, modified enzyme was incubated with [3H]NaBH4 to reduce the carbonyl groups of enzyme-bound 8-BDB-TA-5'-TP and to introduce a radioactive tracer into the modified residues. Following carboxymethylation and digestion with trypsin, the radioactive peptides were separated on a phenylboronate agarose column followed by reverse-phase high-performance liquid chromatography in 0.1% trifluoroacetic acid with an acetonitrile gradient. Gas-phase sequencing gave the cysteine-modified peptides Asn162-Ile-Cys-Lys165 and Cys151-Asp-Glu-Asn-Ile-Leu-Trp-Leu-Asp-Tyr-Lys161, with a smaller amount of Asn43-Thr-Gly-Ile-Ile-Cys-Thr-Ile-Gly-Pro-Ala-Ser-Arg55. Reaction in the presence of the protectants phosphoenolpyruvate, K+, and Mn2+ yielded Asn-Ile-Cys-Lys as the only labeled peptide, indicating that inactivation is caused by modification of Cys151 and Cys48.

Adenosine Diphosphate↗
PubMedDate not supplied

The biochemical characterization of the native pancreatic cholecystokinin receptor using affinity labeling approaches.

Affinity labeling has been a powerful tool for the biochemical characterization of sparse molecules which bind to a ligand probe in a specific, high-affinity manner. The rat pancreatic acinar cell receptor for cholecystokinin (CCK), the major physiologic hormonal stimulant of pancreatic exocrine secretion, has been the target of such investigation. Of interest, affinity-labeling studies have identified two distinct plasma membrane glycoproteins as candidates to represent this receptor. The initial candidate, which was identified using 125I-Bolton Hunter-labeled CCK-33 as probe, migrates on a SDS-polyacrylamide gel as a broad band in the M(r) = 80,000 range. Subsequently, using shorter probes in which the site of covalent attachment was closer to the receptor-binding domain of the probe, a band of M(r) = 85,000-95,000 was specifically labeled. Deglycosylation and protease-peptide mapping demonstrated that these bands represent distinct molecules. Using "intrinsic" probes of the receptor, in which a photolabile residue was sited within the pharmacophoric domain of the ligand, attention was focused on the latter candidate as representing the binding protein. Insight into the relationship between these proteins as they reside in the plasma membrane was contributed by labeling with a "topographical mapping" probe, which incorporates a flexible spacer of variable length between a CCK-like ligand and a photolabile residue. This procedure confirmed that these two minor membrane proteins are spatially associated with each other.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1977

Affinity labeling of adenine nucleotide-related enzymes with reactive adenine nucleotide analogs. II. Affinity labeling of phosphoglycerate kinase with a reactive AMP analog.

Affinity labeling of yeast and B. stearothermophilus phosphoglycerate kinases with a reactive AMP analog, N6-(p-bromoacetaminobenzyl)-AMP was examined. Complete loss of enzyme activity was observed when 1 mol of the reagent had reacted per mol of either enzyme. Results on the effect of pH and substrate addition on the inactivation, titration of SH groups before and after modification, and kinetic studies with AMP analogs suggest that the modification occurs at one amino group at or near the substrate binding site. General affinity labeling of kinases is discussed based on the results obtained.

Adenosine Monophosphate↗
PubMed1977

Affinity labeling of adenine nucleotide-related enzymes with reactive adenine nucleotide analogs. I. Affinity labeling of glyceraldehyde 3-phosphate dehydrogenase and myokinase with a reactive AMP analog.

Rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (GPD) and myokinase (MK) were rapidly inactivated by a reactive AMP analog, N6-(p-bromoacetaminobenzyl)-AMP, under mild conditions. Complete inactivation was observed when 4 and 0.3 mol of the reagent with respect to enzyme were reacted with GPD and MK, respectively. The inactivation of both enzymes were favored at higher pH and the enzymes were protected by addition of adenine nucleotide substrate. Modified GPD or MK had no affinity for AMP-Sepharose, in contrast to the native enzymes. From these results, the inactivation of GPD and MK by the reactive AMP analog can be regarded as an affinity labeling. The posibility that the present AMP analog may be used as a general affinity labeling reagent for various adenine nucleotide-related enzymes is discussed based on the results obtained.

Adenosine Monophosphate↗
PubMed1980

Detection of nucleoside triphosphate binding sites of two types in Escherichia coli RNA-polymerase by affinity labeling.

The affinity labeling of E. coli RNA polymerase by periodate-oxidized uridin triphosphate (o-UTP) has been carried out under the conditions of poly(dA) and poly(dT) transcription. The extent of RNA polymerase labeling proved to be 2.5 times higher under the transcription of poly(dA) as compared to poly(dT). The amount of o-UTP attached to beta beta'-subunits has been found to decrease if RNA polymerase is labeled in the transcribed complex with poly(dT). These results as well as those obtained in our previous study (1), suggest that there are two types of binding sites for nucleoside triphosphates and their analogs in E. coli RNA polymerase.

Affinity Labels↗
PubMed1981

Affinity labelling of liver alcohol dehydrogenase. Effects of pH and buffers on affinity labelling with iodoacetic acid and (R, S)-2-bromo-3-(5-imidazolyl)propionic acid.

Both iodoacetic acid and (R,S)-2-bromo-3-(5-imidazolyl)propionic acid (BrImPpOH) react with liver alcohol dehydrogenase in an affinity labelling mechanism between pH 6.1 and 10.5. The buffer-independent dissociation constants and the first-order rate constants have been determined as a function of pH. With BrImPpOH a pKa close to 9 for the free enzyme is assigned to the zinc-water ionization. The buffers used exerted a protective effect upon the inactivation of the enzyme by iodoacetic acid and BrImPpOH. Phosphate buffer showed a high degree of protection especially at lower pH, while zwitterionic buffers like Mes (4-morpholineethanesulfonic acid), Pipes (1,4-piperazinediethanesulfonic acid), Epps [4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid] and Bicine [N,N-bis(2-hydroxyethyl)glycine] gave less protection to various degrees. An exception was Ches (cyclohexylaminoethanesulfonic acid) which had an anomalously high affinity for the iodoacetate binding site. The dissociation constants of the buffers were calculated for the case of inactivation by both iodoacetic acid and BrImPpOH.

Affinity Labels↗
PubMed2000

Probing the vitamin D sterol-binding pocket of human vitamin D-binding protein with bromoacetate affinity labeling reagents containing the affinity probe at C-3, C-6, C-11, and C-19 positions of parent vitamin D sterols.

The multiple physiological properties of vitamin D-binding protein (DBP) include organ-specific transportation of vitamin D(3) and its metabolites, manifested by its ability to bind vitamin D sterols with high affinity. In the present investigation we probed the vitamin D sterol-binding pocket of human DBP with affinity labeling analogs of 25-hydroxyvitamin D(3) ¿25-OH-D(3) and 1, 25-dihydroxyvitamin D(3) ¿1,25(OH)(2)D(3) containing bromoacetate alkylating probe at C-3 (A-ring), C-6 (triene), C-11 (C-ring), and C-19 (exocyclic methylene) of the parent sterol. Competitive binding assays with DBP showed approximately 22-, 68-, and 2000-fold decrease in the binding of 1,25(OH)(2)-D(3)-11-BE, 25-OH-D(3)-3-BE, and 25-OH-D(3)-6-BE, respectively, compared to that seen with 25-OH-D(3), while there was no significant difference in the DBP-binding affinity of 25-OH-D(3)-19-BE and 25-OH-D(3). Surprisingly, ¿(14)C25-OH-D(3)-11-BE and ¿(14)C1, 25(OH)(2)-D(3)-19-BE failed to label DBP despite high-affinity DBP-binding, indicating the absence of any nucleophilic amino acid in the vicinity of their bromoacetate moiety to form a covalent bond, while these analogs are inside the binding pocket. In contrast, ¿(14)C25-OH-D(3)-6-BE and ¿(14)C25-OH-D(3)-3-BE specifically labeled DBP. BNPS-skatole digestion of DBP labeled with ¿(14)C25-OH-D(3)-3-BE or ¿(14)C25-OH-D(3)-6-BE produced two peptides (M(r) 17,400 and 33,840), with radioactivity associated with the N- and C-terminal peptides, respectively, raising the possibility that either different areas of the same vitamin D sterol-binding pocket, or different domains of DBP might be labeled by these analogs. Successful affinity labeling of recombinant domain I (1-203) of DBP with both reagents indicated that different areas of the same vitamin D-binding pocket (domain I) were labeled. These affinity analogs are potentially useful for "mapping" the vitamin D sterol-binding pocket and developing a functional model.

Acetates↗
PubMed1974

Stimulation of antibody production to the hapten 2,4-dinitrobenzene by affinity labeled murine lymphoid cells. I. Ability of affinity labeled murine lymphoid cells to activate the in vivo immune response.

The ability of meta-nitrobenzenediazonium fluoborate (m-NBDF)-labeled thymus and spleen (S) cells to transfer immunity to 2,4-dinitrophenyl (DNP) into irradiated syngeneic recipients was investigated. There was a significant increase in the number of anti-DNP plaque-forming cells (PFC) when m-NBDF-labeled thymus cells and normal spleen cells, or normal thymus cells and m-NBDF-labeled spleen cells were transferred, but not when both thymus- and S-cell populations were labeled and injected together into irradiated recipients. The ability of these cell populations to cooperate and enhance the in vivo immune response to DNP is discussed. The T cells seem to be actively involved in the development of this response; they participate beyond the mere role of carrying and presenting antigen to the B cells. It is suggested that cell to cell contact between T and B cells may be an important factor in the elicitation of an immune response. In addition, the cellular interaction is affected by irradiating the thymus cell preparation and the initiating interaction required for antibody synthesis probably occurs within 48 h after injecting the cell populations into the syngeneic irradiated recipients.

Animals↗
PubMed2002

Use of fluorescently labeled caspase inhibitors as affinity labels to detect activated caspases.

Activation of caspases is the key event of apoptosis and different approaches were developed to assay it. To detect their activation in situ, we applied fluorochrome labeled inhibitors of caspases (FLICA) as affinity labels of active centers of these enzymes. The FLICA ligands are fluorescein or sulforhodamine conjugated peptide-fluoromethyl ketones that covalently bind to enzymatic centers of caspases with 1:1 stoichiometry. The specificity of FLICA towards individual caspases is provided by the peptide sequence of amino acids. Exposure of live cells to FLICA results in uptake of these ligands and their binding to activated caspases; unbound FLICA is removed by cell rinse. Cells labeled with FLICA can be examined by fluorescence microscopy or subjected to quantitative analysis by cytometry. Intracellular binding sites of FLICA are consistent with known localization of caspases. Covalent binding of FLICA allowed us to identify the labeled proteins by immunoblotting: the proteins that bound individual FLICAs had molecular weight between 17 and 22 kDa, which corresponds to large subunits of the caspases. Detection of caspases activation by FLICA can be combined with other markers of apoptosis or cell cycle for multiparametric analysis. Because FLICA are caspase inhibitors they arrest the process of apoptosis preventing cell disintegration. The stathmo-apoptotic method was developed, therefore, that allows one to assay cumulative apoptotic index over long period of time and estimate the rate of cell entry into apoptosis for large cell populations. FLICA offers a rapid and convenient assay of caspases activation and can also be used to accurately estimate the incidence of apoptosis.

Affinity Labels↗
PubMed1989

Determination of the roles of active sites in F1-ATPase by controlled affinity labeling.

The affinity reagents 3'-O-(5-fluoro-2,4-dinitrophenyl) [alpha-32P]ATP (FDNP-[alpha-32P]ATP) and 3'-O-(5-fluoro-2,4-dinitrophenyl) [8-14C]ATP (FDNP-[14C]ATP) were synthesized and used to characterize the structure and function of the three active sites in F1-ATPase. FDNP-[alpha-32P]ATP was found to bind covalently to F1 up to two DNP-[alpha-32P]ATP labels per F1 in the absence of Mg2+ without decreasing the ATPase activity. However, when MgCl2 was subsequently added to the reaction mixture, the enzyme could be further labeled with concomitant decrease in ATPase activity that is consistent with the complete inactivation of one enzyme molecule by an affinity label at the third ATP-binding site. Partial hydrolysis of the FDNP-[14C]ATP-labeled enzyme and sequencing of the isolated peptide indicated that the affinity label was attached to Lys-beta 301 at all three active sites. Samples of F1 with covalent affinity label on Lys-beta 301 were also used to reconstitute F1-deficient submitochondrial particles. The reconstituted particles were assayed for ATPase and oxidative phosphorylation activities. These results show that the catalytic hydrolysis of ATP either by F1 in solution or by F0F1 complex attached to inner mitochondrial membrane takes place essentially at only one active site, but is promoted by the binding of ATP at the other two active sites, and that ATP synthesis during oxidative phosphorylation takes place at all three active sites [corrected].

Adenosine Triphosphatases↗