The mass spectral analysis of covalently labeled amino acid methylthiohydantoin derivatives derived from affinity-labeled proteins.
Explore the source record for details and available documents.
Affinity Labels↗
SEARCH · PubMed Health
Results for “Affinity Labels”
Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
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↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗
Investigation of type I and type III iodothyronine deiodinases in rat tissues using N-bromoacetyl-iodothyronine affinity labels.
In the present study the hypothesis was tested that N-bromoacetyl-3,3',5-[125I]triiodothyronine (BrAc[125I]T3) is a useful affinity label for both type I and type III iodothyronine deiodinases (ID-I and ID-III). Therefore, the microsomal fractions of various rat tissues were tested for ID-I and ID-III activities, and microsomal proteins were labeled with BrAc[125I]T3 and analyzed by SDS-PAGE. In agreement with previous observations, high ID-I activities were found in liver, kidney and thyroid, and high ID-III activities in brain, in particular fetal brain, and placenta. SDS-PAGE of BrAc[125I]T3-labeled microsomes showed a prominent radioactive approximately 27 kDa protein (p27) in liver, kidney and thyroid, which was previously identified as ID-I, and a approximately 32 kDa protein (p32) in brain, in particular fetal brain, and placenta. A good correlation was found between the affinity labeling of p32 and the inactivation of ID-III by BrAcT3, suggesting that p32 represents ID-III or a subunit thereof. After treatment of microsomes with 0.05% deoxycholate or carbonate buffer (pH 11.5) p32 was still labeled by BrAc[125I]T3, indicating that p32 is a transmembrane protein. Although 3,3',5'-triiodothyronine (rT3) is not a substrate for ID-III, p32 was readily labeled with BrAc[125I]rT3. Labeling of p32 in rat brain microsomes by BrAc[125I]rT3 was not affected by addition of 100 microM unlabeled thyroxine (T4) or T3, whereas deiodination of [125I]T3 by ID-III was inhibited by 91 and 96% in the presence of 1 microM T4 and T3, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Affinity Labels↗
Affinity labeling of Escherichia coli phenylalanyl-tRNA synthetase at the binding site for tRNAPhe.
Periodate-oxidized tRNA(Phe) (tRNA(oxPhe)) behaves as a specific affinity label of tetrameric Escherichia coli phenylalanyl-tRNA synthetase (PheRS). Reaction of the alpha 2 beta 2 enzyme with tRNA(oxPhe) results in the loss of tRNAPhe aminoacylation activity with covalent attachment of 2 mol of tRNA dialdehyde/mol of enzyme, in agreement with the stoichiometry of tRNA binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the PheRS-[14C]tRNA(oxPhe) covalent complex indicates that the large (alpha, Mr 87K) subunit of the enzyme interacts with the 3'-adenosine of tRNA(oxPhe). The [14C]tRNA-labeled chymotryptic peptides of PheRS were purified by both gel filtration and reverse-phase high-performance liquid chromatography. The radioactivity was almost equally distributed among three peptides: Met-Lys[Ado]-Phe, Ala-Asp-Lys[Ado]-Leu, and Lys-Ile-Lys[Ado]-Ala. These sequences correspond to residues 1-3, 59-62, and 104-107, respectively, in the N-terminal region of the 795 amino acid sequence of the alpha subunit. It is noticeable that the labeled peptide Ala-Asp-Lys-Leu is adjacent to residues 63-66 (Arg-Val-Thr-Lys). The latter sequence was just predicted to resemble the proposed consensus tRNA CCA binding region Lys-Met-Ser-Lys-Ser, as deduced from previous affinity labeling studies on E. coli methionyl- and tyrosyl-tRNA synthetases [Hountondji, C., Dessen, P., & Blanquet, S. (1986) Biochimie 68, 1071-1078].
Affinity Labels↗
Determination of lysine residues affinity labeled in the active site of yeast RNA polymerase II(B) by mutagenesis.
In a previous study, yeast RNA polymerase II(B) was affinity labeled with two nucleotide derivatives (III and VIII) (1). In both cases, the labeled site was localized to the C-terminal part of the B150 subunit. The potential target lysyl residues of derivative III were mapped to the conserved domain H, between Asn946 and Met999. In the present work, we have mutagenized to arginine the five lysines present in domain H. Three lysines can be replaced, individually or simultaneously, without affecting cell growth, and each mutated enzyme can still be affinity labeled. Hence one or both of the other two lysyl residues, Lys979 and Lys987, is the target of the affinity reagent. These two lysines were each found to be essential for cell viability. Derivative VIII labeled another domain in addition to domain H. Supported by analogous results obtained for E. coli RNA polymerase using derivative VIII (2), we hypothesized that the second domain labeled by this derivative in the B150 subunit was domain I. Mutagenesis of the unique lysine present in domain I demonstrated that Lys 1102 was the target of derivative VIII. These results indicate that in both prokaryotic and eukaryotic RNA polymerases, domains H and I are in close proximity and participate to the active site.
Affinity Labels↗
Affinity labeling of the ATP-binding site of Ca2+-transporting ATPase of sarcoplasmic reticulum by adenosine triphosphopyridoxal: identification of the reactive lysyl residue.
Adenosine triphosphopyridoxal (AP3PL) was used as an affinity label directed toward the ATP binding site of the Ca2+-transporting ATPase of the rabbit skeletal muscle sarcoplasmic reticulum (SR). The reagent inhibited the ATPase activity competitively with ATP, Ki = 20 microM. Incubation of SR membranes with 100 microM AP3PL followed by treatment with NaBH4 resulted in 90% inactivation of the E-P forming activity as well as of the Ca2+-transporting activity. Adenosine di- and tetraphosphopyridoxals had similar but less pronounced effects on the Ca2+-transport system. AP3PL was bound to ATPase in a one-to-one stoichiometry in parallel with the loss of the enzymatic activities. ATP and ADP prevented the binding of AP3PL and thereby protected the enzyme from inactivation. The SR membranes were labeled with [3H]AP3PL and then digested with thermolysin in order to identify the attachment site of the affinity label. A 3H-labeled peptide (Val-Glu-Pro-Ser-His-Lys* 684-Ser-Lys) was purified to homogeneity by Sephadex LH-20 chromatography and C18-reversed phase HPLC (Lys* denotes the binding site of [3H]AP3PL). These results indicate that the SR-ATPase peptide is folded in such a manner that Lys684 and Asp351, the phosphorylation site, are located very close to each other, since the distance between the 4-formyl group reacting with Lys684 and the gamma-phosphoryl group of the ATP moiety of AP3PL is rather small.
Adenosine Triphosphate↗
Reversible embedment cytochemistry (REC): a versatile method for the ultrastructural analysis and affinity labeling of tissue sections.
Reversible embedment cytochemistry (REC) is a new method for revealing cellular ultrastructure and for improving access of intracellular targets to macromolecular affinity labels. Fully polymerized polymethylmethacrylate was dissolved in dichloromethane and infiltrated into fixed tissue-culture cells and tissues. After evaporation of the solvent, samples were left in hard plastic. Samples were thus embedded without exposure to chemical polymerization reactions that might damage tissue ultrastructure or antigenicity. Glass or diamond knives fitted with water troughs were used to cut sections 30-1000 nm thick. Since polymethylmethacrylate is composed of linear polymers that are not covalently crosslinked, the plastic was easily extracted from the sections by immersion in solvent. Subsequently, various preparative methods, including negative staining, critical point-drying, and platinum-carbon rotary shadowing, were used to provide detailed images of well-preserved cell structure for conventional and high-voltage transmission electron microscopy. Fluorescein-conjugated affinity labels were used to obtain subcellular distributions of target molecules in semi-thick sections of cultured cells and tissues for light microscopy. Colloidal gold-labeled antibodies were used to localize microtubules in sections of cultured cells by electron microscopy. REC is a versatile method that should find wide application in many studies of cellular function.
Affinity Labels↗
Phosphate binding to liver alcohol dehydrogenase studied by the rate of alkylation with affinity labels.
In this work we report that phosphate anions interact with the anion binding site of alcohol dehydrogenase from horse liver. In protection experiments against the two affinity labels, iodoacetic acid and bromo-imidazolylpropionic acid, the dissociation constant for the enzyme-phosphate complex at pH 7.0 is, based on total phosphate, found to be 20 +/- 5 mM. The 1,4-piperazinediethanesulfonate anion has a lower affinity for the anion binding site, the dissociation at pH 7.0 being 130 +/- 20 mM. The anion-independent dissociation constants for the reversible enzyme-affinity label complexes are at pH 7.0, 1.35 +/- 0.2 mM for iodoacetic acid and 0.39 +/- 0.05 mM for bromo-imidazolylpropionic acid. These findings have important implications with respect to past and future work on this well known enzyme.
Affinity Labels↗
Bromoacetamido analogs of indomethacin and mefenamic acid as affinity-labeling agents and mechanistic probes for prostaglandin H2 synthase.
Affinity-labeling agents, 1-[4-(bromoacetamido)benzyl]-5-methoxy-2-methylindole-3-acetic acid (I) and 4-(bromoacetamido)-N-(2,3-dimethylphenyl)anthranilic acid (II), were synthesized on the basis of their respective nonsteroidal anti-inflammatory drugs (NSAIDs), indomethacin and mefenamic acid [Askonas & Penning (1991) Biochemistry 30, 11553-11560]. Compounds I and II are now shown to inhibit homogeneous ram seminal vesicle prostaglandin H2 (PGH2) synthase by two kinetically distinct complexes. They are competitive inhibitors versus arachidonic acid via the formation of high-affinity E.I complexes, and they cause time-dependent inactivation of the holoenzyme via low-affinity E.I complexes. Compounds I and II, unlike classical NSAIDs, were found to inactivate both the cyclooxygenase and peroxidase reactions of the synthase in a parallel manner. Inactivation was accompanied by the incorporation of 2 mol of either radiolabeled I or II per synthase monomer. The covalent bonds that result were stable to boiling in SDS, indicating that I and II offer alternatives to aspirin in locating NSAID binding sites. Incubation of aspirin-treated PGH2 synthase with radiolabeled I reduced the stoichiometry of incorporation to 1.0, suggesting that one of the sites modified corresponds to the cyclooxygenase site. By saturating the cyclooxygenase site with mefenamic acid, I and II only abolished the peroxidase activity of the enzyme, suggesting that the second site of modification corresponds to the peroxidase site. When PGH2 synthase was incubated with mefenamic acid and I or II, only the peroxidase activity was inactivated. Subsequent removal of all drugs by dialysis gave a preparation of PGH2 synthase that could perform the cyclooxygenase reaction, but lacked the ability to cleave ethyl hydroperoxide to ethanol and water.(ABSTRACT TRUNCATED AT 250 WORDS)
Affinity Labels↗
The synthesis and study of some potential affinity labeling reagents for estrogen receptors.
The influence of the following affinity labeling reagents on the binding of tritiated estradiol-17 beta (E) by human and calf uterine cytosols was studied: 11 beta-chloromethylestradiol (ORG4333), 2-azidoestradiol (2A-E), 4-azidoestradiol (4A-E), 3-azidohexestrol (3A-H), estradiol-17 beta 17-bromoacetate (E-17BrAc), 6-[O-carbo-(2'-chloroethoxy)methyl] oximinoestradiol (6-CMOEtC1), 17-[O-carbo-(2'-chloroethoxy) methyl] oximinoestrone (17-CMOEtCl), 2-di (2'-hydroxy-3'-chloropropyl)aminoestradiol (E-Mustard). For the human uterine estrogen receptor the relative binding affinity decreased in the order E greater than ORG 4333 greater than E-17BrAc greater than 3A-H greater than 2A-E greater than 4A-E greater than 6-CMOEtCl greater than E-Mustard greater than 17-CMOEtCl. The binding characteristics of the calf uterine estrogen receptor were qualitatively similar, but quantitatively different. ORG 4333 appeared to form a highly stable association with the receptors, but alkylation of the protein could not be conclusively demonstrated.
Affinity Labels↗