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PubMedDate not supplied

"Cleavable trifunctional" approach to receptor affinity labeling: chemical regeneration of binding to A1-adenosine receptors.

A general approach for reversible affinity labeling of receptors has been developed. The objective is to carry out a series of chemical modifications resulting in a covalently-modified, yet functionally-regenerated, receptor protein that also may contain a reporter group. The ligand recognition site of A1-adenosine receptors in bovine brain membranes was probed to demonstrated the feasibility of this approach. Use of disulfide or ester linkages, intended for cleavage by exposure of the labeled receptor to either reducing reagents or hydroxylamine, respectively, was considered. Binding of the antagonist radioligand [3H]CPX was preserved following incubation of the native receptor with 3 M hydroxylamine, while binding was inhibited by the reducing reagent dithiothreitol (DTT) with an IC50 of 0.29 M. Hydroxylamine displaced specific agonist ([3H]PIA) binding in a noncovalent manner. Specific affinity labels containing reactive isothiocyanate groups were synthesized from XCC (8-[4-](carboxymethyl)-oxy]phenyl]-1,3-dipropylxanthine) and shown to bind irreversibly to A1-receptors. The ligands were structurally similar to previously reported xanthine inhibitors (e.g., DITC-XAC: (1989) J. Med. Chem. 32, 1043) except that either a disulfide linkage or an ester linkage was incorporated in the chain between the pharmacophore and the isothiocyanate-substituted ring. These groups were intended for chemical cleavage by thiols or hydroxylamine, respectively. Radioligand binding to A1-receptors was inhibited by these reactive xanthines in a manner that was not reversed by repeated washing. Hydroxylamine or DTT restored a significant fraction of the binding of [3H]CPX in A1-receptors inhibited by the appropriate cleavable xanthine isothiocyanate derivative.

Affinity Labels↗

PubMed1987

Estrogenic affinity labels: synthesis, irreversible receptor binding, and bioactivity of aziridine-substituted hexestrol derivatives.

To develop an affinity label for the estrogen receptor that would be an estrogen agonist, rather than antagonist, we prepared several aziridine derivatives of the potent nonsteroidal estrogen hexestrol [3R,4S)-3,4-bis(4-hydroxyphenyl)hexane) bearing an aziridine function on the side chain. Three functional groups link the hexestrol ligand and the aziridine: a carbonyl group (ketone or ester), a thioether, or a methylene chain. The apparent competitive binding affinity of these derivatives for the estrogen receptor ranges from 1.8% to 25% that of estradiol, and most of them bind in a time-dependent, irreversible manner with the receptor, although the rate and efficiency of this binding vary widely, often with relatively small changes in structure. This is consistent with the irreversible attachment requiring a precise alignment of activating and reacting residues in the binding site of the receptor. The estrogenic and antiestrogenic activity of these aziridine derivatives was investigated in MCF-7 human breast cancer cells. Most of the compounds are agonists, with one being an antagonist. The derivative (6R,7S)-1-N-aziridinyl-6,7-bis(4-hydroxyphenyl)-5-nonanone (keto-nonestrol aziridine 3) appears to have the most ideal behavior of the estrogenic affinity labeling agents prepared: It is an agonist, and it binds to receptor irreversibly, efficiently, and quite rapidly.

Affinity Labels↗

PubMed1987

Analysis of the carbohydrate composition of the pancreatic plasmalemmal glycoprotein affinity labeled by short probes for the cholecystokinin receptor.

Affinity labeling of the rat pancreatic cholecystokinin (CCK) receptor with decapeptide probes has identified an Mr = 85,000-95,000 protein, distinct from the Mr = 80,000 component previously labeled with 125I-Bolton Hunter-CCK-33. We have characterized the carbohydrate composition of this novel protein labeled with 125I-D-Tyr-Gly-[(Nle28,31)-CCK-26-33] and disuccinimidyl suberate by using chemical and enzymatic deglycosylation and lectin chromatography. The Mr = 85,000-95,000 component was demonstrated to be an N-linked sialoglycoprotein based on neuraminidase digestion to Mr = 75,000-85,000 and endo-beta-N-acetylglucosaminidase F (Endo F) digestion to Mr = 42,000. This was distinct from the Mr = 65,000 product of Endo F digestion of the protein labeled with 125I-Bolton Hunter-CCK-33. Lack of an effect of endo-beta-N-acetylglucosaminidase H demonstrated the absence of N-linked simple oligosaccharides, while products of chemical deglycosylation with hydrogen fluoride and endo-alpha-N-acetylgalactosaminidase supported the absence of O-linked carbohydrate. The presence of at least four oligosaccharide chains on the core protein was suggested by Endo F digestion of the Mr = 85,000-95,000 protein using limiting enzyme conditions. This glycoprotein was retained on wheat germ agglutininagarose and eluted by N,N',N"-triacetylchitotriose. Identification of the Mr = 85,000-95,000 component on the ectodomain of the plasmalemma of intact pancreatic acini confirmed this to be the fully processed form of the CCK-binding protein.

Affinity Labels↗

PubMed1988

Synthesis of 3H-Tyr-D-Ala-Gly-N(Me)Phe chloromethyl ketone--an opioid affinity label.

A radioactive enkephalin affinity reagent, selective for the mu opioid receptor subtype, was synthesized by a fragment condensation method. 3H-BOC-Tyr-D-Ala-Gly-OH was prepared by catalytic tritiation of the protected iodinated tripeptide. The protected tritiated tripeptide and N(Me)Phe-CH2Cl were condensed by the mixed anhydride method. The protecting group was removed by HCl/acetic acid. The tritiated tetrapeptide has a specific radioactivity of 56.8 Ci/mmole (2.1 TBq/mmole).

Affinity Labels↗

PubMed1990

Affinity labelling of a partially purified ecdysteroid receptor with a bromoacetylated 20-OH-ecdysone derivative.

The novel bromoacetyl ecdysteroid IV, (20R,22R)-2 beta,3 beta,14 alpha,20,22,25 xi-hexahydroxy-26-(3- bromoacetoxypropyl)-5 beta-cholest-7-en-6-one, BAEIV, has been synthesized by extending the side chain on C26 of 20-OH-ecdysone. BAEIV meets all the requirements for an affinity-labelling reagent. It reacts with the partially purified ecdysteroid receptors of Drosophila melanogaster rapidly and almost quantitatively. Reactions require only micromolar concentrations of BAEIV. The rate of the affinity-labelling reaction is determined by the association of BAEIV with the ecdysteroid receptor. The value of the apparent reaction rate constant is very similar to that of the association rate constant for the binding of 20-OH-ecdysone to the ecdysteroid receptor. Product analysis of the reaction of [14C]BAEIV with the ecdysteroid receptor revealed two labelled peptides having molecular masses 150 kDa and 90 kDa. The smaller peptide is possibly a proteolytic fragment of the larger peptide. The identification of a 150-kDa peptide by chemical affinity labelling of the ecdysteroid receptor agrees with previously reported photoaffinity-labelling results from our laboratory. The results also demonstrate that the ecdysteroid receptor of D. melanogaster has a molecular mass higher than all other vertebrate steroid hormone receptors studied so far.

Affinity Labels↗

PubMed1987

Establishment of a new short, protease-resistant, affinity labeling reagent for the cholecystokinin receptor.

Proteolytic degradation of radioligands is an important source of artifact in affinity labeling of receptor proteins. To complement our previous characterization of the pancreatic acinar cell cholecystokinin (CCK) receptor, we synthesized D-Tyr-Gly[(Nle28,31)CCK-26-33]. The amino terminal D-enantiomer of tyrosine provided a site for oxidative iodination, a free amino group for cross-linking, and rendered the peptide resistant to aminopeptidases. The decapeptide was oxidatively iodinated and purified by reverse-phase HPLC to 2,000 Ci/mmol, to yield a probe which was equal in potency and efficacy to CCK-8, and which bound to rat pancreatic membranes in a rapid, reversible, temperature-dependent, specific, saturable and high affinity manner. This probe was resistant to aminopeptidase degradation, and maintained its ability to bind to receptor after incubation with pancreatic membranes or dispersed cells. Affinity labeling of pancreatic membranes with this analogue identified an Mr = 85,000-95,000 molecule. This analogue offers several advantages over existing probes and should be useful for future studies of this and other CCK receptors.

Affinity Labels↗

PubMed1983

Co-operative effects in affinity labeling reveal the interaction of tRNA-recognition centers of phenylalanyl-tRNA synthetase.

A mathematical treatment of affinity labeling of the enzymes is presented. The model considered involves a dimeric enzyme with identical ligand binding sites. Equations are derived which describe the kinetics of modification; mutual influence of ligand molecules on association, on the rate of covalent attachment and the possibility of the existence of different sites of modification are taken into account. Experimental data on affinity labeling of phenylalanyl-tRNA synthetase (L-phenylalanine:tRNAPhe ligase (AMP-forming), EC 6.1.1.20) of Escherichia coli MRE-600 with N-bromoacetyl-[14C]phenylalanyl-tRNA are treated in terms of the model suggested. The affinity (association constant value) of the tRNAPhe analog molecule towards the enzyme is only slightly affected by another molecule, whereas the reaction rate constant of covalent attachment decreases significantly. The latter is assumed to be due to acceptor site change in the complex containing two molecules of the tRNAPhe analog.

Affinity Labels↗

PubMed1991

Identification of RNA replicase subunits responsible for initiation of RNA synthesis of tick-borne encephalitis virus by affinity labelling.

Porcine embryo kidney cells infected by tick-borne encephalitis virus (TBEV) were fractionated into nuclear, membrane, and cytoplasmic fractions. To identify proteins involved in the initiation of RNA replication at different stages of infection a highly specific affinity labelling technique was used. In samples of the nuclear fraction taken from cells 45 h after infection (late stage), affinity labelling with aldehyde-containing derivatives of ATP and elongation of this label with [alpha-32P]GTP identified a polypeptide with a molecular mass of about 69 kDa. By means of affinity labelling with aldehyde-containing analogues of GMP, GDP, and GTP as initiation substrates and [alpha-32P]ATP as the elongation substrate, a polypeptide of 100 kDa was selectively modified in the nuclear fraction of cells at the early stages of infection (8 h). These proteins were immunostained with TBEV-specific antibodies, and were identified as the nonstructural TBEV proteins NS3 and NS5, respectively. It was concluded that NS3 and NS5 take part in the initiation of TBEV genome replication at the late and early stages of infection, respectively.

Animals↗

PubMed1982

Interactions between the mitochondrial adenosinetriphosphatase and periodate-oxidized adenosine 5'-triphosphate, an affinity label for adenosine 5'-triphosphate binding sites.

Periodate-oxidized ATP (o-ATP) was prepared as an affinity label of nucleotide binding sites on the chloroform-released ox heart mitochondrial ATPase. In the presence of MgSO4, o-ATP is a substrate for the ATPase. It can act as a reversible, competitive inhibitor of ATPase activity and can also induce an irreversible inhibition of ATPase activity. In parallel with the irreversible inhibition, covalent incorporation of [3H]o-ATP occurs. ATPase has about 1.05 mol of o-ATP bound per mol of ATPase when the enzyme is 50% inhibited. Most of the covalently bound o-ATP is associated with the alpha and beta subunits and is equally distributed between them. The incorporation of o-ATP into the ATPase is reduced, and the irreversible inhibition induced by o-ATP can be prevented totally by MgADP, MgATP, EDTA/ATP, or EDTA. The location, number, and the functional significance of the o-ATP binding sites are discussed. o-ATP can decompose to form an adenosine-containing compound and the tripolyphosphate anion in a beta-elimination reaction mechanism. The structures of the adenine-containing compound and its borohydride reduction product were determined. The adenine-containing elimination product inhibited the mitochondrial ATPase activity at a rate greater than that observed with o-ATP. The nature and mechanism of the inhibition of ATPase activity exerted by o-ATP and the elimination product were examined. The significance of the beta-elimination reaction to the use of periodate-oxidized nucleotides as affinity labels of nucleotide binding sites on other proteins is discussed.

Adenosine Diphosphate↗

PubMed1979

The effect of label affinity on the sensitivity and specificity of a hapten radioimmunoassay: a comparison of three [125I]diphenylhydantoin radioligands with the 14C-labelled drug.

The effect on the sensitivity and specificity of a radioimmunoassay for diphenylhydantoin (DPH)has been investigated using three 125I-labelled tyrosine ester derivatives of DPH having different bridge lengths between the tyrosine moiety and the DPH moiety and 14C-labelled DPH. The results demonstrate that for a hapten which does not completely fill the antibody-binding sites, greatest sensitivity is achieved when the bridge of the iodine label is most dissimilar to that present in the original immunogen, when the hapten and label affinities are nearly equivalent. Greatest specificity is achieved with the label which most resembles the original immunogen. These results illustrate the difficulty of designing satisfactory labels for assays of both high specificity and sensitivity since minimal changes in label structure may produce greatly amplified changes in the subsequent affinity of the label for the antiserum.

Affinity Labels↗

PubMed1984

Further characterization of membrane proteins involved in the transport of organic anions in hepatocytes. Comparison of two different affinity labels: 4,4'-diisothiocyano-1,2-diphenylethane-2,2'-disulfonic acid and brominated taurodehydrocholic acid.

4,4'-Diisothiocyano-1,2-diphenylethane-2,2'-disulfonic acid (H2DIDS) known as an irreversible inhibitor of the anion transport in red blood cells (Cabantchik, Z.I. and Rothstein, A. (1972) J. Membrane Biol. 10, 311-330) blocks also the uptake of bile acids and of some foreign substrates in isolated hepatocytes (Petzinger, E. and Frimmer, M. (1980) Arch. Toxicol. 44, 127-135). [3H]H2DIDS was used for labeling of membrane proteins probably involved in anion transport of rat liver cells. The membrane proteins modified in vitro by [3H]H2DIDS were compared with those labeled by brominated taurodehydrocholic acid. The latter is one of a series of suitable taurocholate derivatives, all able to bind to defined membrane proteins of hepatocytes and also known to block the uptake of bile acids as well as of phallotoxins and of cholecystographic agents (Ziegler, K., Frimmer, M., Möller, W. and Fasold, H. (1982) Naunyn-Schmiedeberg's Arch. Pharmacol. 319, 254-261). The radiolabeled proteins were compared after SDS-electrophoresis with and without reducing agent present, solubilization by detergents, two-dimensional electrophoresis and after separation of integral and peripheral proteins. Our results suggest that the anion transport system of liver cells cannot distinguish between bile acids and the anionic stilbene derivative (DIDS). The labeling pattern for both kinds of affinity labels was very similar. Various combinations of separation techniques gave evidence that the radiolabeled membrane proteins are not subunits of a single native channel protein.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1984

Affinity labeling of the aldehyde site of bacterial luciferase.

2-Bromo[1-14C]1-decanal was synthesized as an affinity labeling probe for the aliphatic aldehyde site of Vibrio harveyi luciferase. In the presence of excess amounts of this probe, the inactivation of bacterial luciferase occurred following apparent first order kinetics. This inactivation was markedly retarded in the presence of decanal but neither butanal (a very poor aldehyde substrate) nor FMN (a reaction product derived from reduced FMN) showed any significant protective effect. Upon mixing luciferase with the affinity labeling probe, a noncovalent complex was formed prior to the covalent attachment. At pH 6 and 23 degrees C, the dissociation constant for the binding step and the rate constant for the covalent modification step were determined to be 23 microM and 1 min-1, respectively. The displacement of a bound aldehyde substrate by this probe added secondarily was also demonstrated. The inactivation of luciferase was correlated with both the incorporation of about 1.2 molecules of the probe and the loss of 0.8 to 1.1 cysteinyl residues/luciferase alpha beta dimer. The presence of an essential sulfhydryl group at the aldehyde site of luciferase has thus been demonstrated. This sulfhydryl group was a constituent residue of the alpha subunit and was near the alpha beta subunit interface. This residue appears to be the same essential cysteinyl group previously identified by chemical modification (Nicoli, M.Z., Meighen, E.A., and Hastings, J.W. (1974) J. Biol. Chem. 249, 2385-2392). The labeled luciferase did not exhibit any significant binding for the reduced FMN substrate.

Affinity Labels↗

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↗

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↗