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PubMed1984

Synthesis of affinity-label chelates: a novel synthetic method of coupling ethylenediaminetetraacetic acid to amine functional groups.

An affinity-label chelate for the enzyme trypsin was synthesized by a novel synthetic technique which takes advantage of the presence of a dangling carboxylate arm in the [Co(EDTA)Cl]2- complex anion. The dangling carboxylate group was coupled to the amino group of p-aminobenzamidine, an effective inhibitor of trypsin activity, via the carbodiimmide reaction to produce a trypsin affinity label at one end and a strong EDTA-like chelating agent at the other, coupled through an amide bond. The cobalt ion can be removed if desired by reduction with Fe2+ + ascorbate, and alternate metal ions inserted in its place. The reaction is general, and affinity labels which contain amino groups can be easily coupled via this procedure, allowing the introduction of a paramagnetic or fluorescent probe into a protein or nucleotide system. The same method has been used to prepare a highly effective chelating gel which is capable of removing calcium and lanthanide ions from the binding protein parvalbumin.

Affinity Labels↗
PubMedDate not supplied

[Study of the possibility of identifying the structural elements of the phenylalanyl-tRNA-synthetase active center by affinity labeling].

The possibility of localization of active sites structural components by affinity labelling was investigated. The modification of E. coli MRE-600 phenylalanyl-tRNA synthetase (E.C.6.1.1.20) (alpha 2 beta 2-type) by the phosphorylating analog of ATP-- [14C]adenosine-5'-trimetaphosphate results in the labelling of both heavy (beta) and light (alpha) enzyme subunits. Analysis of the peptide maps of the tryptic enzyme hydrolysate reveals a great number of peptides containing [14C]radioactivity. The decrease of covalent binding at low concentration of the analog did not abolish the plural labelling. The data permit to consider this kind of analogs as unperspective for localization of specific peptides. Modification of phenylalanyl-tRNA synthetase by tRNAPhe containing the photoreactive group (--CH2CONHC6H5N3) at eighth position of molecule (S8U) results in the labelling of only heavy beta-subunits. These data correspond to the previous results which testify to the disposition of tRNA binding sites on beta-subunits of phenylalanyl-tRNA synthetase. After hydrolysis of the modified phenylalanyl-tRNA synthetase by trypsin six peptides covalently bound with tRNAPhe were revealed. This quantity of modified peptides is higher than the number of tRNA binding sites. Hence the method of affinity labelling has definite limitations for localization of peptides of enzyme active sites.

Affinity Labels↗
PubMed1989

Affinity labeling of the folate-methotrexate transporter from Leishmania donovani.

An affinity labeling technique has been developed to identify the folate-methotrexate transporter of Leishmania donovani promastigotes using "activated" derivatives of the ligands. These "activated" derivatives were synthesized by incubating folate and methotrexate with a 10-fold excess of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) for 10 min at ambient temperature in dimethyl sulfoxide. Preincubation of intact cells with nonradioactive "activated" folate or methotrexate at a concentration of 40 microM inhibited the capacity of wild-type cells to transport submicromolar concentrations of unmodified ligand. When intact wild-type (DI700) Leishmania donovani or preparations of their membranes were incubated with a 0.4 microM concentration of either "activated" [3H]folate or "activated" [3H]methotrexate, the radiolabeled ligands were covalently incorporated into a polypeptide with a molecular weight of approximately 46,000, as demonstrated by SDS-polyacrylamide gel electrophoresis. No affinity labeling of a 46,000-dalton protein was observed when equimolar concentrations of "activated" radiolabeled ligands were incubated with intact cells or membranes prepared from a methotrexate-resistant mutant clone of Leishmania donovani, MTXA5, that is genetically defective in folate-methotrexate transport capability [Kaur, K., Coons, T., Emmett, K., & Ullman, B. (1988) J. Biol. Chem. 263, 7020-7028]. However, some labeling of a 46,000-dalton protein was observed when MTXA5 cells were incubated with higher concentrations of "activated" ligands. Time course studies indicated that maximal labeling of the 46,000-dalton protein occurred within 5-10 min of incubation of intact cells with "activated" ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1985

Determination of the free-energy coupling between ATP and an affinity label attached to rabbit muscle phosphofructokinase.

The smallest enzymatically active form of rabbit muscle phosphofructokinase is a tetramer of four identical or nearly identical monomers. The enzyme is inhibited by ATP, and this inhibition by ATP is relieved by the activating adenine nucleotides adenosine cyclic 3',5'-phosphate, AMP, and ADP. Each monomer contains one binding site specific for the inhibitor ATP and another site specific for the activating adenine nucleotides. The enzyme can also be activated by covalently labeling the activating adenine nucleotide binding sites with the affinity label 5'-[p-(fluorosulfonyl)benzoyl]adenosine. These activator binding sites on the enzyme have been covalently labeled to various degrees, ranging from an average value of less than one label per tetramer to four labels per tetramer, and the free-energy coupling, delta Gxy, between the covalently bound affinity label and ATP binding at the inhibitory site was determined. For enzyme preparations containing four labels per tetramer, delta Gxy is approximately 1 kcal/mol at pH 6.95 and 25 degrees C. A very significant free-energy coupling is observed in those preparations containing an average of one label per tetramer and less, and the change in delta Gxy in going from native tetramers to ones containing an average of two labels per tetramer is twice as great as the change in delta Gxy observed in going from tetramers containing an average of two labels per tetramer to ones containing four labels per tetramer, suggesting that modification of the final two monomers in the tetramer contributes much less to the antagonistic effect on ATP binding than does modification of the first two monomers in the tetramer.

Adenosine↗
PubMed1981

Structural similarities between human receptors for somatomedin C and insulin: analysis by affinity labeling.

Human placental receptors for insulin and somatomedin C (Sm-C) were affinity labeled with [125I]insulin and [125I]Sm-C by using the bifunctional cross-linking agent disuccinimidyl suberate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that both labeled hormones were specifically cross-linked to three protein species with apparent molecular weights of 240 000, 310 000, and 330 000. Following disulfide bond reduction, subunits of approximately 140 000 daltons were evident. Partial reduction of disulfide bonds yielded intermediate-sized species with apparent molecular weights of 180 000, suggesting the existence of an additional, smaller subunit attached to the 140 000-dalton subunit. Limited proteolysis of the hormone-receptor complexes with chymotrypsin, trypsin, and Staphylococcus aureus V-8 protease gave similar but not identical results for each labeled receptor. The distinction between the two receptors was further documented by inhibition of affinity labeling with graded amounts of the native hormones. These data demonstrate a substantial structural similarity between the human Sm-C and insulin receptors paralleling the homology of the native hormones and their actions.

Affinity Labels↗
PubMed1994

Affinity labeling of aryl sulfotransferase IV. Identification of a peptide sequence at the binding site for 3'-phosphoadenosine-5'-phosphosulfate.

2'-O-[(R)-Formyl(adenin-9-yl)-methyl]-(S)-glyceraldehyde 3'-triphosphate (also designated as ATP dialdehyde or ATPDA) was utilized as an affinity label for the 3'-phosphoadenosine 5'-phosphosulfate (PAPS) binding site of an aryl sulfotransferase. The sulfotransferase employed in these studies was rat hepatic aryl sulfotransferase (AST) IV (also known as tyrosine-ester sulfotransferase, EC 2.8.2.9), for which a cDNA had been previously cloned and expressed in Escherichia coli and the resulting enzyme purified to homogeneity. ATPDA was a time-dependent irreversible inhibitor of the recombinant AST IV, and this inhibition was prevented by including either PAPS or adenosine 3',5'-diphosphate (PAP) in the incubation of AST IV with ATPDA. Experiments relating covalent binding of [2,8-3H]ATPDA with catalytic activity indicated that 1 nmol of the affinity label was bound per nmol of AST IV subunit. Incubation of [2,8-3H]ATPDA with the enzyme followed by reduction with sodium cyanoborohydride, proteolysis with trypsin, and separation of the resulting peptides by high pressure liquid chromatography yielded two labeled peptide fractions. Automated sequence analysis showed that both modified peptide fractions were derived from the same sequence in AST IV: 63-Leu-Glu-Lys-Cys-Gly-Arg-68. Both the sequencing results and examination of the two peptide fractions by matrix-assisted laser desorption ionization mass spectrometry indicated that the ATPDA affinity label was bound to the hexapeptide at both lysine 65 and cysteine 66. These affinity labeled amino acids are located within a region of sequence in AST IV that shows considerable homology with various sulfotransferases that possess diverse specificities for acceptor substrates, and this may provide insight into PAPS binding in other sulfotransferases.

Adenosine Triphosphate↗
PubMed1986

Synthesis of diazomethyl beta-D-galactopyranosyl and beta-D-glucopyranosyl ketones. Potential affinity-labeling reagents for carbohydrate-binding proteins.

3,7-Anhydro-1-deoxy-1-diazo-D-glycero-L-manno-2-octulose (6a; diazomethyl beta-D-galactopyranosyl ketone) and 3,7-anhydro-1-deoxy-1-diazo-D-glycero-D-gulo-2-octulose (6b; diazomethyl beta-D-glucopyranosyl ketone) have been prepared. Readily available C-glycosyl compounds possessing the appropriate stereo-chemistry and hydroxyl-group protection, viz., per-O-acetyl-2,6-anhydroheptononitriles and per-O-acetyl-2,6-anhydroheptonamides, were employed as precursors to per-O-acetyl-2,6-anhydroheptonic acids. These key intermediates were then converted into mixed carboxylic-carbonic acid anhydrides, and these caused to react with diazomethane, to give the corresponding per-O-acetyl-3,7-anhydro-1-deoxy-1-diazo-2-octuloses. Zemplén deacetylation gave, stereospecifically, the crystalline target-molecules in good overall yield. It is proposed that such C-glycosyl compounds as 6a and 6b, which possess the diazoacetyl functional groups as their "aglycon", will be useful as enzyme-activated irreversible inhibitors (suicide substrates) of glycosidases, and as photoaffinity-labeling reagents and classical affinity-labeling reagents for carbohydrate-binding proteins.

Affinity Labels↗
PubMed1989

Identification of cysteine 530 as the covalent attachment site of an affinity-labeling estrogen (ketononestrol aziridine) and antiestrogen (tamoxifen aziridine) in the human estrogen receptor.

Radiosequence analysis of peptide fragments of the estrogen receptor (ER) from MCF-7 human breast cancer cells has been used to identify cysteine 530 as the site of covalent attachment of an estrogenic affinity label, ketononestrol aziridine (KNA), and an antiestrogenic affinity label, tamoxifen aziridine (TAZ). ER from MCF-7 cells was covalently labeled with [3H]TAZ or [3H]KNA and purified to greater than 95% homogeneity by immunoadsorbent chromatography. Limit digest peptide fragments, generated by prolonged exposure of the labeled receptor to trypsin, cyanogen bromide, or Staphylococcus aureus V8 protease, were purified to homogeneity by high performance liquid chromatography (HPLC), and the position of the labeled residue was determined by sequential Edman degradation. With both aziridines, the labeled residue was at position 1 in the tryptic peptide, position 2 in the cyanogen bromide peptide, and position 7 in the V8 protease peptide. This localizes the site of labeling to a single cysteine at position 530 in the receptor sequence. The identity of cysteine as the site of labeling was confirmed by HPLC comparison of the TAZ-labeled amino acid (as the phenylthiohydantoin and phenylthiocarbamyl derivatives) and the KNA-labeled amino acid (as the phenylthiocarbamyl derivative) with authentic standards prepared by total synthesis. Cysteine 530 is located in the hormone binding domain of the receptor, near its carboxyl terminus. This location is consistent with earlier studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to analyze the size of the proteolytic fragments containing the covalent labeling sites for TAZ and KNA and the antigen recognition sites for monoclonal antibodies. The fact that both the estrogenic and antiestrogenic affinity labeling agents react covalently with the same cysteine indicates that differences in receptor-agonist and receptor-antagonist complexes do not result in differential covalent labeling of amino acid residues in the hormone binding domain.

Affinity Labels↗
PubMed1981

Nicotinamide adenine dinucleotide binding and promotion of enzyme activity: model based on affinity labeling of 3 alpha, 20 beta-hydroxysteroid dehydrogenase with a nucleoside.

5'-[p-(Fluorosulfonyl)benzoyl]adenosine (FSA) was used to affinity-label the NADH binding region of 3 alpha, 20 beta-hydroxysteroid dehydrogenase (3 alpha, 20 beta-HSD) to further test our hypothesis [Sweet, F., & Samant, B. R. (1980) Biochemistry 19, 978-986] that 3 alpha and 20 beta activities occur at the same active site. Incubation of 3 alpha, 20 beta-HSD (0.45 microM) with FSA (125 microM) at pH 7.0 and 0 degrees C caused simultaneous loss of 3 alpha and 20 beta activities by a first-order kinetic process, with t1/2 = 300 min for both activities. Dinucleotides and adenosine mononucleotides which acted as competitive inhibitors protected 3 alpha, 20 beta-HSD against inactivation by FSA in a concentration-dependent manner, in the order reduced nicotinamide dinucleotide phosphate greater than oxidized nicotinamide dinucleotide phosphate greater than adenosine diphosphate-ribose greater than adenosine diphosphate greater than adenosine monophosphate (AMP) greater than adenosine. Oxidized and reduced nicotinamide mononucleotides (NMH and NMNH) and steroid substrates did not protect 3 alpha, 20 beta-HSD against affinity labeling by FSA. Although NMN was not a competitive inhibitor of 3 alpha, 20 beta-HSD, NMN with AMP and also AMP with NMNH produced positive cooperativity for competitive inhibition of 3 alpha, 20 beta-HSD. The results from FSA affinity labeling of the cofactor region confirm that both 3 alpha and 20 beta activities share the same active site of 3 alpha, 20 beta-HSD and suggest a model of cofactor binding and promotion of enzyme activity. The adenosine 5'-phosphate component anchors the NAD or NADH to an adenosine domain in the cofactor binding region. The nicotinamide nucleotide component then carries out the hydrogen-transfer reaction at a neighboring domain near the steroid binding region.

20-Hydroxysteroid Dehydrogenases↗
PubMed1983

Rabbit muscle phosphofructokinase. 2. Inactivation by the affinity label 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine.

The reaction of the fluorescent affinity label 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine with rabbit skeletal muscle phosphofructokinase results in an inactivation of the enzyme and in the covalent incorporation of up to one label/monomer. The substrates, MgATP and fructose 6-phosphate, each protect against inactivation of the enzyme, but neither diminishes the extent of covalent incorporation of the label, indicating that the inactivation is not the result of covalent incorporation of the label. Dithiothreitol reactivates the inactivated enzyme but does not reduce the extent of incorporation of the label. A determination of the number of free sulfhydryl groups on the enzyme as a function of the extent of inactivation by the reagent suggests that the inactivation is associated with the loss of two free sulfhydryl groups per phosphofructokinase monomer. The inactivation reaction appears to involve the reversible formation of an enzyme-reagent complex (Kd = 1.11 mM) prior to the conversion of the complex to inactive enzyme (k1 = 0.98 min-1). In view of the protection afforded by either substrate and the evidence suggesting the formation of an enzyme-reagent complex prior to inactivation, it would appear that the inactivation results from a reagent-mediated formation of a disulfide bond between two cysteinyl residues in close proximity, possibly in or near the catalytic site of the enzyme. The site of covalent attachment of the label appears to be the binding site specific for the activating adenine nucleotides cAMP, AMP, and ADP. The extent of covalent incorporation of the label at this site is diminished in the presence of cAMP, and phosphofructokinase modified at this site by this affinity label is no longer subject to activation by cAMP.

Adenosine↗
PubMed1984

The synthesis and reaction of a specific affinity label for the hydrophobic drug-binding domains of calmodulin.

An affinity-labeling reagent for the two hydrophobic drug-binding domains of calmodulin has been prepared and its reaction with calmodulin characterized. The reagent, 10-(3-propionyloxysuccinimide)-2-(trifluoromethyl)phenothiazine, was shown to be very specific labeling reagent for these domains. Its specificity was demonstrated by the following observations. 1) Previous reports have shown that Ca2+ is required for phenothiazine binding to calmodulin, and here we show that the affinity-labeling reagent reacts with and inactivates calmodulin in the presence of Ca2+, but not in its absence. 2) Inclusion of trifluoperazine, fluphenazine, W-7, or 10-(3-aminopropyl)-2-(trifluoromethyl)phenothiazine in the reaction mixture protected calmodulin from inactivation by the reagent. 3) Inactivation by the reagent yielded calmodulin that was no longer retained on a phenothiazine-Sepharose column under conditions in which unreacted calmodulin was retained. 4) The measured stoichiometry of the reaction in the presence of excess reagent was 2.1 mol of reagent per mol of calmodulin which agrees well with previous reports of two high-affinity phenothiazine-binding sites on calmodulin. 5) The stoichiometry of the reaction was further confirmed by tryptic peptide maps which show two phenothiazine-labeled peptides unique to the fully reacted protein. 6) The spectral properties of the reagent, while attached to calmodulin, change in the presence of Ca2+ in a manner consistent with the known effects of Ca2+ binding by calmodulin on these hydrophobic domains. The specificity of the reagent makes it useful for further characterization of these hydrophobic binding domains on calmodulin.

Affinity Labels↗
PubMed1991

Activation of reconstituted Escherichia coli outer-membrane phospholipase A by membrane-perturbing peptides results in an increased reactivity towards the affinity label hexadecanesulfonyl fluoride.

The activity of the Escherichia coli outer-membrane phospholipase (OM PLA) is strictly regulated in its natural habitat, the E. coli outer membrane. OM PLA can be reconstituted in phospholipid bilayers, resulting in low specific activity of the enzyme compared to its activity on mixed lipid/detergent micelles. The enzyme can be activated by the addition to these vesicles of the membrane-perturbing peptides polymyxin B, melittin or cardiotoxin resulting in hydrolysis of mainly the sn-1 ester bond of the phospholipids as is also observed in vivo. We used the affinity label hexadecanesulfonyl fluoride to probe the influence of lipid environment on the activity of OM PLA. In detergent and substrate micelles, the rate constant for the sulfonylation of the active-center serine of the purified OM PLA by the affinity label hexadecanesulfonyl fluoride depends on amphiphile concentration. We have reported a similar influence of amphiphile concentration on the activity of the enzyme [Horrevoets, A. J. G. et al. (1989) Biochemistry 28, 1139-1147]. Analysis of the rates of inactivation of OM PLA by hexadecanesulfonyl fluoride in vesicles composed of various phospholipids indicated that activation of the enzyme by membrane-perturbing peptides can be accurately quantified with this affinity label. Our results show that the affinity label hexadecanesulfonyl fluoride can be used to monitor the state of activation of OM PLA in different lipid environments, including non-hydrolyzable substrate analogues. Implications for the in vivo situation are discussed.

Affinity Labels↗
PubMed1989

Affinity labeling of the active center and ribonucleoside triphosphate binding site of yeast DNA primase.

A highly selective affinity labeling procedure has been applied to map the active center of DNA primase from the yeast Saccharomyces cerevisiae. Enzyme molecules that have been modified by covalent attachment of benzaldehyde derivatives of adenine nucleotides are autocatalytically labeled by incubation with a radioactive ribonucleoside triphosphate. The affinity labeling of primase requires a template DNA, is not affected by DNase and RNase treatments, but is sensitive to proteinase K. Both the p58 and p48 subunits of yeast DNA primase appear to participate in the formation of the catalytic site of the enzyme, although UV-photocross-linking with [alpha-32P]ATP locates the ribonucleoside triphosphate binding site exclusively on the p48 polypeptide. The fixation of the radioactive product has been carried out also after the enzymatic reaction. Under this condition the RNA primers synthesized by the DNA polymerase-primase complex under uncoupled DNA synthesis conditions are linked to both DNA primase and DNA polymerase. When DNA synthesis is allowed to proceed first, the labeled RNA chains are fixed exclusively to the DNA polymerase polypeptide. These results, in accord with previous data, have been used to propose a model illustrating the interactions and the putative roles of the polypeptides of the DNA polymerase-primase complex.

Adenosine Triphosphate↗
PubMed1975

Affinity labeling of catechol O-methyltransferase by N-haloacetyl derivatives of 3,5-dimethoxy-4-hydroxyphenylethylamine and 3,4-dimethoxy-5-hydroxyphenylethylamine. Kinetics of inactivation.

In an attempt to elucidate the relationship between the chemical structure and the catalytic function of catechol O-methyltransferase (COMT), several classes of affinity labeling reagents have been synthesized and their interaction with COMT has been studied. Earlier studies have shown that various N-haloacetyl derivatives of 3,5-dimethoxy-4-hydroxyphenylethylamine were effective affinity labeling reagents for this enzyme. In this report we have shown that N-haloacetyl derivatives of the isomeric 3,4-dimethoxy-5-hydroxyphenylethylamine also rapidly and irreversibly inactivate COMT ant they satisfy many of the criteria established for affinity labeling reagents. This latter group of agents appear to modify a nucleophilic residue at the active site of COMT different from that modified by the 3,5-dimethoxy-4-hydroxyphenylethylamine series. Evidence to support this conclusion has been obtained by comparing the kinetics of COMT inactivation and the substrate protection profiles for these two classes of affinity labeling reagents.

Animals↗
PubMed1981

Affinity labelling of the human uterine progesterone receptor with 21-, 16 alpha- and 11 alpha-bromoacetoxyprogesterones.

This report describes the use of 21-, 16 alpha- and 11 alpha -[2'-3H]bromoacetoxyprogesterone as affinity labels to characterize the human uterine progesterone receptor (HPR). These three derivatives can bind to and displace progesterone bound to the HPR. This affinity labelling was inhibited by an excess of radioinert progesterone and could not be demonstrated if bovine serum albumin was used in place of the HPR. Bromoacetic acid alone did not affinity label the HPR. Polyacrylamide gel electrophoresis under denaturing conditions showed that all three derivatives bound to a 45,000 molecular weight protein.

Affinity Labels↗
PubMed1977

Affinity labelling of human transcortin.

The binding site of transcortin has been studied by using bromoacetyltestosterone and bromoacetylated derivatives of progesterone which were monohydroxylated at different positions of the steroid nucleus. Specificity of affinity labelling was demonstrated by the displad cortisol analog was added to a [3H]cortisol-transcortin complex solution. The binding site crevice was found to be very narrow in the vicinity of the A and B rings of steroid since 2alpha-hydroxyprogesterone, 6alpha- or 6beta-bromoacetoxyprogesterone and dexamethasone could not displace bound cortisol. A specific affinity labelling was obtained with 11alpha-bromoacetoxyprogesterone, 16alpha-bromoacetoxyprogesterone and 17beta-bromoacetyltestosterone. The results of the affinity labelling by these hormone analogs suggested that one methionine and one histidine residues were located within the active site:methionine might interact with the 11beta-hydroxyl group and histidine with the 20 keto group of cortisol.

Affinity Labels↗
PubMedDate not supplied

Glyoxylate for affinity labelling of 6-phosphogluconate dehydrogenase.

In order to find a new reagent for the affinity labelling, 6-phosphogluconate dehydrogenase was treated with glyoxylate, a versatile metabolite with a carboxyl and a reactive aldehydic group. High concentrations of glyoxylate inhibit the enzyme, while in the presence of the reducing agent cyanoborohydride, the enzyme is irreversibly inactivated by only millimolar glyoxylate. This indicates the formation of a Schiff base between the aldehydic group of glyoxylate and one enzyme lysine residue. The kinetics and substrate competition suggest that inactivation is due to affinity labelling. In the first step the inhibitor carboxylic group binds to the substrate carboxyl binding site, and in the second slower step the aldehydic group binds a nearby lysine. We have also found that other enzymes are inactivated by the combined actions of glyoxylate and cyanoborohydride, with a saturation kinetics. Hence, glyoxylate can be helpful to identify specific lysines at the carboxyl binding sites in proteins.

Affinity Labels↗
PubMed1987

[14C]chloroacetylcholine as an advantageous affinity label of the acetylcholine receptor.

The alkylating agent [14C]chloroacetylcholine perchlorate [( 14C] ClACh) was synthesized and used for affinity labelling of the nicotinic acetylcholine receptor from Torpedo marmorata. Solubilized and affinity-purified receptor proteins were reduced and alkylated according to the bromoacetylcholine-method. Covalent binding of [14C] ClACh to the cholinergic receptor proved to be specific and saturable, and occurred exclusively to the alpha-subunit. Halogen substitution of acetylcholine by chlorine and insertion of a 14C-isotope instead of the widely used 3H resulted in favourable properties of the affinity label.

Acetylcholine↗