Christian Hirth

Sequence determination of a peptide fragment from electric eel acetylcholinesterase, involved in the binding of quaternary ammonium

Specific photoaffinity labelling of purified electric eel acetylcholinesterase by 3H‐labelled p‐(N,N‐dimethyl‐amino) benzenediazonium fluoroborate allows the identification of a labelled peptide fragment which is described as being involved in the binding of quaternary ammonium ions on this enzyme. Denaturation and proteolytic cleavage of the inactivated enzyme gave a mixture of peptide fragments. The purification of one labelled fragment, containing over 15% of the radioactivity incorporated in the enzyme, led to the following sequence: Gly‐Ser‐X‐Phe. The relatively low amount of this tetrapeptide did not allow us to determine the nature of the labelled residue X.

A photoaffinity ligand of the acetylcholine-binding site predominantly labels the region 179–207 of the α-subunit on native acetylcholine receptor from Torpedo marmorata

Regions of the Torpedo marmorata acetylcholine receptor (AChR) α‐subunit involved in the binding of acetylcholine were probed with two different covalent ligands. The sulfhydryl‐directed affinity reagent 4‐(N‐maleimido)phenyltrimethylammonium iodide labeled a single α‐subunit cyanogen bromide fragment on the reduced AChR which was identified as α 179–207. The novel photoaffinity ligand p‐(N,N‐dimethylamino)‐benzenediazonium fluoroborate, on the other hand, labeled three distinct α‐chain cyanogen bromide fragments on the unmodified AChR in a carbamylcholine‐protectable manner. The major radiolabeled species was purified and identified by sequence analysis as α 179–207. The acetylcholine‐binding site on the native AChR may thus involve several distinct portions of the α‐chain, with the region α 179–207 making a major contribution to the site.

A μ-opioid receptor-filter assay: Rapid estimation of binding affinity of ligands and reversibility of long-lasting ligand-receptor complexes

A filter-associated binding technique, originally described by Leysen and Gommeren [J. Receptor Res. 4, 817 (1984); Drug Dev. Res. 8, 119 (1986)], was focused on the study of mu-opioid receptor sites. The interesting binding features of 3H-Sufentanil, a mu-selective radioligand, permitted such a filter procedure to be performed. Its application was 3-fold. A 5-min binding assay allowed us to verify that specific 3H-Sufentanil binding to filter-absorbed rat brain membranes was endowed with the known kinetic and equilibrium binding properties of membrane-bound mu-opioid receptor sites. Moreover, the filtration technique allowed us to get rapid information about the dissociation rates of unlabelled compounds from the receptor sites. In practice, membranes, preincubated with high concentrations of cold drugs, were absorbed to filters. Dissociation was achieved by repeatedly applying buffer samples on the filter, and monitored by the recovery in free specific 3H-Sufentanil binding sites. Finally, such a dissociation procedure, improved by a washing buffer at high ionic strength, was found to be much more efficient and attractive than the classical dilution-centrifugation procedure, especially for slowly-dissociating compounds. Special attention was paid to the discrimination between pseudo-irreversible binding of drugs and stable covalent labelling of mu-opioid receptor sites (either by affinity or photoaffinity probes), particularly when unlabelled ligands were the only tools available.

Pathogenie de l'hepatite toxique de la souris provoquee par le fv 3 (frog virus 3). Inhibition de la synthese des macromolecules du foie.

Abstract Pathogenesis of the toxic hepatitis of mice provoked by FV 3 (frog virus 3): inhibition of liver macromolecular synthesis Intraperitoneal inoculation of FV 3 (frog virus 3) into mice produces an acute degenerative hepatitis. This hepatitis is due to a toxic effect of the virus which is not able to multiply at 37 °C. In order to elucidate the pathogeny of the lesions, the synthesis of the liver macromolecules has been studied. The following observations were made: (1) a rapid inhibition of the synthesis of RNA, DNA and cellular proteins, (2) an inhibition of [ 3 H]orotic acid incorporation in the nucleoplasmic and nucleolar RNA, (3) an inhibition of [ 3 H]orotic acid incorporation in the cytoplasmic ribosomal and messenger RNA as early as 3 h after infection, (4) a considerable decrease of the RNA polymerases activity of isolated nuclei, (5) a late breakdown of the polysomes. These macromolecular perturbations may explain the morphological and physiological alterations noticed.

m-sulfonate benzene diazonium chloride : a powerful affinity label for the γ-aminobutyric acid binding site from rat brain

Abstract: m‐Sulfonate benzene diazonium chloride (MSBD) was used to affinity‐label the γ‐aminobutyric acid (GABA) binding site from rat brain membranes. To assess the irreversibility of the labeling reaction, we used an efficient ligand dissociation procedure combined to a rapid [3H]muscimol binding assay, both steps being performed on filter‐adsorbed membranes. Inactivation of specific [3H]‐muscimol binding sites by MSBD and its prevention by GABA were both time‐ and concentration‐dependent. The time course of MSBD labeling was shortened as the pH of the incubation medium was increased from 6.2 to 8. These data suggest that MSBD can efficiently label the GABA binding site through alkylation of a residue having an apparent dissociation constant around neutrality.

p-Butyroxybenzenediazonium fluoroborate, substrate of acetylcholinesterase and butyrylcholinesterase, discriminates between the two enzymes by a specific affinity labelling

p-Butyroxybenzenediazonium fluoroborate 1 was shown to be a substrate of both acetylcholinesterase (AcChE) and butyrylcholinesterase (BuChE) with Michaelis constants of 6.10(-5) M and 1.3. 10(-4)M, respectively. Upon incubation in the dark, 1 was able to discriminate between the two enzymes AcChE was efficiently inactivated in a time-dependent manner while BuChE remained unaffected. Kinetic analysis of the inactivation of AcChE (i) by various concentrations of 1 indicated that it behaves as an affinity label, (ii) at three different pH levels suggested that the pKa of the labelled residue was higher than 7 and (iii) in the presence of different selective ligands for either the active site (edrophonium) or the peripheral site (propidium) indicated that 1 alkylated the active site rather than the peripheral one. Differences of reactivity between AcChE and BuChE suggest a different positioning and/or a different chemical environment of the substrate within two active sites.

Marquage irreversible des recepteurs des opioides a l'aide de sels d'aryldiazonium derives du fentanyl

The synthesis of several fentanyl derivatives is reported. The binding affinity of these compounds to membrane opioid receptors is also presented. Several potentially irreversible aryldiazonium ligands were tested for irreversible inactivation of opioid receptors. Compound 13 was shown to inactivate up to 50 % of rat brain opioid receptors, in a ligand-protectable manner. It is therefore a potentially efficient affinity probe for these receptors.

3-chloroacetylpyridine adenine dinucleotide phosphate, an alkylating analogue of NADP+

An alkylating analogue of NADP+ the 3-chloroacetylpyridine adenine dinucleotide phosphate was prepared from 3-diazoacetylpyridine adenine dinucleotide phosphate which was obtained by enzymatic transglucosidation of NADP+. The 3-diazoacetylpyridine adenine dinucleotide phosphate proved to be more unstable when compared to the corresponding NAD+ analogue. The alkylation of several dehydrogenases using this alkylating analogue is mentioned.

Light-induced catalytic inhibition of acetylcholine esterase.

Catalytic enzyme inhibitors are defined as chemically-inert substrate analogs. Inhibition of the enzyme is due to exclusive active-site alkylation by a species generated after the enzymatic reaction. These inhibitors, first discovered by Bloch and his group [l] have the potential advantage of being more specific than the classical affinity-labelling inhibitors. Most of these inhibitors have been developed for enzymes involving proton abstraction in the first catalytic steps [2]. More recently glucotriazene [3] and N-nitrosolactam [4] have been used as catalytic active-site inhibitors of @galactosidase and chymotrypsin, respectively, by in-situ generation of a carbonium ion from an alkyl diazonium. As well, acylated chymotripsin carrying a photolabile group in the active site was described some years ago [S]. In this paper we describe a new type of inhibitor which combines the reaction mechanisms of enzyme catalysed transformation and product photolability. We relate here the irreversible, light-promoted, catalytic inhibition of acetylcholine esterase (EC 3.1.1.7) by methyl (acetoxymethyl) nitrosamine (DMN-OAc) L (flg.1).

Novel Photoactivatable Agonist of the Nicotinic Acetylcholine Receptor of Potential Use for Exploring the Functional Activated State

Abstract: The nicotinic acetylcholine receptor (AChR) exhibits at least four different conformational states varying in affinity for agonists such as acetylcholine (ACh). Photoaffinity labeling has been previously used to elucidate the topography of the AChR. However, to date, the photosensitive probes used to explore the cholinergic binding site photolabeled only closed or desensitized states of the receptor. To identify the structural modifications occurring at the ACh binding site on allosteric transition associated with receptor activation, we have investigated novel photoactivatable 4‐diazocyclohexa‐2,5‐dienone derivatives as putative cholinergic agonists. Such compounds are fairly stable in the dark and generate highly reactive carbenic species on irradiation. In binding experiments using AChRs from Torpedo marmorata, these ligands had affinities for the ACh binding site in the micromolar range and did not interact with the noncompetitive blocker site (greater than millimolar affinity). Irreversible photoinactivation of ACh binding sites was obtained with the ligand 1b (up to 42% at 500 µM) in a protectable manner. In patch‐clamp studies, 1b was shown to be a functional agonist of peripheral AChR in TE 671 cells, with the interesting property of exhibiting no or very little desensitization even at high concentrations.