Does "butyrylization" of acetylcholinesterase through substitution of the six divergent aromatic amino acids in the active center gorge generate an enzyme mimic of butyrylcholinesterase?

The active center gorge of human acetylcholinesterase (HuAChE) is lined by 14 aromatic residues, whereas in the closely related human butyrylcholinesterase (HuBChE) 3 of the aromatic active center residues (Phe295, Phe297, Tyr337) as well as 3 of the residues at the gorge entrance (Tyr72, Tyr124, Trp286) are replaced by aliphatic amino acids. To investigate whether this structural variability can account for the reactivity differences between the two enzymes, gradual replacement of up to all of the 6 aromatic residues in HuAChE by the corresponding residues in HuBChE was carried out. The affinities of the hexamutant (Y72N/Y124Q/W286A/F295L/F297V/Y337A) toward tacrine, decamethonium, edrophonium, huperzine A, or BW284C51 differed by about 5-, 80-, 170-, 25000-, and 17000-fold, respectively, from those of the wild-type HuAChE. For most of these prototypical noncovalent active center and peripheral site ligands, the hexamutant HuAChE displayed a reactivity phenotype closely resembling that of HuBChE. These results support the accepted view that the active center architectures of AChE and BChE differ mainly by the presence of a larger void space in BChE. Nevertheless, reactivity of the hexamutant HuAChE toward the substrates acetylthiocholine and butyrylthiocholine, or covalent ligands such as phosphonates and the transition state analogue m-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA), is about 45-170-fold lower than that of HuBChE. Most of this reduction in reactivity can be related to the combined replacements of the three aromatic residues at the active center, Phe295, Phe297, and Tyr337. We propose that the hexamutant HuAChE, unlike BChE, is impaired in its capacity to accommodate certain tetrahedral species in the active center. This impairment may be related to the enhanced mobility of the catalytic histidine His447, which is observed in molecular dynamics simulations of the hexamutant and the F295L/F297V/Y337A HuAChE enzymes but not in the wild-type HuAChE.

Bis-catechol-substituted redox-reactive analogues of hexamethonium and decamethonium: stimulated affinity-dependent reactivity through iron peroxide catalysis.

Symmetrically bis-catechol-substituted analogues (1 and 2, respectively) of hexamethonium and decamethonium were synthesized and investigated as redox-activated affinity reagents toward the neurotoxin-binding sites of the nicotinic acetylcholine receptor (nAcChR), purified from Torpedo californica electroplax. These reagents bound to nAcChR with Kd = 1.8 x 10(-8) and 2.3 x 10(-7) M for 1 and 2, respectively. In the presence of a metal, Fe(II)/Fe(III), and peroxide, both reagents produced a rapid and efficient half-of-sites inactivation of neurotoxin-binding sites in the nAcChR in a concentration-dependent manner, which paralleled the extent of receptor binding of the reagents. In the absence of Fe(II)/Fe(III) peroxide, redox-dependent inactivation occurred for both 1 and 2 more slowly and only at concentrations much higher (10(3)-10(4) times) than those necessary to produce significant binding to nAcChR. However, receptor inactivation in the absence of added metal peroxide was still more efficient for 1 and 2 than observed previously for [(trimethylammonio)methyl]catechol (3), the prototypic redox-dependent affinity reagent after which 1 and 2 were patterned. Thus, the new reagents reported are expected to provide more efficient and selective conditions for redox-dependent inactivation at nAcChR and other macromolecular sites to which such reagents may be directed.

Differential effects of "peripheral" site ligands on Torpedo and chicken acetylcholinesterase.

Comparison of the effect of three 'peripheral' site ligands, propidium, d-tubocurarine, and gallamine, on acetylcholinesterase (acetylcholine hydrolase; EC 3.1.1.7) of Torpedo and chicken shows that all three are substantially more effective inhibitors of the Torpedo enzyme than of the chicken enzyme. In contrast, edrophonium, which is directed to the "anionic" subsite of the active site, inhibits the chicken and Torpedo enzymes equally effectively. Two bisquaternary ligands, decamethonium and 1,5-bis(4-allydimethylammoniumphenyl)pentan-3-one dibromide, which are believed to bridge the anionic subsite of the active site and the "peripheral" anionic site, are much weaker inhibitors of the chicken enzyme than of Torpedo acetylcholinesterase, whereas the shorter bisquaternary ligand hexamethonium inhibits the two enzymes similarly. The concentration dependence of activity towards the natural substrate acetylcholine is almost identical for the two enzymes, whereas substrate inhibition of chicken acetylcholinesterase is somewhat weaker than that of the Torpedo enzyme. The experimental data can be rationalized on the basis of the three-dimensional structure of the Torpedo enzyme and alignment of the chicken and Torpedo sequences; it is suggested that the absence, in the chicken enzyme, of two aromatic residues, Tyr-70 and Trp-279, that contribute to the peripheral site of Torpedo acetylcholinesterase is responsible for the differential effects of peripheral site ligands on the two enzymes.

Quaternary ligand binding to aromatic residues in the active-site gorge of acetylcholinesterase.

Binding sites of Torpedo acetylcholinesterase (EC 3.1.1.7) for quaternary ligands were investigated by x-ray crystallography and photoaffinity labeling. Crystal structures of complexes with ligands were determined at 2.8-A resolution. In a complex with edrophonium, and quaternary nitrogen of the ligand interacts with the indole of Trp-84, and its m-hydroxyl displays bifurcated hydrogen bonding to two members of the catalytic triad, Ser-200 and His-440. In a complex with tacrine, the acridine is stacked against the indole of Trp-84. The bisquaternary ligand decamethonium is oriented along the narrow gorge leading to the active site; one quaternary group is apposed to the indole of Trp-84 and the other to that of Trp-279, near the top of the gorge. The only major conformational difference between the three complexes is in the orientation of the phenyl ring of Phe-330. In the decamethonium complex it lies parallel to the surface of the gorge; in the other two complexes it is positioned to make contact with the bound ligand. This close interaction was confirmed by photoaffinity labelling by the photosensitive probe 3H-labeled p-(N,N-dimethylamino)benzenediazonium fluoroborate, which labeled, predominantly, Phe-330 within the active site. Labeling of Trp-279 was also observed. One mole of label is incorporated per mole of AcChoEase inactivated, indicating that labeling of Trp-279 and that of Phe-330 are mutually exclusive. The structural and chemical data, together, show the important role of aromatic groups as binding sites for quaternary ligands, and they provide complementary evidence assigning Trp-84 and Phe-330 to the "anionic" subsite of the active site and Trp-279 to the "peripheral" anionic site.

Acetylcholine receptor-mediated membrane current in oocytes injected with Electrophorus electricus mRNA: analyses of nicotine, succinylcholine, and decamethonium responses on the basis of the minimal model.

Nicotinic acetylcholine receptor was synthesized in Xenopus oocytes after injection of the mRNA purified from Electrophorus electricus electroplax. Nicotine, succinylcholine, and decamethonium (agonist)-elicited membrane currents in the injected oocytes were measured electrophysiologically by the voltage-clamping method. The following four different measurements were made to establish the relationship between the agonist concentration and the membrane current: 1) the agonist-induced membrane current before desensitization, 2) the agonist-induced membrane current after desensitization equilibrium, 3) the fraction of the active form of the receptors after desensitization equilibrium, 4) the rate of recovery of desensitized receptors upon removal of the agonist. These results were analyzed on the basis of the minimal model proposed from receptor-mediated ion translocation measurements. The equilibrium and rate constants of the model were evaluated for nicotine, succinylcholine, and decamethonium, and could explain the observed electrical responses in the injected oocyte, i.e. the characteristics of the receptor response caused by these agonists.

[Pharmacologic characterization of alpha bungarotoxin binding sites on microcellular lung carcinoma cell lines]. / Farmakoloska karakterizacija alfa bungarotosksin vezajucih mjesta na stanicnim linijama mikrocelularnog karcinoma pluca.

Alpha Bungarotoxin binding protein belongs to the group of nicotinic receptors regarding their pharmacologic characteristics. Presence of alpha Bungarotoxin binding sites was confirmed on six cell lines of human microcellular lung carcinomas. The binding sites are saturable, specific and show high affinity for alpha Bungarotoxin. The presence of the binding sites was found on human microcellular lung carcinoma cultured in the mice of "Nude" clones. Using pharmacologically active substances like succinilcholine, decamethonium, atropine and propranolol, the nicotinic characteristics of alpha Bungarotoxin binding sites were confirmed. The presence of alpha 5 subunits in the alpha Bungarotoxin binding protein of human microcellular lung cells was established by Northern blot experiments classifying alpha Bungarotoxin binding protein on microcellular cells as a neuronal nicotinic receptor regarding its structure.

[The study of complex-formation of DNA with the antimicrobial drug decamethoxine]. / Izuchenie kompleksoobrazovaniia DNK s antimikrobnym preparatom dekametoksinom.

The interaction of effective antibacterial drug decametoxyn with natural DNA was studied by UV-spectroscopy. Decametoxyn shows a specificity to nucleotides: it decreases the cooperativity of melting and the thermal stability of DNA parts enriched by AT pairs. The characteristics of the helix-coil transition on the DNA parts enriched by GC-pairs are invariable. Interaction with AT-pairs results in their partial or complete melting at room temperature, followed by intermolecule aggregation. Interacting with phosphates decametoxyn manifests itself not as a dication but as two single-charged ions.

Localization of mechanoreceptors in the protozoan, Stentor coeruleus.

Mechanoreceptor channels were localized by using the ligands, tubocurarine (TC), decamethonium (Deca), and gallamine (Gall), which have been shown to bind specifically to these channels. The binding of radioactively labeled TC (TC*) was found to be directly proportional to the cell surface area suggesting that the channels are uniformly distributed over the cell surface. Intracellular TC and Gall injections did not depress mechanical stimulus sensitivity though these drugs did depress sensitivity when applied extracellularly at the same concentrations; therefore, the ligand binding sites are on or near the external surface of the cell. Autoradiographs revealed that radioactively labeled Deca (Deca*) bound to the pigmented stripes but not to the ciliary stripes or membranellar band. Further, Stentor induced to shed their membranellar band through exposure to 8% urea were more sensitive to mechanical stimuli than were controls; therefore, the membranellar cilia do not appear to contain mechanoreceptor channels. Collectively, these data indicate that the mechanoreceptor channels are located in the somatic surface covering the pigmented stripes. The density of mechanoreceptor channels in the plasma membrane covering the somatic surface is tentatively estimated to be between 5500 and 14,500 microns-2 based on the density of TC* binding, the apparent number of TC molecules binding per mechanoreceptor channel, and data suggesting that only one fifth to one fourth of the bound TC* is bound to structures in the plasma membrane.

Nicotine, cotinine, and anabasine inhibit aromatase in human trophoblast in vitro.

Epidemiologic studies suggest that women who smoke have lower endogenous estrogen than nonsmokers. To explore the possible link between cigarette smoking and decreased endogenous estrogens, we have examined the effects of constituents of tobacco on estrogen production in human choriocarcinoma cells and term placental microsomes. In choriocarcinoma cell cultures, nicotine, cotinine (a major metabolite of nicotine), and anabasine (a minor component of cigarette tobacco) all inhibited androstenedione conversion to estrogen in a dose-dependent fashion. Removal of nicotine, cotinine, and anabasine from the culture medium resulted in the complete reversal of the inhibition of aromatase. In the choriocarcinoma cell cultures, a supraphysiologic concentration of androstenedione (73 microM) in the culture medium blocked the inhibition of aromatase caused by nicotine, cotinine, and anabasine. In preparations of term placental microsomes, nicotine, cotinine, and anabasine inhibited the conversion of testosterone to estrogen. Kinetic analysis demonstrated the inhibition to be competitive with respect to the substrate. These findings suggest that some nicotinic alkaloids directly inhibit aromatase. This mechanism may explain, in part, the decreased estrogen observed in women who smoke.

Interaction of cholinergic ligands with the purified acetylcholine receptor protein. I. Equilibrium binding studies.

We have studied the binding equilibria of two fluorescent ligands and several nonfluorescent cholinergic ligands with the purified acetylcholine receptor from Electrophorus electricus. The assay was based on the specifically cholinergic and reversible quenching of fluorescence observed upon complex formation between the receptor protein and N-7-(4-nitrobenzo-2-oxa-1,3-diazole)-5-aminopentanoic acid beta-(N-trimethylammonium) ethyl ester. This way, a large body of accurate, true equilibrium data was obtained. We find 1) all ligands studied compete for the same number of binding sites at the receptor; 2) agonists compete for half of these sites with high affinity and for the other half of these sites with significantly lower affinity; 3) antagonists compete for all of these sites with one affinity; and 4) in the presence of disulfide reducing agents, the binding patterns of some agonists and antagonists are changed in accordance with the electrophysiological changes observed under the same conditions with Rana pipiens Sartorius muscle fibers. Our studies indicate that the mechanism of ligand recognition is still functional at equilibrium and is not subject to the presence of an intact membrane environment. Furthermore, the existence of two types of agonist sites at every receptor molecule excludes most of the presently discussed two-state models as the basis for a mechanism of receptor-ligand interaction. To explain sigmoidal dose-response curves, a two-site model is already sufficient.

The isolation of endogenous modulators of the affinity of acetylcholinesterase to cholinergic ligands.

By help of a batchwise affinity chromatography procedure the binding of cholinergic ligands to AChE obtained from caudate nucleus from calf brain was studied. The affinity of edrophonium to a crude as compared to a pure enzyme was about 50 times higher. After addition of material isolated from the crude preparation the enzyme was changed to the high affine form. The dissociation constant of the crude enzyme-edrophonium complex determined in the affinity chromatographic experiments was 1.5 X 10(-5) M and in enzymatic experiments 1.8 X 10(-7) M. It is proposed that there is present in mammalian neuronal tissue a factor that increases the affinity of certain cholinergic ligands to a site other than the catalytic site on AChE.

Properties and affinity purification of the mixed-type putative acetylcholine receptor from wild and a mutant strain of hose flies.

Binding of decamethonium to a soluble preparation from house fly head (either wild or a mutant strain) showed a single kind of binding with values for wild strain of Kd = 0.095 micrometers and Bmax = 0.22 nmol/mg protein. The mutant had a four-fold greater affinity and a seven-fold lesser amount. The binding was blocked by both nicotinic and muscarinic drugs. The decamethonium binding migrated in sucrose gradients as a single peak, with sedimentation coefficient s20,w = 12.5 S and therefore a molecular weight of 342 000. Purification by affinity chromatography was achieved with only partial loss of activity, andthe purified material demonstrated a single band on analytical disc gel electrophoresis. Electrophoresis in sodium dodecyl sulphate gels showed two subunits of molecular wegiths 94 000 and 64 000. Both subunits had an isoelectric point of 4.8.

[Study of skeletal muscle cholinoreceptors using alkylating agents]. / Izuchenie kholinoretseptorov skeletnoi myshtsy s pomoshch'iu alkiliruiushchikh agentov.

The ability of decamethonium containing beta-clorethylamino groups to alkylate the nicotinic cholinoreceptors of the frog tonic muscles was studied. D-tubocurarine prevented the action of the alkylating decamethonium (AD). The latter equally inhibited the effects of carbacholine and tetramethylammonium. The degree of alkylation did not change with pH varying from 6 to 11. AD did not produce any parallel shifts, but inhibited at once the maximal response to carbacholine both of the frog intact muscle and of a single tonic fibre. It is suggested that decamethonium blocks the cholinoreceptors anionic sites, which are represented by the carboxylate, or phosphate anions. The frog tonic muscle probably fails to posses any spare receptors.