An enzymatic method for measuring picomole quantities of acetylcholine and choline in CNS tissue

A rapid and sensitive enzymatic assay for measuring picomole quantities of both acetylcholine (ACh) and choline (Ch) in tissue extracts has been developed. After ACh and Ch were extracted into 15% 1 n formic acid/85% acetone by the procedure of Toru and Aprison, lipids were removed by a heptane-chloroform extraction. All quaternary ammonium compounds were isolated by precipitation with periodide. After the precipitate (including ACh and Ch) was dissolved in a known volume of water, aliquots were taken for both assays. In the ACh assay, endogenous Ch was removed after conversion to choline phosphate by choline kinase, whereas ACh was subsequently hydrolyzed by base. In the presence of [¹⁴C]acetyl-CoA and choline acetyltransferase, the choline moiety was converted into [¹⁴C]ACh. The labeled ACh was extracted into sodium tetraphenylboron/butenenitrile and then counted in a scintillation counter. In the Ch assay, the first enzyme reaction step is omitted and only the second is used. The lower limit of sensitivity in both assays is 20 pmoles. Once the tissue has been carried through the extraction step, over eighty determinations can be made in one day. In vivo levels of ACh and Ch in the cerebrum of rats are reported for totally frozen rats and for rats sacrificed by the near-freezing procedure of Takahashi and Aprison. Mean ACh values in the two groups statistically were the same (26.5 ± 2.2 and 25.3 ± 1.7 nmoles/g, respectively) whereas the mean Ch values were significantly different (25.7 ± 0.9 and 64.0 ± 3.6 nmoles/g, respectively). The difference in the Ch levels as well as the importance of specifying the conditions that effect the measurement of ACh and Ch are discussed.     

Evidence for the extreme overestimation of choline acetyltransferase in human sperm, human seminal plasma and rat heart: a case of mistaking carnitine acetyltransferase for choline acetyltransferase

Detection of choline acetyltransferase (ChAc) in a number of non-neuronal tissues has been extremely overestimated. There are two major types of errors encountered. Type 1 error occurs when endogenous substrates (e.g. L-carnitine) are acetylated by acetyltransferase enzymes (e.g. carnitine acetyltransferase ( CarAc ) ) yielding an acetylated product mistaken for acetylcholine (AcCh). In the past, human sperm and human seminal plasma putative ChAc activity has been extremely overestimated due to Type 1 error. This study demonstrates (1) an endogenous acetyltransferase and substrate activity in human sperm and human seminal plasma forming an acetylated product that is not AcCh but probably acetylcarnitine ( AcCar ); (2) that the addition of 5 mM choline substrate does not significantly increase acetyltransferase activity; (3) that boiled seminal plasma contains an endogenous acetyltransferase substrate which is not choline, but probably L-carnitine. Type 2 error occurs when endogenous carnitine acetyltransferase synthesizes true AcCh, resulting in mistaken evidence for ChAc. This is demonstrated by the fact that the choline substrate Km-value for the neuronal or true ChAc from mouse brain is 0.73 +/- 0.06 mM while the Km-value of choline substrate for purified CarAc from pigeon breast muscle is 108 +/- 4 mM. Type 2 error has occurred for the estimation of putative ChAc in rat heart. The rat heart ChAc was measured in previous studies utilizing a concentration of 30 mM choline substrate. While saturation of neuronal ChAc is observed at 2-5 mM choline, saturation of the rat heart CarAc enzyme is not reached until over 800 mM. Purified CarAc significantly synthesizes AcCh at 30 mM choline. Thus, putative ChAc has been greatly overestimated in the scientific literature for mammalian sperm, human seminal plasma and rat heart.     

Copper(II) and zinc(II) complexes of the peptides Ac-HisValHis-NH2 and Ac-HisValGlyAsp-NH2 related to the active site of the enzyme CuZnSOD

Copper(II) and zinc(II) complexes of the peptides Ac-HisValHis-NH2 and Ac-HisValGlyAsp-NH2 related to the active site of the enzyme CuZnSOD were studied by potentiometric and spectroscopic (UV-Vis, CD and EPR) techniques. The results reveal that both ligands have effective metal binding sites, but the tripeptide is a much stronger complexing agent than the tetrapeptide. The formation of a macrochelate via the coordination of the imidazolyl residues is suggested in the copper(II)-Ac-HisValHis-NH2 system in the acidic pH range, while a 4N complex predominates at physiological pH. The interaction of Ac-HisValHis-NH2 with zinc(II) results in the formation of a precipitate indicating polynuclear complex formation. Both copper(II)-Ac-HisValHis-NH2 and copper(II)-HisValHis systems exhibit catalytic activity toward the dismutation of superoxide anion at physiological pH, but the saturated coordination sphere of the metal ions in both systems results in low reactivity as compared to the native enzyme.     

General occurrence of binding to acetylcholinesterase-substrate complex in noncompetitive inhibition and in inhibition by substrate

To assess the relative importance of binding to enzyme-substrate complex (E.S) and to acetylenzyme (EA), noncompetitive inhibition has been studied in hydrolysis by acetylcholinesterase (AcChE) of cationic and uncharged substrates - acetylcholine (AcCh), 3,3-dimethylbutyl acetate, n-butyl acetate, 2-(methylammonio)ethyl acetate, 2- (N,N-diethyl-N-n-butylammonio)ethyl acetate (DEBAAc) and 2-(methylsulfonyl)ethyl acetate. For the N-trimethyl quaternary ions related to AcCh, tetramethylammonium ion, choline and choline ethyl ether, noncompetitive inhibition (Ki(nonc) is more favorable with the slower substrates than with AcCh, i.e., when E.S greater than EA, and is attributed to formation of enzyme-substrate-inhibitor complexes, E.S.I'. Noncompetitive inhibition by tetraethyl-, tert-butyl- and isopropylammonium ions, and acetamidocholine and its lower dimethyl analogue, is also attributed to E.S.I' complexes. Peripheral binding of these inhibitors decreases acylation more than deacylation. Some tertiary dimethylamonio ions have more favorable Ki(nonc) values with AcCh, decreasing deacylation more than acylation. The substrate DEBAAc is a more effective noncompetitive than competitive inhibitor in hydrolysis of AcCh, indicating that it binds more strongly in a peripheral site than in the active site of the free enzyme. In its hydrolysis by AcChE, it acts as its own noncompetitive inhibitor, by this non-productive binding. Formation of E.S.I' complexes is a general characteristic of hydrolysis by AcChE and decrease in rates at high concentrations of AcCh and related substrates is attributed to peripheral regulatory site binding, formation of E.S.S' complexes, rather than to binding to the acetylenzyme.     

Inhibition of iron-molybdenum cofactor binding to component I of nitrogenase

Tetrathiomolybdate inhibits iron-molybdenum cofactor (FeMo cofactor) binding to component I of nitrogenase. Molybdenum-iron cluster (a subcomponent of FeMo cofactor) and tetrathiomolybdate inhibited FeMo cofactor activation of inactive nitrogenase component I in extracts of Azotobacter vinelandii and Klebsiella pneumoniae mutant strains defective in the biosynthesis of FeMo cofactor. Addition of tetrathiotungstate, the tungsten analog of tetrathiomolybdate, to the mutant extracts had no significant inhibitory effect on subsequent activation by FeMo cofactor.     

High-performance liquid chromatography followed by peroxyoxalate chemiluminescence detection of acetylcholine and choline utilizing immobilized enzymes

A sensitive and selective method for the simultaneous determination of acetylcholine (ACh) and choline (Ch) is reported. ACh and Ch were separated on a reversed-phase column, passed through an immobilized enzymes (acetylcholine esterase and choline oxidase) column, and converted to hydrogen peroxide. The generated hydrogen peroxide was detected by the peroxyoxalate chemiluminescence reaction. The linear determination ranges were from 10 pmol to 10 nmol. The detection limit for both cholines was 1 pmol.     

Patterns of tolerance to heavy metals among methane-utilizing bacteria

The susceptibility of obligate methane-utilizing bacteria, including 14 reference strains and 175 environmental isolates, to five readily available heavy metal pollutants was determined. The chloride salts of Hg(II), Cd(II), Cu(II), Cr(III) and Zn(II) were tested in a system free from organic matter. Methane-utilizers appeared to have relatively discrete metal tolerance patterns with resistances to all metals varying with isolation site. Methanotrophs proved to be quite sensitive to mercury and cadmium but relatively resistant to copper, chromium and zinc     

Synthesis, antimicrobial activity and chemotherapeutic potential of inorganic derivatives of 2-(4'-thiazolyl)benzimidazole[thiabendazole]: X-ray crystal structures of [Cu(TBZH)2Cl]Cl.H2O.EtOH and TBZH2NO3 (TBZH=thiabendazole)

Thiabendazole (TBZH) reacts with iron(III) nitrate causing protonation of the ligand to yield the nitrate salt [TBZH(2)NO(3)] (1). Reaction of TBZH with copper(II) acetate results in the deprotonation of the ligand yielding [Cu(TBZ)2.(H2O)2] (2). Reactions of TBZH with the chloride, nitrate and butanedioate salts of copper(II) yields [Cu(TBZH)2Cl]Cl.H2O.EtOH (3), [Cu(TBZH)(2)(NO(3))(2)] (4) and [Cu(TBZH)(O(2)C-CH(2)CH(2)-CO(2))] (5), respectively. The TBZH acts as a neutral chelating ligand in 3-5. Molecular structures of 1 and 3 were determined crystallographically. In 1, the asymmetric unit contains one TBZH(2)(+) cation and one NO(3)(-) anion. The structure of 3 comprises a five coordinate copper centre with the metal bound to two chelating TBZH ligands and one chloride. The geometry is best described as trigonal bipyramidal. Hydrogen bonding connects the complex cation with the uncoordinated chloride anion and the water and ethanol solvate molecules. Compound 1 and the copper complexes 2-5, the metal free ligands and a number of simple copper(II) salts were each tested for their ability to inhibit the growth of Candida albicans. The metal free TBZH and its nitrate salt (1) exhibited very poor activity. Complex 2, in which the TBZH is present as an anionic ligand (TBZ(-)), exhibits moderate activity towards the pathogen. Chelation of the neutral TBZH to copper centres (complexes 3-5) results in potent anti-candida activity. The dimethyl sulphoxide (DMSO) soluble complexes 3 and 4, along with metal free TBZH were assessed for their cancer chemotherapeutic potential towards two human epithelial-derived cancer model cell lines. Complexes 3 and 4 displayed similar dose-dependent cytotoxicity in both cell lines with IC(50) values of approximately 50 microM, which were found to be significantly lower than that for metal free TBZH.     

Comparative efficacy of several potential treatments for copper mobilization in copper-overloaded rats

d-Penicillamine (DPA) is effective in the treatment of Wilson’s disease, whereas zinc salts are also used as a therapy for this disorder of copper transport. Recently, it has been shown that the copper chelators 1,4,7,11-tetraazaundecane tetrahydrochloride (TAUD) and tetraethylenepentamine pentahydrochloride (TETREN) could be useful for copper mobilization in rats. Because these agents could be potential clinical alternatives to DPA for patients with Wilson’s disease who are intolerant to this drug, we examined whether oral administration of TAUD and TETREN could be effective in mobilizing copper in experimental copper-overloaded rats. The efficacy of a combined administration of zinc and DPA, TAUD, or TETREN was also assessed. Rats were copper loaded with 0.125% copper acetate in water for 12 wk. After this period, DPA, TAUD, and TETREN were administered by gavage at 0.67 mmol/kg/d for 5 d, and zinc was given at 2.5 mg Zn/kg/d. Twelve weeks of copper loading resulted in a 32-fold increase in total hepatic copper. TETREN was the most effective chelator in increasing the urinary excretion of copper. However, it did not reduce significantly the hepatic copper levels. In turn, combined administration of zinc and chelating agents significantly reduced the amount of copper found in the feces. Although TAUD and TETREN showed a similar or higher efficacy to DPA in mobilizing copper, concurrent treatment of chelating agents and zinc salts should be discarded according to the current results.     

Hydrolysis of a mRNA 5'-cap model substrate, 5',5'-ApppA by di- and trinuclear zinc(II) complexes of a polyamino-polyol ligand

Copper(II) and zinc(II) complexes of a polyamino-polyol ligand 1,3,5-trideoxy-1,3,5-tris(methylamino)-cis-inositol (tmci) have been investigated as potential candidates for the selective elimination of the 5'-cap structure of mRNA. A cap-model compound ApppA has been utilised as substrate for studying the effect of the different metal ion complex catalysts on the hydrolysis of the triphosphate bridge. Kinetic experiments have been performed by the variation of pH, metal-to-ligand ratio and total concentrations of the metal ion and ligand. The zinc(II) complexes of tmci have been proved to possess a remarkable activity for the hydrolysis of ApppA. The observed rate enhancement compared to the uncatalysed reaction was found to be 12,000-fold, in the presence of 4.5mM zinc(II) and 1.5mM tmci at pH approximately 7.5. In contrast with the copper(II) containing systems, an extra product has also been formed during the cleavage process, beside the expected AMP and ADP. According to the ESI-MS characterisation of the samples, the additional product is a covalent phosphoester adduct of AMP and the ligand. The formation of this species is initiated by a nucleophilic attack of a zinc(II)-bound alcohol or alkoxo group on one of the alpha phosphate groups of ApppA, which leads to the formation of a phosphodiester bond. In an alternative pathway, the substrate is cleaved into AMP and ADP. According to the pH-potentiometric studies, performed with the tmci-zinc(II) system, di- and trinuclear complexes are responsible for the accelerated ApppA hydrolysis. The copper(II)-tmci 2:1 system showed only a modest kinetic activity. The rate acceleration significantly increased when threefold excess of copper(II) was applied. Although, the detailed investigations above pH approximately 6.6 have been prevented by precipitate formation during the addition of the substrate into the reaction solution, the activity of the copper(II)-tmci 3:1 system does not exceed that of the zinc(II) complexes. Due to the specific mechanism leading to the covalent extra product, the zinc(II) complexes of tmci provide a comparable rate enhancement for ApppA hydrolysis to the widely studied lanthanide or copper(II) species, in spite of the fact that they are stronger Lewis acids.     

LABELLING OF ACETYLCHOLINE IN THE BRAIN OF MICE FED ON A DIET CONTAINING DEUTERIUM LABELLED CHOLINE: STUDIES UTILIZING GAS CHROMATOGRAPHY—MASS SPECTROMETRY

—A method to achieve labelling of the acetylcholine stores of the brain under ideal physiological conditions is described. To this end, mice fed on a choline free diet were supplied with deuterium labelled choline in the drinking water. Labelled and unlabelled choline in plasma and in the brain as well as labelled and unlabelled acetyicholine in the brain were measured by a gas chromatographic-mass spectrometric method. It was found that after 1–25 days on the deuterium choline diet, substantial amounts of the plasma choline and brain acetylcholine were displaced by deuterium choline and deuterium acetylcholine, respectively. Already on the first day, the mole ratio of deuterium choline/total choline in plasma was 0·22, and it approached a maximum of 0·57 on the 14th day. The mole ratios of deuterium acetylcholine/total acetylcholine in the brain were slightly but significantly lower than those of deuterium choline/total choline in plasma 1–14 days, but asymptotically approached the mole ratios of deuterium Ch/total Ch in plasma by 25 days. Intact brains submitted to incubation at room temperature for 10 min increased their total choline content by about 500 per cent. Concurrently, in brains from animals kept on a deuterium choline diet for 1–2 days, the level of deuterium choline rose only by 50 per cent after incubation. Deuterium choline levels increased, however, by 200–300 per cent in the brains from animals kept on the deuterium diet for longer time periods. On the basis of these data it is suggested that: (a) choline in plasma is partly supplied from the food and partly from endogenous sources; (b) plasma choline rapidly equilibrates (less than one day) with a pool of Ch in the brain which is responsible for biosynthesis of acetylcholine; (c) the size of this choline pool is in the order of 34–40 nmol/g.     

Reaction of N,N-diethyldithiocarbamate and other bidentate ligands with Zn, Co and Cu bovine carbonic anhydrases. Inhibition of the enzyme activity and evidence for stable ternary enzyme-metal-ligand complexes

The reactions with N,N-diethyldithiocarbamate (DDC) of zinc, cobalt and copper carbonic anhydrase from bovine erythrocytes were investigated. The native zinc enzyme was inhibited by DDC, but no removal of zinc could be detected even at a very high [ligand]/[protein] ratio. At identical pH values a larger inhibitory effect was found for the cobalt enzyme. The metal was removed by DDC from the protein at pH less than 7.0. No cobalt removal occurred at pH 10, where a stable ternary complex with the enzyme-bound Co(II) was detected. Its optical and EPR spectra are indicative of five-coordinate Co(II). The reaction of the Cu(II) enzyme with stoichiometric chelating agent was marked by the appearance of an electronic transition at 390 nm (epsilon = 4300 M-1 X cm-1). Metal removal from the copper enzyme readily occurred as the ligand was in excess over the metal, with parallel appearance of a band at 440 nm, which was attributed to the free Cu(II)-DDC complex. Also, in the case of the copper enzyme an alkaline pH was found to stabilize the ternary adduct with the diagnostic 390 nm band. EPR spectra showed that the ternary adduct is a mixture of two species, both characterized by the presence in the EPR spectrum of a superhyperfine structure from two protein nitrogens and by a low g parallel value, indicative of coordination to sulfur ligands. It is suggested that the two species contain the metal as penta- and hexacoordinated, respectively. Measurements of the longitudinal relaxation time, T1, of the water protons suggested that water coordination is retained in the latter case. Hexacoordination with retention of water is also proposed for the Cu(II) derivatives with the bidentate oxalate and bicarbonate anions, unlike the corresponding Co(II) derivatives, which are pentacoordinated. Different coordination of Co(II) and Cu(II) adducts may be relevant to the difference of activity of the two substituted enzymes.     

Dipole Moment of Acetylcholine and Its Relevance to the Chemical Synaptic Transmission

The dipole moment of acetylcholine (AcCh) has been measured in chloroform and a value of 8.49 D was obtained. Such a value actually represents the total dipole moment of the ion pair (AcCh)⁺(Cl)^-. The dipole moment of the (AcCh)⁺ cation alone turned out to be 2.65 D whereas its theoretical value obtained after a vectorial calculation was 1.65 D. The discrepancy was related to an interaction between AcCh and the solvent. The meaning of the measured value is discussed on the basis of a recent theory of chemical synaptic transmission based on the assumption of a much higher dipole moment value for the AcCh molecule.     

The ultrastructural ionic fixation technique localizing acetylcholinesterase activity, reveals simultaneously acetylcholine-like cation in the synaptic vesicles of the motor nerve terminals

A new cytochemical technique is proposed for side by side localization of acetylcholine and of acetylcholinesterase activity of motor end-plate at ultrastructural level. The technique is based on the simultaneous "ionic fixation" of vesicular acetylcholine and of histochemical copper thiocholine precipitate with phosphomolybdic acid: the molybdic heteropolyanion forms insoluble salts with these two quaternary ammonium cations, providing in situ "acetylcholine phosphomolybdate" and "copper thiocholine phosphomolybdate". Both of them are osmium resistant; the electron dense precipitates allow for a fine localization of acetylcholine and acetylcholinesterase activity at electron microscopic level.     

An enzyme-reactor for electrochemical monitoring of choline and acetylcholine: applications in high-performance liquid chromatography, brain tissue, microdialysis and cerebrospinal fluid.

A sandwich-type enzyme reactor in which the enzymes are physically immobilized in a minimal dead space between two cellulose membranes, resulting in improved sensitivity, was developed for the electro-chemical detection of choline (Ch) and acetylcholine (ACh). The reactor contains the enzymes choline oxidase with or without acetylcholine esterase, for the detection of ACh and Ch, respectively. For the HPLC analysis of Ch and ACh the detection system was coupled post column. Levels of Ch and ACh of rat striatum tissue and human cerebrospinal fluid were found to be similar to those determined with published methods. Because of low back pressure--a further advantage of the reactor--the detection system could also be directly coupled to the outlet of a microdialysis device, allowing the on-line real-time measurement of extracellular brain Ch. The versatility of the enzyme reactor for the monitoring of analytes in HPLC eluates, flow injection analysis, with or without prepurification, is emphasized. The usefulness of the reactor-detector system in biomedical applications is illustrated by the measurement of increases of rat striatal extracellular Ch following cardiac arrest.     

Aqueous dissolution of Alzheimer's disease Abeta amyloid deposits by biometal depletion.

Zn(II) and Cu(II) precipitate Abeta in vitro into insoluble aggregates that are dissolved by metal chelators. We now report evidence that these biometals also mediate the deposition of Abeta amyloid in Alzheimer's disease, since the solubilization of Abeta from post-mortem brain tissue was significantly increased by the presence of chelators, EGTA, N,N,N',N'-tetrakis(2-pyridyl-methyl) ethylene diamine, and bathocuproine. Efficient extraction of Abeta also required Mg(II) and Ca(II). The chelators were more effective in extracting Abeta from Alzheimer's disease brain tissue than age-matched controls, suggesting that metal ions differentiate the chemical architecture of amyloid in Alzheimer's disease. Agents that specifically chelate copper and zinc ions but preserve Mg(II) and Ca(II) may be of therapeutic value in Alzheimer's disease.     

Complexes of zinc, copper, and nickel with the nonprotein amino acid L-alpha-amino-beta-methylaminopropionic acid: a naturally occurring neurotoxin

The non-protein amino acid L-alpha-amino-beta-methylaminopropionic acid (L-MeDAP) causes motor neuron dysfunction in macaques. The amino acid is a potent chelator of divalent metal ions such as copper and zinc. Binding constants nickel(II) for copper(II) and zinc(II) with L-MeDAP have been measured. Some copper(II) complexes of L-MeDAP have been synthesized and characterized by their electronic, infrared, and epr spectra. These results are used to comment on the possibility that metal ion complexation in the central nervous system is involved in the expression of the meurotoxicity of L-MeDAP.     

Effects of thio-group modification and Ca2+ on agonist-specific state transitions of a central nicotinic acetylcholine receptor.

Agonist-binding affinities of central nervous system nicotinic acetylcholine receptors (nAcChR) are sensitive to the duration of exposure to agonist. These agonist-induced changes in receptor state may be mimicked by appropriate modification of receptor thio groups and/or by manipulation of solvent ionic composition. In the absence of Ca2+, the concentration of acetylcholine (AcCh) necessary to prevent half of specific 3H-labeled alpha-bungarotoxin binding is approximately 1 mM for nAcChR treated with dithiothreitol (DTT) or DTT-N-ethylmaleimide (low-affinity states) and approximately 40 microM for nAcChR treated with DTT-5,5'-dithiobis(2-nitrobenzoic acid) or for native nAcChR pretreated with AcCh (high-affinity states). Addition of Ca2+ results in an increase in the effectiveness of AcCh toward blocking toxin binding. None of these treatments alters toxin or antagonist binding nor are there observed differences in Hill numbers for agonist binding. Agonists competitively inhibit toxin binding to low-affinity states, but noncompetitive inhibition is observed for binding to high-affinity states. Values of AcCh dissociation constants estimated from these data fall within the range of values determined physiologically with nAcChR from other systems. The data indicate that the redox state of brain nAcChR thio groups and Ca2+ may mediate physiologically important changes in the receptor state during activation and desensitization.     

Preferences of kanamycin A towards copper(II). Effect of the resulting complexes on immunological mediators production by human leukocytes

The widespread presence of pathogenic bacteria is a cause of permanent demand for investigating the properties of antimicrobial agents. The chemical basis of several toxic effects induced by antibiotics still remains unclear. Aminoglycosides, highly ototoxic and nephrotoxic drugs, are capable of copper(II) ions chelating. In this study we established the affinity of kanamycin A towards copper(II), in contrast with other metal ions: iron(III), nickel(II), cobalt(II) and zinc(II) by means of potentiometry. Circular dichroism spectroscopy was applied to monitor the competition of copper(II) partition between kanamycin A and human serum albumin. We show, that the drug is able to digest Cu(II) ions from HSA to some extent and comparing the stability constants for metal and antibiotic with those, obtained for the N-terminal Asp-Ala-His-Lys (DAHK) sequence, which constitutes a copper(II) binding domain within albumin, we demonstrate that the Cu(II)-kanamycin A complex formation is possible also in blood plasma. Bioassays and immunoassay were used to find out the possibility of Cu(II)-kanamycin A complexes to induce cytokines: tumor necrosis factor (TNF), interferon (IFN) and interleukin-10 (IL-10) in human peripheral blood leukocytes. The effect on the cytokines release was dose and time dependent and the interdependence between IL-10 and TNF stimulation was found. We report that Cu(II)-aminoglycoside systems can act as moderate inducers of TNF-alpha, IFN-alpha/beta and IL-10 released from human leukocytes. We have also found that these complexes are non-toxic for human A549 cells.