Production in Large Quantities of Actinorhodin and Undecyl-prodigiosin Induced by afsB in Streptomyces lividans

Paraquat inhibited the acetylcholinesterase activity of human erythrocytes and electric organs of Electrophorus electricus. The inhibition of acetylcholinesterase activity was reversible, as shown from the following two experimental results: [I] The degree of inhibition was not affected by changing the preincubation time of the enzyme and paraquat before the addition of the substrate. [II] The enzyme, preincubated with paraquat and subsequently freed from inhibitor by gel filtration on Sephadex G-25, showed the same activity as the untreated enzyme. Paraquat gave effective protection against the inhibition by an irreversible anionic site inhibitor, dibenamine, but not by irreversible esteratic site inhibitors, dichlorvos and methanesulfonyl chloride. These results indicate that paraquat functions as a reversible inhibitor for the anionic site. The inhibitory powers and Hill coefficients of paraquat and diquat were compared with the other quaternary ammonium compounds. Although secondary to edrophonium, paraquat strongly inhibited acetylcholinesterases of human erythrocytes and electric eel, and showed higher inhibition selectivity for both acetylcholinesterases than for human plasma butyrylcholinesterase. The Hill coefficients concerning the interaction of paraquat with acetylcholinesterases of human erythrocytes and electric eel were given as 0.83 and 0.73, respectively. This indicates negative cooperativity between these enzymes and paraquat, which is similar to the case with d-tubocurarine. On the other hand, diquat showed weak inhibitory power and low inhibition selectivity, and its Hill coefficients were almost 1.0, indicating a competitive inhibition mode.     

Identification of a modifier site on acetylcholinesterase with affinity for d-tubocurarine

Decamethonium and d-tubocurarine displace N-methylacridinium ion, a potent fluorescent inhibitor of acetylcholinesterase, from the surface of the enzyme. Decamethonium is competitive with N-methylacridinium which indicates that the binding sites for these ligands overlap. However, the displacement of N-methylacridinium ion by d-tubocurarine requires the existence of a binding site for d-tubocurarine in addition to the active site. Since the affinities for d-tubocurarine at both sites are comparable, two well defined ligand binding sites must exist for each catalytic site that is titratable by 7-dimethylcarbamyl-N-methylquinolinium iodide.     

Identification of covalently bound inositol in the hydrophobic membrane-anchoring domain of Torpedo acetylcholinesterase

The hydrophobic, membrane-bound form of Torpedo acetylcholinesterase is specifically solubilized by a phosphatidylinositol-specific phospholipase C, suggesting that acetylcholinesterase is bound to the membrane via a direct and specific interaction with phosphatidylinositol (Futerman et al., Biochem. J. (1985) 226, 369-377). Here we demonstrate the presence of covalently bound inositol in the membrane-anchoring domain of purified Torpedo acetylcholinesterase. Upon removal of this domain, levels of inositol are reduced to only 15-20% of those found in the intact enzyme. The results presented strongly support our suggestion that phosphatidylinositol is indeed involved in anchoring acetylcholinesterase to the plasma membrane.     

Effects of selected cholinergic and anticholinergic drugs on Brugia malayi (Nematoda)

1. Several drugs were tested as inhibitors of the body movements of adult Brugia malayi. 2. Atropine, carbachol, DDNS (a fluorescent acetylcholine analog), diethylcarbamazine, and physostigmine caused significant reduction in motor activity. 3. Glutamate, hexamethonium, muscarine, norepinephrine, serotonin and d-tubocurarine had no effect. Three novel phosphonium compounds were tested as inhibitors of Brugia and vertebrate acetylcholinesterase. 4. Two of these produced preferential inhibition of the enzyme from Brugia.     

Differential perturbation of erythrocyte membrane function by structurally related polycyclic aromatic hydrocarbons

Examination of the interaction of a number of structurally related polycyclic aromatic hydrocarbons with the erythrocyte plasma membrane indicated that the presence and position of methyl groups on the lipophilic hydrocarbon nucleus determined whether the compound acted as an inhibitor of membrane function. 7,12-Dimethylbenz(a)anthracene, a potent carcinogen, acted as a noncompetitive inhibitor of membrane acetylcholinesterase. The inhibition depended on the anion composition of the buffer at the time of exposure of the cells to inhibitor, i.e., it was only manifest in the presence of an anion gradient. The temperature dependence of the intact cell enzyme in the presence of inhibitor was influenced by the temperature at which the compound was added prior to assay and may involve the perturbation of tightly associated lipids. Glucose exchange across the membrane was inhibited by the same compounds which inhibit acetylcholinesterase. The temperature dependence of the exchange was not grossly altered by the presence of 7,12-dimethylbenz(a)anthracene. The observed inhibition of two membrane functions by the polycyclic aromatic hydrocarbons does not correlate simply with their theoretical octanol/water partition coefficients, water solubilities, or ability to confer membrane stabilization against osmotic hemolysis. This demonstration of differential inhibition by compounds having the same overall hydrophobicity was unexpected and suggests a more complex mode of interaction with the cell membrane.     

Differential perturbation of erythrocyte membrane-associated transport and enzyme activities by structurally related lipophilic compounds

The alteration of two erythrocyte plasma membrane functions, acetylcholine hydrolysis and glucose exchange, by a series of structurally related small lipophilic compounds which exhibit similar antihemolytic behavior was studied. 2-Methyldimethylaminoazobenzene is a more potent inhibitor of acetylcholinesterase than the 3′-methyl analogue, while the unsubstituted compound fails to inhibit. Esterase inhibition by the 2-methyl compound is noncompetitive and dependent on the anion composition of the assay buffer. The temperature dependence of acetylcholinesterase activity in the presence of the 2-methyl compound suggests that interaction with inhibitor is influenced by the state of lipids tightly bound to the enzyme. Glucose exchange is inhibited to the same extent by both methyl derivatives but not by the unsubstituted dye, and the temperature dependence in the presence of inhibitor is not grossly altered. The lack of correlation between inhibition of membrane function and stabilization of erythrocytes against osmotic hemolysis is discussed.     

Acetylcholinesterase: A probe for the study of antiarrhythmic drug-membrane interactions

Structural consequences of antiarrhythmic drug interaction with erythrocyte membranes were analyzed in terms of resulting changes in the activity of membrane-associated acetylcholinesterase. When enzyme inhibitory effects of drugs were compared at concentrations producing an equivalent degree of erythrocyte antihemolysis, a number of distinct groupings emerged, indicating that the molecular consequences of drug-membrane interaction are not identical for all agents examined. Differences in drug-induced acetylcholinesterase inhibition in intact erythrocytes, erythrocyte membranes and a brain synaptic membrane preparation emphasized the role of membrane structural organization in determining the functional consequences of antiarrhythmic interaction in any given system. While the inhibitory actions of lidocaine, D-600 and bretylium in intact red cells were not altered by an increased transmembrane chloride gradient, enhanced enzyme inhibition by quinidine and propranolol was observed under these conditions. The diverse perturbational actions of these membrane-stabilizing antiarrhythmics observed here may be indicative of a corresponding degree of complexity in the mechanisms whereby substances modify the potential-dependent properties of excitable tissues.     

Differential perturbation of erythrocyte membrane function by structurally related polycylic aromatic hydrocarbons

Examination of the interaction of a number of structurally related polycyclic aromatic hydrocarbons with the erythrocyte plasma membrane indicated that the presence and position of methyl groups on the lipophilic hydrocarbon nucleus determined whether the compound acted as an inhibitor of membrane function. 7,12-Dimethylbenz(a)anthracene, a potent carcinogen, acted as a noncompetitive inhibitor of membrane acetylcholinesterase. The inhibition depended on the anion composition of the buffer at the time of exposure of the cells to inhibitor, i.e., it was only manifest in the presence of an anion gradient. The temperature dependence of the intact cell enzyme in the presence of inhibitor was influenced by the temperature at which the compound was added prior to assay and may involve the perturbation of tightly associated lipids. Glucose exchange across the membrane was inhibited by the same compounds which inhibit acetylcholinesterase. The temperature dependence of the exchange was not grossly altered by the presence of 7, 12-dimethylbenz(a)anthracene.The observed inhibition of two membrane functions by the polycyclic aromatic hydrocarbons does not correlate simply with their theoretical octanol/ water partition coefficients, water solubilities, or ability to confer membrane stabilization against osmotic hemolysis. This demonstration of differential inhibition by compounds having the same overall hydrophobicity was unexpected and suggests a more complex mode of interaction with the cell membrane.     

Effect of membrane fluidity upon binding of Electrophorus acetylcholinesterase to lipid vesicles

A previous report (Watkins, M.S., Hitt, A.S. and Bulger, J.E. (1977) Biochem. Biophys. Res. Commun. 79, 640-647) has indicated that the asymmetric forms of Electrophorus acetylcholinesterase bind exclusively to sphingomyelin vesicles through interaction with the collagen-like 'tail' portion of the enzyme. We report here that acetylcholinesterase also binds to phosphatidylcholine vesicles containing saturated fatty acyl chains and to egg phosphatidylcholine vesicles containing cholesterol. This suggests preferential binding of acetylcholinesterase to membranes of lower fluidity. Surface charge of vesicles and density of zwitterionic lipid headgroups do not significantly affect binding of native acetylcholinesterase. The presence of chondroitin sulfate or hyaluronic acid slightly increases the binding of native acetylcholinesterase to sphingomyelin vesicles, while the presence of 1 M NaCl, bovine serum albumin, or tissue fractions enriched in basement membrane diminish binding. The dissociation constant for native acetylcholinesterase and sphingomyelin vesicles is (1.0-1.5) X 10(-7) M, as measured by a flotation binding assay. The globular, 11S form of acetylcholinesterase also binds to lipid vesicles, although not to the same degree as native acetylcholinesterase. This suggests that the collagen tail of the enzyme enhances binding, but is not essential for binding to occur. These results are consistent with the location of acetylcholinesterase on the surface of the postsynaptic plasma membrane in vivo.     

Effect of erythropoietin on the different ATPases and acetylcholinesterase of rat RBC membrane

The effect of Ep on different ATPases and acetylcholinesterase of rat RBC membrane was studied. Starvation caused a slight decrease in Mg2+-, Ca2+-, and Na+ + K+-ATPases. However, these enzyme activities were markedly increased on Ep treatment of starved rats. Specific activities of all three ATPases increased linearly with increasing concentration of Ep. Under identical conditions the hormone failed to stimulate the ATPase activity of liver plasma membrane. Desensitization by fluoride of allosteric inhibition of erythrocyte membrane-bound Na+ + K+-ATPase was observed under starvation which showed a return to normal n values on Ep administration. The enzyme from normal animals was inhibited almost completely at 0.1 mM fluoride whereas enzyme from starved and Ep-treated animals showed only about 50% inhibition at that fluoride concentration. Ep increased the acetylcholinesterase activity of normal RBC membrane to a small extent whereas the stimulation was much higher under starvation. The fluoride inhibition curve of this enzyme changed from sigmoidal to hyperbolic under starvation which again changed to allosteric on administration of Ep. These changes were closely correlated to n values. Red blood cells of Ep-treated animals became more susceptible to osmotic shock under the experimental conditions.     

Nonquantum release of acetylcholine in frog neuromuscular junction after blocking of axoplasmic transport with colchicine

Standard microelectrode techniques were used to evaluate the effect of d-tubocurarine chloride on membrane potential of junctional and extrajunctional areas of muscle fibers in a potassium-free Ringer solution. The experiments were made on frogs after inactivation of acetylcholinesterase. d-Tubocurarine chloride hyperpolarized the membrane of muscle fibers only in the junctional area. Blockade of axoplasmic transport with colchicine did not affect the magnitude of the hyperpolarization response of the membrane end plate to the presence of d-tubocurarine chloride, but at the same time it significantly reduced the membrane rest potential of muscle fibers, and gave rise to the appearance of extrajunctional sensitivity to acetylcholine. It is concluded that the blockade of axoplasmic transport does not affect the pattern of non-quantum acetylcholine release from nerve terminals. Therefore, this is unlikely to cause denervation-like changes in the muscle under the conditions described.     

Action of phosphatidylinositol specific phospholipase C on platelets: nonlytic release of acetylcholinesterase, effect on thrombin and PAF induced aggregation

Phosphatidylinositol (PI) specific phospholipase C treatment of rabbit platelets caused 95% release of acetylcholinesterase in the supernatant and 4 to 6% hydrolysis of membrane PI in 2 min. Under these conditions there was no cell lysis as monitored by lack of lactate dehydrogenase activity in the medium. The phospholipase C had no activity towards phosphatidylinositol-4- phosphate and phosphatidylinositol-4,5-bis phosphate. Platelets pretreated with the phospholipase C responded normally to thrombin and platelet activating factor. It is concluded that acetylcholinesterase exists in specific interaction with PI in platelet membranes. Further, the membrane protein release phenomenon caused by the PI-specific phospholipase C did not effect the physiological responsiveness of platelets. Possible implications of these findings to the linkage between PI and membrane enzyme are also discussed.     

Characterization of acetylcholinesterase in Caco-2 cells

Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was solubilized from cultured Caco-2 cells. It was established that this enzyme activity is acetylcholinesterase by substrate specificity (acetylthiocholine, acetyl-beta-methylthiocholine>propionylthiocholine>butyrylthiocholine), substrate inhibition, and specificity of inhibitors (BW284c51>iso-OMPA). The acetylcholinesterase activity increased proportional to the degree of differentiation of the cells. Most of the enzyme was membrane bound, requiring detergent for solubilization, and the active site faced the external fluid. Only one peak of activity, which corresponded to a monomeric form, could be detected on linear sucrose density gradients. The sedimentation of this form of the enzyme was shifted depending on whether Triton X-100 or Brij 96 detergent was used. These results indicate that the epithelial-derived Caco-2 cells produce predominantly an amphiphilic, monomeric form of acetylcholinesterase that is bound to the plasma membrane and whose catalytic center faces the extracellular fluid.     

Inactivation of rat brain acetylcholinesterase by pyridoxal 5'-phosphate

Effects of pyridoxal 5'-phosphate on the activity of crude and purified acetylcholinesterase from cerebral hemispheres of adult rat brain were examined. Acetylcholinesterase was completely inactivated by incubation with 0.5 mM pyridoxal 5'-phosphate. The enzyme activity remained unaltered in the presence of analogs of pyridoxal 5'-phosphate, pyridoxal, pyridoxamine and pyridoxamine 5'-phosphate. The inhibition of acetylcholinesterase activity by pyridoxal 5'-phosphate appeared to be of a noncompetitive nature, as determined by Lineweaver-Burk analysis. The inhibitory effect of pyridoxal 5'-phosphate on acetylcholinesterase appeared to be a general one, as the activity of the enzyme from the brains of immature chick and egg-laying hen, and from different tissues of the adult male rats, exhibited a similar pattern in the presence of the inhibitor. The inhibitory effects of pyridoxal 5'-phosphate could be reversed upon exhaustive dialysis of the pyridoxal 5'-phosphate-treated acetylcholinesterase preparations. We propose that the effects of pyridoxal 5'-phosphate are due to its interaction with acetylcholinesterase, and that it can be employed as a useful tool for studying biochemical aspects of this important brain enzyme.     

Acetylcholine receptor inhibition by d-tubocurarine involves both a competitive and a noncompetitive binding site as determined by stopped-flow measurements of receptor-controlled ion flux in membrane vesicles

The issue of whether d-tubocurarine, the classical acetylcholine receptor inhibitor, inhibits the receptor by a competitive or noncompetitive mechanism has long been controversial. d-Tubocurarine, in this study, has been found to be both a competitive (KC = 120 nM) and a noncompetitive (KNC = 4 microM) inhibitor of receptor-mediated ion flux at zero transmembrane voltage in membrane vesicles prepared from Electrophorus electricus electroplax. A spectrophotometric stopped-flow method, based on fluorescence quenching of entrapped anthracene-1,5-disulfonic acid by Cs+, was used to measure both the rate coefficient of ion flux prior to receptor inactivation (desensitization) and the rate coefficient of the rapid inactivation process. Inhibition by d-tubocurarine of the initial rate of ion flux decreased with increasing acetylcholine concentration, consistent with competitive inhibition, but the inhibition by micromolar concentrations of d-tubocurarine could not be overcome with saturating concentrations of acetylcholine, consistent with noncompetitive inhibition. A minimum mechanism is proposed in which d-tubocurarine competes for one of the two acetylcholine activating sites and also binds to a noncompetitive site. The present data do not distinguish between one or two competitive sites, although one successfully accounts for all of the data. By variation of the acetylcholine concentration, the two types of sites could be studied in isolation. Binding of d-tubocurarine to the noncompetitive site does not change the rate of rapid receptor inactivation, whereas binding of d-tubocurarine to the competitive site decreases the rate of rapid inactivation by displacing acetylcholine, in agreement with the observation that d-tubocurarine does not inactivate (desensitize) the E. electricus receptor by itself.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrical excitability of artificial enzyme membranes: II. Electrochemical and enzyme properties of immobilized acetylcholinesterase membranes

This paper deals with aspects of the reciprocal interaction between enzyme activity and the microenvironment or the potential difference in artificial proteinaceous membranes bearing cross-linked acetylcholinesterase. The potential difference resulting from asymmetric substrate injection into the system is recorded as a function of time. The influence of the membrane charge density on both enzyme activity and potential difference is studied by varying the external solution pH. The enzyme specific potential is initiated by local change of pH at the membrane level and the dependence on the buffer strength is studied. The recorded potential difference appears to be the result of the reciprocal interaction between enzyme reaction and the diffusion of substrate or products.     

Inhibition of erythrocyte acetylcholinesterase by n-butanol at high concentrations

Erythrocyte acetylcholinesterase (AChE) is bound to the membrane by a complex glycosylphosphatidylinositol anchor, so the effect of alcohol on AChE activity may reflect direct and/or membrane-mediated effects. The indication of a direct interaction between n-butanol and AChE molecules is the activation/inhibition of AChE by occupation of the enzyme's active and/or regulatory sites by alcohol. The activation of AChE can occur only at low concentrations of alcohols, while at high concentrations AChE is inhibited. In this work the mechanism of inhibition of erythrocyte AChE by n-butanol at high concentrations was studied. The values of activity, calculated assuming parabolic competitive inhibition, which implies that one or two molecules of inhibitor bind to the enzyme, fit well to the experimental values. From the values of the inhibition constants it was concluded that at high n-butanol concentrations two alcohol molecules usually interact with AChE.     

Fluorinated aldehydes and ketones acting as quasi-substrate inhibitors of acetylcholinesterase.

1. The inhibition of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) by compounds containing trifluoromethyl-carbonyl groups was investigated and related to the effects observed with structurally similar, non-fluorinated chemicals. 2. Compounds that in aqueous solution readily form hydrates inhibit acetylcholinesterase in a time-dependent process. On the other hand non-hydrated, carbonyl-containing compounds showed rapid and reversible, time-independent enzyme inactivation when assayed under steady state conditions. 3. m-N,N,N-Trimethylammonium-acetophenone acts as a rapid and reversible, time-independent, linear competitive inhibitor of acetylcholinesterase (Ki = 5.0 . 10(-7) M). 4. The most potent enzyme inhibitor tested in this series was N,N,N,-trimethylammonium-m-trifluoroacetophenone. It gives time-dependent inhibition and the concentration which inactivates eel acetylcholinesterase to 50% of the original activity after 30 min exposure is 1.3 . 10(-8) M. The bimolecular rate constant for this reaction is 1.8 . 10(6) 1 . mol-1 . min-1. The enzyme-inhibitor complex is very stable as the inhibited enzyme after 8 days of dialysis is reactivated to 20% only. This compound represents a quasi-substrate inhibitor of acetylcholinesterase.     

The inhibitory effects of polyoxyethylene detergents on human erythrocyte acetylcholinesterase and Ca2+ + Mg2+ ATPase

The activities of acetylcholinesterase and Ca2+ + Mg2+ ATPase were measured following treatment of human erythrocyte membranes with nonsolubilizing and solubilizing concentrations of Triton X-100. A concentration of 0.1% (v/v) Triton X-100 caused a significant inhibition of both enzymes. The inhibition appears to be caused by perturbations in the membrane induced by Triton X-100 incorporation. No acetylcholinesterase activity and little Ca2+ + Mg2+ ATPase activity were detected in the supernatant at 0.05% Triton X-100 although this same detergent concentration induced changes in the turbidity of the membrane suspension. Also, no inhibition of soluble acetylcholinesterase was observed over the entire detergent concentration range. The inhibition of these enzymes at 0.1% Triton X-100 was present over an eightfold range of membrane protein in the assay indicating an independence of the protein/detergent ratio. The losses in activities of these two enzymes could be prevented by either including phosphatidylserine in the Triton X-100 suspension or using Brij 96 which has the same polyoxyethylene polar head group but an oleyl hydrophobic tail instead of the p-tert-octylphenol group of Triton X-100. The results are discussed in regard to the differential recovery of enzyme activities over the entire detergent concentration range.     

Inactivation of rat brain acetylcholinesterase by pyridoxal 5′-phosphate

Effects of pyridoxal 5′-phosphate on the activity of crude and purified acetylcholinesterase from cerebral hemispheres of adult rat brain were examined. Acetylcholinesterase was completely inactivated by incubation with 0.5 mM pyridoxal 5′-phosphate. The enzyme activity remained unaltered in the presence of analogs of pyridoxal 5′-phosphate, pyridoxal, pyridoxamine and pyridoxamine 5′-phosphate. The inhibition of acetylcholinesterase activity by pyridoxal 5′-phosphate appeared to be of a noncompetitive nature, as determined by Lineweaver-Burk analysis. The inhibitory effect of pyridoxal 5′-phosphate on acetylcholinesterase appeared to be a general one, as the activity of the enzyme from the brains of immature chick and egg-laying hen, and from different tissues of the adult male rats, exhibited a similar pattern in the presence of the inhibitor. The inhibitory effects of pyridoxal 5′-phosphate could be reversed upon exhaustive dialysis of the pyridoxan 5′-phosphate-treated acetylcholinesterase preparations. We propose that the effects of pyridoxal 5′-phosphate are due to its interaction with acetylcholinesterase, and that it can be employed as a useful tool for studying biochemical aspects of this important brain enzyme.