Immobilized electric eel acetylcholinesterase. I. Kinetics of acetylcholinesterase trapped in polyacrylamide membranes.

Techniques are described for the trapping of electric eel acetylcholinesterase in polyacrylamide gel. The activity of the trapped enzyme was substantially reduced, the effect being due to inhibition by acrylamide, but the emzyme immobilized in polyacrylamide was considerable more stable than that in free solutionma kinetic study was made of the hydrolysis of acetylthiocholine, covering a range of membrane thicknesses, enzyme concentrations, substrate concentrations and temperatures. The results were interpreted with reference to the theoretical treatment of Sundaram, Tweedale and Laidler, and of Kobayaski and Laidler, and provided support for those treatments; Clear evidence was obtained for diffusion control with the thicker membranes. An activation energy was obtained for the diffusion of the substrate within the membrane, by combining the temperature results for thick and thin membranes at low substrate concentrations. The results lead to the conclusion that the in vivo kinetics of acetylcholinesterase are largely diffusion-free in muscle filaments, but are substantially diffusion-controlled in fibrils and fibers.     

Molecular structure of elongated forms of electric eel acetylcholinesterase.

Molecular forms of acetylcholinesterase extracted from fresh electric organ tissue of the electric eel are elongated structures in which a multi-subunit head is connected to a fibrous tail. The principal form, 18 S acetylcholinesterase, is of molecular weight approximately 1,050,000, contains about 12 catalytic subunits in its head, has a tail approximately 500 Å long, and aggregates reversibly at low ionic strength. Trypsin converts it to an 11 S globular tetramer devoid of the tail and lacking the capacity to aggregate in low-salt solutions.Amino acid analysis shows that elongated forms of acetylcholinesterase contain significant amounts of hydroxyproline and hydroxylysine, characteristic components of collagen, which are absent from 11 S acetylcholinesterase.Collagenase converts 18 S acetylcholinesterase to a 20 S form which no longer aggregates in low salt. Purified 20 S acetylcholinesterase has about half the hydroxyproline and hydroxylysine contents of the 18 S enzyme, and physicochemical measurements indicate the formation of a more symmetrical molecular structure without marked reduction in molecular weight.Sodium dodecyl sulfate/polyacrylamide gel electrophoresis without reducing agent shows that in 18 S acetylcholinesterase half the catalytic subunits are present as dimers linked by disulfide bonds. The remaining subunits migrate as larger molecular species which contain significant amounts of hydroxylysine, are specifically modified by collagenase and are converted to dimers and monomers by trypsin.Sodium dodecyl sulfate/acrylamide gel electrophoresis with reducing agent reveals, in 18 S acetylcholinesterase, two polypeptides of molecular weights 45,000 and 47,000 which are absent in the 11 S tetramer. They are readily digested by collagenase under conditions which do not affect the catalytic subunits, with concomitant formation of a new 30,000 polypeptide.The above data can be rationalized by a model in which 18 S acetylcholinestorase contains three subunit tetramers, each linked by disulfides to one strand of a collagen triple helix. Sodium dodecyl sulfate detaches those subunit dimers which are not covalently linked to the tail; trypsin attacks the distal portion of the collagen triple helix releasing discrete tetramers, and collagenase specifically attacks the triple helix near its midpoint, producing a shortened structure in which the residual tail still holds the tetramers together, but destroying the capacity for self-association at low ionic strength. This latter property may be related to the postulated role of the tail in anchoring acetylcholinesterase to the fibrillar matrix of the basement membrane.     

Chemical modification of electric eel acetylcholinesterase by tetranitromethane

The reaction of acetylcholinesterase (acetylcholinehydrolase, EC 3.1.1.7) with tetranitromethane has been studied. The reaction caused a decrease in enzyme activity as measured with the substrate acetylthiocholine under conditions where hydrolysis of the neutral substrate indophenyl acetate was unaltered. The inactivation of acetylcholinesterase by tetranitromethane was greatly accelerated by the quaternary oximes pyridine-2-aldoxime methyl nitrate or toxogonin, though not by other quaternary inhibitors tested and not by an aliphatic oxime. The enhanced inactivation by tetranitromethane in the presence of pyridine-2-aldoxime methyl nitrate was blocked by the enzyme inhibitor decamethonium.The oxime-induced inactivation of acetylcholinesterase by tetranitromethane was accompanied by significant changes in the immunological properties of the enzyme as demonstrated by complement fixation. The reaction also resulted in the disappearance of tyrosine and appearance of nitrotyrosine.     

Inhibition of electric eel acetylcholinesterase by triarylmethane dyes

The effects of three cationic triarylmethane dyes - pararosaniline (PR), malachite green (MG), methyl green (MetG) - on electric eel AChE (eAChE) activity were tested at 25 degrees C, in 100mM MOPS buffer (pH 8) containing 0.125mM 5-5-dithio-bis(2-nitrobenzoic acid), 20-120muM acetylthiocholine and 0-20muM dye. All three dyes caused reversible, linear- or hyperbolic-mixed inhibition of esteratic activity. The respective inhibitory parameters for PR, MG and MetG were K(i)=8.4+/-0.67, 1.9+/-0.51 and 0.27+/-0.017muM; alpha (competitive coefficient)=5.8+/-2.0, 4.8+/-1.8 and 2.7+/-0.32; beta (noncompetitive coefficient)=0, 0 and 0.20+/-0.011. The data were consistent with ligand binding at the peripheral site and a remote effect on substrate binding and turnover.     

Fine structural localization of acetylcholinesterase in electroplaque of the electric eel

The electroplaques composing the electric organ of the eel, Electrophorus electricus, have been utilized for the dual purpose of demonstrating the subcellular sites of acetylcholinesterase activity and as a model for comparison of the several cytochemical methods available. Fresh tissue and tissue fixed by immersion in formalin, hydroxyadipaldehyde, or glutaraldehyde was reacted with the Cu-thiocholine method, the Cu-ferrocyanide thiocholine method, or the thiolacetic acid (TAA) method using Pb, Ag, or Au as capture reagents. Controls were obtained by omission of substrate, or by addition to complete media of varying concentrations of different cholinesterase inhibitors. Reactions were run at 0–5°C at a pH range of 5.0–7.1 for 0.25 to 120 min. Regardless of the capture metal, the localization obtained with TAA as substrate was identical with that observed with acetylthiocholine, the majority of precipitate being deposited on or near the external innervated surface of the plaque and within the tubulovesicular organelles opening onto the innervated surface. Both of the thiocholine methods and the Pb-TAA method showed reaction product in synaptic vesicles of the nerve endings innervating the plaque which was uninhibitable by 10^-4 M physostigmine. All methods also showed some inhibitor-sensitive deposition of reaction product in the mucoid material forming the immediate extracellular environment of the innervated surface.     

Kinetic properties of soluble and membrane-bound acetylcholinesterase from electric eel

Using electric eel acetylcholinesterase (AChE) which was either membrane-bound (AChEm) or solubilized (AChEs), similar kinetics were seen in the absence of inhibitor or in the presence of edrophonium, trimethylammonium ion or paraoxon. Thus, both forms of the enzyme appear to behave similarly toward various inhibitors. However, in the presence of a probe sensitive to allosteric effects or changes in membrane fluidity, the two forms exhibit altered behavior. In the presence of F-, the relative rate of substrate hydrolysis by AChEm was reduced more rapidly than with AChEs, whether or not paraoxon was present. When inhibition by paraoxon (10(-7)-10(-4) M) was studied in the presence of F-, AChEs had a Hill coefficient of 1.0, whereas with AChEm the Hill coefficient changed from 0.8 to 1.5.     

Aging of soman-inhibited acetylcholinesterase: inhibitors and accelerators

The influence of 27 possible effectors, mostly bispyridinium salts, upon the dealkylation (aging) of soman-inhibited acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was examined at pH 7.6 and 25 degrees C. In the absence of effectors, the rate constant of the aging process was 4.0. 10(-2) min-1. At 2 mM, the strongest inhibitor reduced the rate to 0.8. 10(-2) min-1, whereas it was raised to 8.2. 10(-2) min-1 by the most potent accelerator.     

Immobilized electric eel acetylcholinesterasemii. II. Flow kinetics of acetylcholinesterase chemically attached to nylon tubing.

Acetylcholinesterase has been attached covalently to the inner surface of nylon tubing. An experimental study has been carried out on the flow kinetics; solutions of acetylthiocholine at various concentrations were passed through tubing at various flow rates, and measurements made of the rates of formation of product. The results were analyzed in the light of the theoretical treatment of Kobayashi and Laidler, four different methods of analysis being employed. It is found that at lower substrate concentrations and flow rates the reactions are largely diffusion controlled. The Km(app) values are substantially higher than the Km value for diffusion-free conditions, but approach it as the flow rate is increased, when the diffusion layer becomes less important. The results are entirely consistent with the Kobayaski-Laidler theory, and provide guidelines for the design of open tubular heterogeneous enzyme reactors, both for industrial and analytic purposes.     

A simple and rapid chromatographic method for the immobilization of acetylcholinesterase from electric eel.

Acetylcholinesterase from electric eel is selectively immobilized on Amberlite IR-120 resin equilibrated with Al³⁺ ions. Immobilized acetylcholinesterase activity is stable at least for 85 days in the wet state at 10°C and for 180 days in the dry state at room temperature. Activity determinations in the presence of eserine sulfate, decamethonium bromide, quinidine sulfate and butyryl thiocholine iodide suggested that the immobilized enzyme exhibited essentially the same properties as did the free enzyme.     

Kinetics for the inhibition of acetylcholinesterase from the electric eel by some organophosphates and carbamates

The inhibition kinetics for some organophosphates (paroxon, diisopropylfluorophosphate, sarin, VX, soman and soman isomers) and carbamates (physostigmine, neostigmine, pyridostigmine and carbaryl) in the reaction with acetylcholinesterase from electric eel have been studied. Dissociation constants and rate constants for the irreversible step were determined. The great differences in inhibitory power of the organophosphates were almost entirely due to differences in affinity. A possible correlation between affinity and bonding rate is discussed.     

Anatomy of acetylcholinesterase catalysis: reaction dynamics analogy for human erythrocyte and electric eel enzymes

The anatomy of catalysis (i.e., reaction dynamics, thermodynamics and transition state structures) is compared herein for acetylcholinesterases from human erythrocytes and Electrophorus electricus. The two enzymes have similar relative activities for the substrate o-nitrochloroacetanilide and o-nitrophenyl acetate. In addition, with each substrate K values and solvent deuterium kinetic isotope effects for kES and kE are similar for the two enzymes. Solvent isotope effects in mixed isotopic buffers indicate that the acylation stages of o-nitrochloroacetanilide turnover by the two enzymes are rate-limited by virtual transition states that are weighted averages of contributions from transition states of serial chemical and physical steps. Similar experiments show that the transition states for Vmax of o-nitrophenyl acetate turnover by the two enzymes are stabilized by simple general acid-base (i.e., one-proton) catalysis. These comparisons demonstrate that acetylcholinesterases from diverse sources display functional analogy in that reaction dynamics and transition state structures are closely similar.