Interaction of Proflavine and Acriflavine with Acetylcholinesterase

Abstract

1.Purified acetylcholinesterase from toluene-treated organs of the electric eel (form B) was inhibited in a noncompetitive manner by the acridine derivatives proflavine and acriflavine. With proflavine secondary plots of slopes and intercepts revealed hyperbolic inhibition patterns. The secondary plots of acriflavine showed a biphasic inhibition pattern with two different slopes (slope, > slope,). A similar biphasic inhibition pattern was observed in the Dixon plot. From the two slopes two apparent Ki-values were obtained. Between 0 and 1 pM acriflavine (low concentrations of acriflavine) the apparent inhibition constant was 0.6 pM, a t acriflavine concentrations greater than 1 pM (high concentrations of acriflavine) the apparent inhibition constant was 2.2 pM. The apparent K , values for acetylthiocholine were 52 pM and 99 respectively. The relative V were 0.37 IU and 0.26 IU respectively. At high concentrations of acriflavine the double-reciprocal plot of substrate concentration versus enzyme activity became hyperbolic and the Hill coefficient for acetylthiocholine was 0.6. However, in presence of 2 mM hexamethonium it increased to 0.9.2. Double inhibitor studies were carried out with acriflavine and atropine, d-( +)tubocurarine, hexamethonium, decamethonium, nicotine or tensilone. At a constant concentration of acriflavine, atropine, d.( +)tubocurarine, hexamethonium and to a lesser extent decamethonium decreased the inhibition by acriflavine. Nicotine and tensilone showed no such effect. Yonetani-Theorell plots with atropine, d-( +)tubocurarine, hexamethonium or decamethonium as constant inhibitors and acritlavine as variable inhibitor revealed a decrease in slopes, a t acriflavine concentrations above 1 p3f. At acriflavine concentrations below 1 pM slopes, did not significantly differ from each other for the pairs of inhibitors investigated. From the Yonetani-Theorell plots a-values for acriflavine (fixed inhibitor) and nicotine and tensilone (variable inhibitors) were determined. For slopes, the values ranged between 10 and 100 depending on substrate concentration, for slope, the values were 1-10 a t 0.1 mM substrate and reached infinity at 5 mM substrate.3. Addition of proflavine and acriflavine to acetylcholinesterase produced a bathochromic and hypochromic shift in the visible absorption spectrum. A maximum difference in the spectra was obtained at 465 nm for proflavine and at 472 nm for acriflavine. Spectral titration of acetylcholinesterase with proflavine revealed normal binding with proflavine concentrations up to 22 pM. At higher concentrations the change in absorbance was significantly greater than expected for a simple stoichiometry of one mole of dye bound per mole of active site. The Hill coefficients were 1.03 and 2.26 respectively. The binding of acriflavine followed a theoretical binding curve up to a concentration of 10 @. At higher concentrations the change in absorbance was again greater. For the acriflavine binding the Hill Coefficients were 1.05 and 1.75 respectively. Addition of a seco...

Quinacrine (mepacrine) action at frog end‐plate.

“…(Wermuth & Brodbeck, 1973), quinacrine possessed significant anticholinesterase activity at the rather high concentrations used in the e.p.c. and m.e.p.c.…”

Changes in the cooperativity of diaphragm-associated acetylcholinesterase induced by monovalent (Na+, Li+) cations

“…This hypothesis has been also supported by the studies of several authors (Kitz et al 1970;Rosenbery and Bernhard 1971;Kato 1972;Kato et al 19726). The kinetic properties of AChE are markedly affected by the presence of both organic (Wermuth and Brodbeck 1973;Pattison and Bemhard 1978) and inorganic ions (Tomlinson et al 1980, 198 1, ABBREVIATIONS: A C E , acetylcholinesterase; Khl, Michaelis constant;DTNB, 5,5'-dithionitrobenzoic acid. and 1982;Kouniniotou-Krontiri and Tsakiris 1984b;Hughes and Bennett 1985;Perez-Guillermo et al 1987).…”